CN104685064A - Highly multiplex PCR methods and compositions - Google Patents

Abstract

The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.

Description

Highly multiplexed PCR methods and compositions

Cross References to Related Applications

This application claims the benefit of and priority to US Utility Application No. 13/683,604, filed November 21, 2012, and US Provisional Application No. 61/675,020, filed July 24, 2012. U.S. Utility Application No. 13/683,604 is a continuation-in-part of U.S. Utility Application No. 13/300,235, filed November 18, 2011, a continuation-in-part of U.S. Utility Application No. 13/110,685, filed May 18, 2011 application, and claims the benefit of U.S. Provisional Application No. 61/675,020, filed July 24, 2012. U.S. Utility Application No. 13/110,685 required U.S. Provisional Application No. 61/395,850 filed May 18, 2010; U.S. Provisional Application No. 61/398,159 filed June 21, 2010; filed February 9, 2011 U.S. Provisional Application No. 61/462,972, filed March 2, 2011; U.S. Provisional Application No. 61/448,547, filed March 2, 2011; and U.S. Provisional Application No. 61/516,996, filed April 12, 2011. US Utility Application No. 13/300,235 claims the benefit of US Provisional Application No. 61/571,248, filed June 23, 2011. All of these applications are hereby incorporated by reference in their entirety for the teachings therein.

Statement Regarding Federally Sponsored Research or Development

This work was supported by grant number 5R44HD60423-3 awarded by the National Institutes of Health. The US Government may have rights in any patents issued based on this application.

technical field

The present invention generally relates to methods and compositions for the simultaneous amplification of multiple related nucleic acid regions in one reaction volume.

Background technique

To increase detection throughput and allow for more efficient use of nucleic acid samples, multiple oligonucleotide primers can be combined with a sample and then subjected to the polymerase chain reaction in a process known in the art as multiplex PCR (PCR) conditions to perform simultaneous amplification of multiple target nucleic acids in related samples. The use of multiplex PCR can significantly simplify experimental procedures and shorten the time required for nucleic acid analysis and detection. However, when multiple pairs are added to the same PCR reaction, non-target amplification products such as amplified primer-dimers can be generated. The risk of producing such products increases with the number of primers. These non-target amplicons significantly limit the use of the amplification products for further analysis and/or detection. Therefore, improved methods are needed to reduce the formation of non-target amplicons during multiplex PCR.

The improved multiplex PCR method will be suitable for various applications such as non-invasive prenatal genetic diagnosis (NPD). Specifically, current methods of prenatal diagnosis can alert physicians and parents to abnormalities in the growing fetus. Without prenatal diagnosis, at birth, one in 50 babies will have a serious physical or mental disability, and as many as one in 30 will have some form of congenital malformation. Unfortunately, standard methods are either inaccurate or involve invasive procedures that risk miscarriage. Methods based on maternal blood hormone levels or ultrasound measurements are non-invasive, but they are also less accurate. Methods such as amniocentesis, chorionic villus biopsy and fetal blood sampling are highly accurate, but they are invasive and carry significant risks. In the United States, amniocentesis is performed in about 3% of all pregnancies, but its use has declined in frequency over the past fifteen years.

Normal humans have two sets of 23 chromosomes in each healthy diploid cell, one copy from each parent. Aneuploidy, a condition in which cells contain too many and/or too few chromosomes in the nucleus, is thought to be responsible for a high proportion of implantation failures, miscarriages, and genetic disorders. Chromosomal abnormality testing can identify conditions such as Down syndrome, Klinefelter's syndrome and Turner syndrome in an individual or embryo, among others, in addition to increasing the chances of a successful pregnancy . Chromosomal abnormality testing is especially important as is the age of the mother, it is estimated that at least 40% of embryos between the ages of 35 and 40 are abnormal, and over the age of 40 more than half of all embryos are abnormal.

It has recently been discovered that cell-free fetal DNA and intact fetal cells can enter the maternal blood circulation. Therefore, analysis of this genetic material may allow for early NPD. Improved methods are needed to improve sensitivity and specificity and reduce the time and cost required for NPD.

Contents of the invention

In one aspect, the invention features a method of amplifying a target locus in a nucleic acid sample. In some embodiments, the method involves (i) hybridizing the nucleic acid sample to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different targets simultaneously. The pool of test primers for the locus is contacted to produce a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions to produce an amplification product comprising the amplicon of interest. In some embodiments, the method further comprises determining the presence or absence of at least one target amplicon (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% %, 99%, or 99.5% of target amplicons). In some embodiments, the method further comprises determining at least one target amplicon (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of target amplicons).

In various embodiments of any aspect of the invention, at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the amplification products are target amplicons. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.05 % of amplification products were primer dimers. In some embodiments, the library of test primers comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 test primer pairs, wherein each pair of primers includes Forward test primer and reverse test primer for seat. In some embodiments, the library of test primers comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 individual test primers that hybridize to different target loci, wherein the individual Primers are not part of a primer pair.

In various embodiments of any aspect of the invention, the concentration of each test primer is less than 100, 75, 50, 25, 10, 5, 2 or 1 nM. In various embodiments, the test primers have a GC content of between 30% and 80%, such as between 40% and 70% or between 50% and 60%, inclusive. In some embodiments, the range of GC content of the test primers (eg, maximum GC content minus minimum GC content, eg, a range of 80%-60% = 20%) is less than 30%, 20%, 10%, or 5%. In some embodiments, the test primer has a melting temperature ( _Tm ) between 40°C to 80°C, eg, 50°C to 70°C, 55°C to 65°C, or 57°C to 60.5°C, inclusive. In some embodiments, the range of melting temperatures of the test primers is less than 20°C, 15°C, 10°C, 5°C, 3°C, or 1°C. In some embodiments, the test primers are between 15 and 100 nucleotides in length, such as between 15 and 75 nucleotides, 15 to 40 nucleotides, 17 to 35 nucleotides, 18 to 30 nucleotides, between 20 and 65 nucleotides, inclusive. In some embodiments, a test primer includes a non-target-specific label, eg, a label that forms an internal loop structure. In some embodiments, the label is between two DNA binding regions. In various embodiments, the test primer includes a 5' region specific for the locus of interest, an inner region that is not specific for the locus of interest and forms a loop structure, and a 3' region specific for the locus of interest. In various embodiments, the 3' region is at least 7 nucleotides in length. In some embodiments, the 3' region is between 7 and 20 nucleotides, such as between 7 and 15 nucleotides or 7 and 10 nucleotides in length, inclusive. In various embodiments, the test primer includes a 5' region that is not specific for the locus of interest (e.g., a marker or universal primer binding site), followed by a region that is specific for the locus of interest, a region that is not specific for the locus of interest, and a region that is not specific for the locus of interest. The inner region that is specific and forms the loop structure and the 3' region that is specific for the locus of interest. In some embodiments, test primers range in length from less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the amplicon of interest is between 50 and 100 nucleotides, such as between 60 and 80 nucleotides or 60 to 75 nucleotides in length, inclusive. In some embodiments, target amplicons range in length from less than 50, 25, 15, 10, or 5 nucleotides.

In various embodiments of any aspect of the invention, the primer extension reaction conditions are polymerase chain reaction conditions (PCR). In various embodiments, the length of the annealing step is greater than 3, 5, 8, 10 or 15 minutes. In various embodiments, the length of the extending step is greater than 3, 5, 8, 10 or 15 minutes.

In various embodiments of any aspect of the invention, test primers are used to simultaneously amplify at least 1,000 different target loci in a sample comprising maternal DNA and fetal DNA from a pregnant mother of a fetus to determine the presence or absence There are fetal chromosomal abnormalities. In various embodiments, the method comprises joining universal primer binding sites to DNA molecules in the sample; amplifying the joined DNA molecules using at least 1,000 specific primers and one universal primer, producing a first set of amplified and amplifying the first set of amplification products using at least 1,000 pairs of specific primers to generate a second set of amplification products. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different primer pairs are used.

In various embodiments of any aspect of the invention, the test primers are used to simultaneously amplify at least 1,000 different target loci in a sample comprising DNA from a putative father of a fetus and to simultaneously amplify Target loci in samples of maternal DNA and fetal DNA of the pregnant mother to determine whether the putative father is the biological father of the fetus.

In various embodiments of any aspect of the invention, the test primers are used to simultaneously amplify at least 1,000 different loci of interest in a cell or cells from an embryo to determine the presence or absence of a chromosomal abnormality. In various embodiments, cells from a set of two or more embryos are analyzed, and one embryo is selected for in vitro fertilization.

In various embodiments of any aspect of the invention, the test primers are used to simultaneously amplify at least 1,000 different target loci in a forensic nucleic acid sample. In various embodiments, the length of the annealing step is greater than 3, 5, 8, 10 or 15 minutes.

In various embodiments of any aspect of the invention, the method involves simultaneously amplifying at least 1,000 different target loci in a control nucleic acid sample using test primers to generate a first set of target amplicons and simultaneously amplifying the test target loci in the nucleic acid sample to generate a second set of target amplicons; and comparing the first set and the second set of target amplicons to determine whether the target loci are present in one sample but not in the other sample, Or whether the target locus is present at different levels in the control and test samples. In various embodiments, the test sample is from an individual suspected of having an associated disease or phenotype (e.g., cancer) or an increased risk of an associated disease or phenotype; and wherein one or more of the target loci comprises Sequences (eg, polymorphisms or other mutations) associated with increased risk of phenotypes or associated diseases or phenotypes. In various embodiments, the method involves simultaneously amplifying at least 1,000 different target loci in a control sample comprising RNA using test primers to generate a first set of target amplicons and simultaneously amplifying in a test sample comprising RNA to generate a second set of target amplicons; and comparing the first set and the second set of target amplicons to determine the presence or absence of differences in RNA expression levels between the control sample and the test sample. In various embodiments, the RNA is mRNA. In various embodiments, the test sample is from an individual suspected of having an associated disease or phenotype (e.g., cancer) or an increased risk of an associated disease or phenotype (e.g., cancer); and wherein one or more of the target loci comprises an Sequences (eg, polymorphisms or other mutations) associated with or associated with increased risk of a relevant disease or phenotype. In some embodiments, the test sample is from an individual diagnosed with an associated disease or phenotype (e.g., cancer); and wherein a difference in RNA expression levels between the control sample and the test sample indicates that the target locus comprises a gene associated with the associated disease or phenotype. Sequences (eg, polymorphisms or other mutations) associated with increased or decreased risk of type.

In some embodiments of any aspect of the invention, test primers are selected from a library of candidate primers based on one or more parameters, eg, primers are selected using any of the methods of the invention. In some embodiments, test primers are selected from a pool of candidate primers based at least in part on the ability of the candidate primers to form primer dimers.

In one aspect, the invention features a method of selecting a test primer from a pool of candidate primers. In various embodiments, the selecting involves (i) calculating in silico undesired scores for most or all possible combinations of two candidate primers from the library, wherein each undesired score is based, at least in part, on two the likelihood of dimer formation between the candidate primers; (ii) removal of the candidate primer with the highest undesirable score from the pool of candidate primers; and (iii) if the candidate primer removed in step (ii) is a member of a primer pair , then the other member of the primer pair is removed from the pool of candidate primers; and (iv) optionally repeating steps (ii) and (iii), thereby selecting a pool of test primers. In some embodiments, the selection method is performed until the undesired scores of the remaining candidate primer combinations in the library are all at or below a minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to a desired number. In various embodiments, a non-ideal score is calculated for at least 80%, 90%, 95%, 98%, 99%, or 99.5% of the possible candidate primer combinations in the library. In various embodiments, the remaining candidate primers in the library are capable of simultaneously amplifying at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci. In various embodiments, the method further comprises (v) contacting a nucleic acid sample comprising the target locus with the remaining candidate primers in the library to generate a reaction mixture; and (vi) subjecting the reaction mixture to primer extension reaction conditions to generate a reaction mixture comprising Amplification product of target amplicon.

In one aspect, the invention features a method of selecting a test primer from a pool of candidate primers. In various embodiments, the selection of test primers is selected from a library of candidate primers, involving (i) calculating in silico the undesired scores for most or all possible combinations of two candidate primers from said library, where each undesired The score is based at least in part on the likelihood of dimer formation between the two candidate primers; (ii) removing from the pool of candidate primers those portions of the maximum number of combinations of the two candidate primers that have an undesirable score above a first minimum threshold (iii) if the candidate primer removed in step (ii) is a member of a primer pair, then remove another member of the primer pair from the library of candidate primers; and (iv) optionally repeat steps ( ii) and (iii), thereby selecting a pool of test primers. In some embodiments, the selection method is performed until the undesired scores of remaining candidate primer combinations in the library are all at or below a first minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to a desired number. In various embodiments, a non-ideal score is calculated for at least 80%, 90%, 95%, 98%, 99%, or 99.5% of the possible candidate primer combinations in the library. In various embodiments, the remaining candidate primers in the library are capable of simultaneously amplifying at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci. In various embodiments, the method further comprises (v) contacting a nucleic acid sample comprising the target locus with the remaining candidate primers in the library to generate a reaction mixture; and (vi) subjecting the reaction mixture to primer extension reaction conditions to generate a reaction mixture comprising Amplification product of target amplicon.

In various embodiments of any aspect of the invention, the selection method involves reducing the first minimum threshold used in step (ii) to a lower second minimum threshold and optionally repeating steps (ii) and (iii) to further reduce the number of remaining candidate primers in the library. In some embodiments, the selection method involves increasing the first minimum threshold used in step (ii) to a higher second minimum threshold and optionally repeating steps (ii) and (iii). In some embodiments, the selection method is performed until the undesired scores of the remaining candidate primer combinations in the library are all at or below a second minimum threshold, or until the number of remaining candidate primer combinations in the library is reduced to a desired number until.

In various embodiments of any aspect of the invention, the method involves, prior to step (i), identifying or selecting primers that hybridize to the locus of interest. In some embodiments, multiple primers (or primer pairs) hybridize to the same target locus, and the selection method is used to select a primer (or a primer pair) for this target locus based on one or more parameters . In various embodiments, the method involves removing from the pool, prior to step (ii), a primer pair that produces a target amplicon that overlaps a target amplicon produced by another primer pair. In various embodiments, candidate primers are selected from a group of two or more candidate primers based on one or more other parameters with an equal score of undesirability for removal from the pool of candidate primers. In some embodiments, the remaining candidate primers in the library are used as a pool of test primers in any of the methods of the invention. In some embodiments, the resulting library of test primers includes any of the primer libraries of the invention.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: the heterozygosity rate of the target locus, the correlation between the sequence at the target locus (e.g. , polymorphism), disease prevalence associated with sequence at the target locus (e.g., polymorphism), specificity of candidate primers for the target locus, size of candidate primers, target amplification The melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: heterozygosity rate for the target locus, specificity of the candidate primers for the target locus , the size of the candidate primer, the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; At least 1,000 different target loci in samples of maternal DNA and fetal DNA from pregnant mothers to determine the presence or absence of fetal chromosomal abnormalities. In various embodiments, the method comprises joining universal primer binding sites to DNA molecules in the sample; amplifying the joined DNA molecules using at least 1,000 specific primers and one universal primer, producing a first set of amplified and amplifying the first set of amplification products using at least 1,000 pairs of specific primers to generate a second set of amplification products. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different primer pairs are used. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: heterozygosity rate for the target locus, specificity of the candidate primers for the target locus , the size of the candidate primer, the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; at least 1,000 different target loci in a sample of DNA from the putative father and simultaneously amplify the target loci in a sample including maternal DNA and fetal DNA from the pregnant mother of the fetus to determine whether the putative father is the the biological father of the fetus. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: heterozygosity rate for the target locus, specificity of the candidate primers for the target locus , the size of the candidate primer, the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; and the test primers are used to simultaneously amplify At least 1,000 different target loci in a cell or cells to determine the presence or absence of chromosomal abnormalities. In various embodiments, cells from a set of two or more embryos are analyzed, and one embryo is selected for in vitro fertilization. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: heterozygosity rate for the target locus, specificity of the candidate primers for the target locus , the size of the candidate primer, the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; and the test primers are used to simultaneously amplify the forensic nucleic acid sample At least 1,000 different target loci in . In various embodiments, the length of the annealing step is greater than 3, 5, 8, 10 or 15 minutes. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: the heterozygosity rate of the target locus, the correlation between the sequence at the target locus (e.g. , polymorphism), disease prevalence associated with sequence at the target locus (e.g., polymorphism), specificity of candidate primers for the target locus, size of candidate primers, target amplification The melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; and the method involves using test primers to simultaneously amplify at least 1,000 different target loci to generate a first set of target amplicons and simultaneously amplify target loci in the test nucleic acid sample to generate a second set of target amplicons; and comparing the first and second set of target amplicons to determine the target Whether the locus is present in one sample but not the other, or whether the locus of interest is present at different levels in the control and test samples. In various embodiments, the test sample is from an individual suspected of having a relevant disease or phenotype or an increased risk of a relevant disease or phenotype; Sequences (eg, polymorphisms) associated with increased risk of phenotypes or with associated diseases or phenotypes. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In various embodiments of any aspect of the invention, the undesired score is based at least in part on one or more parameters selected from the group consisting of: the heterozygosity rate of the target locus, the correlation between the sequence at the target locus (e.g. , polymorphism), disease prevalence associated with sequence at the target locus (e.g., polymorphism), specificity of candidate primers for the target locus, size of candidate primers, target amplification the melting temperature of the target amplicon, the GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon; and the method involves using test primers to simultaneously amplify 1,000 of a control sample comprising RNA different target loci to generate a first set of target amplicons and simultaneously amplifying target loci in a test sample comprising RNA to generate a second set of target amplicons; and comparing the first and second set of target amplicons To determine the presence or absence of differences in RNA expression levels between control samples and test samples. In various embodiments, the RNA is mRNA. In various embodiments, the test sample is from an individual suspected of having an associated disease or phenotype (e.g., cancer) or an increased risk of an associated disease or phenotype (e.g., cancer); and wherein one or more of the target loci comprises an Sequences (eg, polymorphisms or other mutations) associated with or associated with increased risk of a relevant disease or phenotype. In some embodiments, the test sample is from an individual diagnosed with an associated disease or phenotype (e.g., cancer); and wherein a difference in RNA expression levels between the control sample and the test sample indicates that the target locus comprises a gene associated with the associated disease or phenotype. Sequences (eg, polymorphisms or other mutations) associated with increased or decreased risk of type. In various embodiments, at least 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci are amplified.

In one aspect, the invention features primer libraries. In some embodiments, primers are selected from a pool of candidate primers using any of the methods of the invention. In some embodiments, the library comprises primers that hybridize simultaneously to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci. In some embodiments, the library comprises primers that simultaneously amplify at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci. In some embodiments, the library comprises simultaneously amplifying at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci such that less than 60%, 40 %, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.05% of the amplification products are primers that are primer dimers. In some embodiments, the library comprises simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci such that at least 50%, 60% , 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the amplification products are primers for the target amplicon. In some embodiments, the library comprises simultaneously amplifying target loci such that among 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci at least Primers that amplify 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the targeted loci. In some embodiments, the primer library comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 primer pairs, wherein each pair of primers includes a forward test primer and a reverse test primer Primers, where each pair of test primers hybridizes to one locus of interest. In some embodiments, the library of primers comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 individual primers that each hybridize to a different target locus, wherein the individual primers Not part of the primer pair.

In various embodiments of any aspect of the invention, the concentration of each primer is less than 100, 75, 50, 25, 10, 5, 2 or 1 nM. In various embodiments, the primers have a GC content of between 30% and 80%, such as between 40% and 70% or between 50% and 60%, inclusive. In some embodiments, the GC content of the primers ranges from less than 30%, 20%, 10%, or 5%. In some embodiments, the primer has a melting temperature between 40°C and 80°C, eg, 50°C to 70°C, 55°C to 65°C, or 57°C to 60.5°C, inclusive. In some embodiments, the melting temperature of the primer ranges from less than 15°C, 10°C, 5°C, 3°C, or 1°C. In some embodiments, the length of the primer is between 15 and 100 nucleotides, such as between 15 and 75 nucleotides, 15 to 40 nucleotides, 17 to 35 nucleotides, 18 to 30 nucleotides or between 20 and 65 nucleotides inclusive. In some embodiments, a primer includes a non-target-specific label, such as a label that forms an internal loop structure. In some embodiments, the label is between two DNA binding regions. In various embodiments, the primers include a 5' region specific for the locus of interest, an inner region that is not specific for the locus of interest and forms a loop structure, and a 3' region specific for the locus of interest. In various embodiments, the 3' region is at least 7 nucleotides in length. In some embodiments, the 3' region is between 7 and 20 nucleotides, such as between 7 and 15 nucleotides or 7 and 10 nucleotides in length, inclusive. In various embodiments, the primers include a 5' region that is not specific for the locus of interest (e.g., another marker or universal primer binding site), followed by a region specific for the locus of interest, An inner region that is not specific and forms a loop structure, and a 3' region that is specific for the locus of interest. In some embodiments, primers range in length from less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the amplicon of interest is between 50 and 100 nucleotides, such as between 60 and 80 nucleotides or 60 to 75 nucleotides in length, inclusive. In some embodiments, target amplicons range in length from less than 50, 25, 15, 10, or 5 nucleotides.

In one aspect, the invention provides a kit comprising any one of the library of primers of the invention for amplifying a target locus in a nucleic acid sample. In some embodiments, the kit includes instructions for using the library to amplify the locus of interest.

In one aspect, the invention features methods for determining the ploidy state of chromosomes in a gestating fetus. In some embodiments, the methods involve simultaneously hybridizing a nucleic acid sample with primers to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different polymorphic loci The libraries are contacted to produce a reaction mixture; wherein the nucleic acid sample comprises maternal DNA from the mother of the fetus and fetal DNA from the fetus. In some embodiments, the reaction mixture is subjected to primer extension reaction conditions to generate amplification products; the amplification products are measured with a high-throughput sequencer to generate sequencing data; alleles at polymorphic loci are calculated in silico based on the sequencing data Gene counting; creating in silico multiple ploidy hypotheses each for the different possible ploidy states of the chromosome; for each ploidy hypothesis, constructing in silico for predicted allele counts at polymorphic loci on the chromosome a joint distribution model; determining in silico the relative probability of each of the ploidy hypotheses using the joint distribution model and the allele counts; and deciphering the ploidy state of the fetus by selecting the ploidy state corresponding to the hypothesis with the greatest probability.

In one aspect, the invention features methods for determining the ploidy state of chromosomes in a gestating fetus. In one embodiment, a method for determining the ploidy state of chromosomes in a gestating fetus comprises obtaining a first DNA sample comprising maternal DNA from the mother of the fetus and fetal DNA from the fetus; preparing a second DNA sample by isolating the DNA; A sample in order to obtain a prepared sample; measure DNA in the prepared sample at multiple polymorphic loci on a chromosome; calculate alleles at the multiple polymorphic loci on a computer from DNA measurements made on the prepared sample Gene counting; creating in silico multiple ploidy hypotheses each for the different possible ploidy states of the chromosome; for each ploidy hypothesis, in silico the predicted alleles at the multiple polymorphic loci on the chromosome constructing a joint distribution model from the counts; determining in silico the relative probability of each of the ploidy hypotheses using the joint distribution model and the allele counts measured on the prepared sample; and by selecting the ploidy state corresponding to the hypothesis with the greatest probability , to interpret the ploidy status of the fetus.

In one aspect, the invention features a method of testing for a non-normal distribution of chromosomes in a sample comprising a mixture of maternal and fetal DNA. In some embodiments, the method involves (i) simultaneously hybridizing the sample to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target genes A primer library for a locus is contacted to generate a reaction mixture; wherein the target locus is from a plurality of different chromosomes; and wherein the plurality of different chromosomes includes at least one first chromosome suspected of having a non-normal distribution in the sample and at least A second chromosome that is assumed to be normally distributed in the sample; (ii) subjecting the reaction mixture to primer extension reaction conditions to generate amplification products; (iii) sequencing the amplification products to obtain multiple pairs of loci of interest. Sequence markers that are accurate; wherein the length of the sequence markers is sufficient to be assigned to a specific target locus; (iv) assigning multiple sequence markers to their corresponding target loci in silico; (v) determining in silico the relationship with the first chromosome and (vi) comparing in silico the numbers from step (v) to determine the presence or absence of the second chromosome A non-normal distribution of chromosomes.

In one aspect, the invention provides methods for detecting the presence or absence of fetal aneuploidy. In some embodiments, the method involves (i) simultaneously hybridizing a sample comprising a mixture of maternal and fetal DNA to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, Primer pools of 75,000 or 100,000 different non-polymorphic target loci are contacted to generate a reaction mixture; wherein the target loci are from a plurality of different chromosomes; (ii) subjecting the reaction mixture to primer extension reaction conditions to generate Amplification products of amplicons; (iii) quantification in silico of relative frequencies of target amplicons from first and second associated chromosomes; (iv) comparison in silico of target amplicons from first and second associated chromosomes the relative frequency of the amplicon; and (v) identifying the presence or absence of aneuploidy based on the compared relative frequencies of the first and second associated chromosomes. In some embodiments, the first chromosome is a chromosome suspected of being euploid. In some embodiments, the second chromosome is a chromosome suspected of aneuploidy.

In one aspect, a method for determining the presence or absence of fetal aneuploidy in a maternal tissue sample comprising fetal and maternal genomic DNA comprising (a) obtaining obtaining a mixture of fetal and maternal genomic DNA from a tissue sample; (b) performing massively parallel DNA sequencing on DNA fragments randomly selected from the mixture of fetal and maternal genomic DNA in step (a) to determine the identity of said DNA fragments sequence; (c) identifying the chromosome to which the sequence obtained in step (b) belongs; (d) using the data from step (c) to determine the amount of at least one first chromosome in the mixture of maternal and fetal genomic DNA, wherein said at least one first chromosome is assumed to be euploid in the fetus, (e) using the data from step (c) to determine the amount of a second chromosome in the mixture of maternal and fetal genomic DNA, wherein said The second chromosome is suspected to be aneuploid in the fetus; (f) calculate the fraction of fetal DNA in the mixture of fetal and maternal DNA; (g) if the second chromosome of interest is euploid, then use the Calculate the expected distribution of the quantity of the second target chromosome; (h) if the second target chromosome is aneuploid, use the first quantity in step (d) and the fetal DNA calculated in step (f) in the fraction in the mixture of fetal and maternal DNA, calculating a predicted distribution of the amount of the second chromosome of interest; and (i) determining the second chromosome as determined in step (e) using a maximum likelihood method or a maximum posterior probability method is more likely to be part of the distribution calculated in step (g) or the distribution calculated in step (h); thereby indicating the presence or absence of fetal aneuploidy.

In various embodiments of any aspect of the invention, the method further comprises obtaining genotype data from one or both parents of the fetus. In some embodiments, obtaining genotype data from one or both parents of the fetus comprises preparing DNA from the parents, wherein said preparing comprises DNA preferentially enriched at multiple polymorphic loci to obtain prepared parental DNA, either The prepared parental DNA is optionally amplified and the parental DNA is measured at multiple polymorphic loci in the prepared sample.

In various embodiments of any aspect of the invention, genetic data obtained from one or both parents is used to construct a joint distribution model for predicted allele count probabilities at multiple polymorphic loci on a chromosome. In some embodiments, the sample (eg, the first sample) has been isolated from maternal plasma and wherein the genotype data is obtained from the mother by estimating the maternal genotype data from DNA measurements taken on preparing the sample.

In one aspect, a diagnostic kit for assisting in determining the ploidy state of chromosomes in a gestating fetus is disclosed, wherein the diagnostic kit is capable of performing the preparation and measurement steps of any one of the methods of the invention.

In various embodiments of any aspect of the invention, the allele counts are probabilistic rather than binary. In some embodiments, measuring DNA at multiple polymorphic loci in the prepared sample is also used to determine whether the fetus has inherited one or more disease-linked haplotypes.

In various embodiments of any aspect of the invention, the joint distribution model for allele count probabilities is constructed by using data on the probability of chromosomal crossovers at different locations in the chromosome for based on model correlation. In some embodiments, the steps of constructing a joint distribution model for allele counts and determining the relative probability of each hypothesis are performed using a method that does not require the use of a reference chromosome.

In various embodiments of any aspect of the invention, determining the relative probability of each hypothesis utilizes the estimated fraction of fetal DNA in the prepared sample. In some embodiments, DNA measurements of prepared samples used to calculate allele count probabilities and determine the relative probabilities of each hypothesis comprise raw genetic data. In some embodiments, selecting the ploidy state corresponding to the hypothesis with the greatest probability is performed using maximum likelihood estimation or maximum a posteriori probability estimation.

In various embodiments of any aspect of the invention, deciphering the ploidy status of the fetus further comprises comparing the relative probabilities of each of the ploidy hypotheses determined using the joint distribution model and allele count probabilities to those calculated using statistical techniques. Relative probability combinations for each of the ploidy hypotheses, the statistical techniques taken from the group consisting of: read count analysis, comparing heterozygosity rates, statistics available only when using parental genetic information, for certain parental backgrounds The probability of a normalized genotype signal for , statistics calculated using the estimated fetal fraction of the sample (eg, the first sample) or the prepared sample, and combinations thereof.

In various embodiments of any aspect of the invention, a confidence estimate of the called ploidy state is calculated. In some embodiments, the method further comprises taking a clinical action based on the called ploidy state of the fetus, wherein the clinical action is selected from one of terminating the pregnancy or maintaining the pregnancy.

In various embodiments of any aspect of the invention, the method can be between 4 weeks and 5 weeks of gestation, between 5 weeks and 6 weeks of gestation, between 6 weeks and 7 weeks of gestation, between 7 weeks and 8 weeks of gestation Between gestation, between 8 and 9 weeks of gestation, between 9 and 10 weeks of gestation, between 10 and 12 weeks of gestation, between 12 and 14 weeks of gestation, between 14 and 20 weeks of gestation, between 20 Fetuses performed between 40 weeks and 40 weeks gestation, in the first trimester, in the second trimester, in the third trimester, or a combination thereof.

In various embodiments of any aspect of the invention, the method is used to generate a report showing the determined ploidy state of chromosomes in a gestating fetus. In some embodiments, a kit designed for use with any of the methods of the invention to determine the ploidy state of a target chromosome in a gestating fetus is disclosed, the kit comprising a plurality of internal normal A forward primer and optionally a plurality of internal reverse primers, wherein each of the primers is designed for hybridization to the target chromosome and optionally an additional chromosome immediately upstream of one of the polymorphic sites and/or A downstream DNA region, wherein the hybridizing region is separated from the polymorphic site by a small number of bases, wherein the small number is selected from the group consisting of: 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 60 and combinations thereof.

In one aspect, the invention features a method for determining whether a hypothetical father is the biological father of a fetus developing in a pregnant mother. In some embodiments, the method involves (i) simultaneously amplifying a plurality of polymorphic loci on the genetic material from the putative father, including at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000 , 50,000, 75,000, or 100,000 different polymorphic loci, thereby generating a first set of amplification products; (ii) simultaneously amplifying corresponding multiple polymorphic loci on the DNA mixture sample derived from the pregnant mother's blood sample to generating a second set of amplification products; wherein the mixed sample of DNA includes fetal DNA and maternal DNA; (iii) determining in silico that the presumed father is the biological father of the fetus based on the first and second sets of amplification products, using genotype measurements. the probability of the father; and (iv) determining whether the hypothetical father is the biological father of the fetus using the determined probability that the hypothetical father is the biological father of the fetus. In various embodiments, the method further comprises simultaneously amplifying corresponding multiple polymorphic loci on the genetic material from the mother to generate a third set of amplification products; wherein based on the first, second, and third sets of amplification Product that uses genotype measurements to determine the probability that the putative father is the biological father of the fetus.

In one aspect, the invention provides a method of estimating the relative likelihood that each embryo in a set of embryos will develop as desired. In some embodiments, the method involves simultaneously hybridizing samples from each embryo to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different genes of interest The primer pools of the loci are contacted to generate a reaction mixture for each embryo, wherein the samples are each derived from one or more cells from the embryo. In some embodiments, each reaction mixture is subjected to primer extension reaction conditions to generate amplification products. In some embodiments, the method comprises determining in silico one or more characteristics of at least one cell from each embryo based on the amplification product; and based on the one or more characteristics of the at least one cell from each embryo, The relative likelihood that each embryo will develop as desired is estimated in silico.

In one aspect, the invention features a method of measuring the amount of two or more target loci in a nucleic acid sample. In some embodiments, the method involves (i) using PCR to amplify a nucleic acid sample comprising a first standard locus, a second standard locus, a first target locus, and a second target locus to form an amplification product; wherein the first standard locus and the first target locus have the same number of nucleotides but differ in sequence at one or more nucleotides; and wherein the second standard locus and the second target locus have the same number of nucleotides but their sequences differ at one or more nucleotides; (ii) sequencing the amplified product to determine the comparison of the amplified first standard locus compared to the amplified second standard locus The standard ratio of the relative amount of; wherein the standard ratio indicates the difference in PCR efficiency of the amplification of the first standard locus and the second standard locus; (iii) determining the comparison of the amplified first target locus compared to a target ratio based on the relative amount of amplified second target locus; and (iv) adjusting the target ratio of step (iii) based on the standard ratio of step (ii) to determine the first target locus and the second target in the sample Relative amount of loci. In various embodiments, the methods involve determining the absolute amounts of a first locus of interest and a second locus of interest in a sample. In various embodiments, the method further comprises determining the presence or absence of the target locus in the sample (e.g., at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci). In various embodiments, the methods involve using any of the primer libraries of the invention. In various embodiments, the method involves simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci.

In one aspect, the invention features methods of quantitatively measuring a plurality of genetic targets in a sample for analysis. In some embodiments, the method comprises (i) mixing genetic material derived from a sample for analysis with a plurality of target-specific amplification reagents and a plurality of standard sequences corresponding to targets of the target-specific amplification reagents; (ii ) amplifying the target region of the genetic material and the standard sequence to generate the target amplicon and the standard sequence amplicon; and (iii) measuring the amount of the target amplicon and the standard sequence amplicon produced. In some embodiments, the genetic material is present in a gene bank. In some embodiments, the genetic target is a polymorphic locus (eg, a SNP). In some embodiments, the measurement of the quantity is achieved by counting the sequence. In some embodiments, the method further comprises determining the estimated copy number of at least one chromosome in the sample from which the gene bank was derived, wherein said determining involves comparing the number of sequence reads of the amplicon of interest to that of a standard amplicon. Number of sequence reads. In some embodiments, standard sequences and gene libraries include universal priming sites capable of being primed by the same primers. In some embodiments, the mixing step includes at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 parts of different target-specific amplification reagents and at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 standard sequences. In various embodiments, the methods involve using any of the primer libraries of the invention. In various embodiments, the method involves simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target regions. In some embodiments, the relative amounts of each of the standard sequences are known. In some embodiments, the relative amounts of each of the sequences have been calibrated against a reference genome. In some embodiments, the sample for analysis includes a mixture of fetal and maternal genomes. In some embodiments, the sample for analysis is derived from blood of a pregnant woman or derived from plasma. In some embodiments, the reference genome has at least one aneuploidy, such as an aneuploidy at chromosome 13, 18, 21, X or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention features a mixture comprising a plurality of genetic standard sequences, wherein the relative amount of each genetic standard sequence in the mixture has been determined by calibration to a reference genome. In various embodiments, the mixture includes at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 gene standard sequences. In various embodiments, the gene standard sequence includes a first universal priming site, a second universal priming site, a first target-specific priming site, a second target-specific priming site and A marker sequence between the generic priming sites, wherein the first target-specific priming site and the second target-specific priming site are located between the first and second universal priming sites. In various embodiments, said calibration involves using any of the primer libraries of the invention. In various embodiments, the calibration involves simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000 or 100,000 different target regions. In some embodiments, the reference genome has at least one aneuploidy, such as an aneuploidy at chromosome 13, 18, 21, X or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention features a method of generating a set of calibrated genetic standard sequences. In some embodiments, the method includes (i) forming an amplification reaction mixture comprising a gene pool prepared from a reference genome, a plurality of target-specific amplification primer reagent sets, and a corresponding target-specific amplification reagent set. multiple gene standard sequences; (ii) amplify the gene pool and gene standard sequences to generate target sequence amplicons and gene standard sequence amplicons; (iii) measure target sequence amplicons and gene standard sequence amplicons the number of accumulators; and (iv) determining the relative amount of each of the gene standard sequences relative to each other, thereby calibrating the plurality of gene standard sequences. In various embodiments, at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 gene standard sequences are used. In various embodiments, the methods involve using any of the primer libraries of the invention. In various embodiments, the method involves simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different sequences. In some embodiments, the reference genome has at least one aneuploidy, such as an aneuploidy at chromosome 13, 18, 21, X or Y. In some embodiments, the reference genome is diploid.

In one aspect, the invention provides a set of genetic standard sequences that have been calibrated according to any of the methods of the invention. In one aspect, the invention provides a set of genetic standard sequences that can be calibrated before, during or after performing the method.

In one aspect, the invention features a method of measuring the copy number of a gene associated with at least one allele having a deletion. In some embodiments, the method comprises (i) combining genetic material derived from a sample for analysis with an amplification reagent specific for a gene of interest comprising a deletion allele that is not capable of significantly amplifying the gene of interest, corresponding to (ii) amplify the relevant gene sequence, the standard sequence corresponding to the relevant gene, the reference sequence and the reference sequence corresponding to the reference sequence; standard sequences of sequences to generate related gene amplicons, reference sequence amplicons, and standard sequence amplicons; and (iii) measuring the number of generated target amplicons and standard sequence amplicons. In some embodiments, the measurement of quantity is achieved by counting sequence reads. In some embodiments, the method further comprises determining the estimated copy number of at least one chromosome in the sample from which the gene bank was derived, wherein said determining involves comparing the number of sequences of target amplicons to the sequences of standard amplicons quantity. In some embodiments, standard sequences and gene libraries include universal priming sites capable of being primed by the same primers. In some embodiments, the relative amounts of each of the sequences have been calibrated against a reference genome. In various embodiments, at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 gene standard sequences are used. In various embodiments, the methods involve using any of the primer libraries of the invention. In various embodiments, the method involves simultaneously amplifying 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different target regions. In some embodiments, the reference genome is diploid. In some embodiments, the sample for analysis is derived from blood.

In some embodiments of any aspect of the invention, preferentially enriching DNA at a locus of interest (e.g., a plurality of polymorphic loci) in a sample (e.g., a first sample) comprises obtaining a plurality of precircularized Probes, wherein each probe targets one of the loci (e.g., polymorphic loci), wherein the 3' and 5' ends of the probes are preferably designed for hybridization to DNA regions separated by polymorphic sites of a genetic locus, wherein said small number is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or combinations thereof; hybridize pre-circularized probes to DNA of a sample (e.g., first sample); fill hybridization using a DNA polymerase Gaps between probe ends; Circularization of precircularized probes; and Amplification of circularized probes.

In some embodiments of any aspect of the invention, DNA preferentially enriched at loci of interest (e.g., a plurality of polymorphic loci) comprises obtaining a plurality of junction-mediated PCR probes, wherein each PCR probe targets To one of the loci of interest (e.g., polymorphic loci), and wherein the upstream and downstream PCR probes are designed for hybridization to DNA preferably separated by a small number of bases from the polymorphic locus of the locus A region of DNA on one strand, wherein said small amount is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 to 25, 26 to 30, 31 to 60, or combinations thereof; hybridizing the ligation-mediated PCR probe to DNA of a sample (e.g., the first sample); filling the ligation-mediated PCR probe with a DNA polymerase gap between needle ends; ligation of the ligation-mediated PCR probe; and amplification of the ligated ligation-mediated PCR probe.

In some embodiments of the various aspects of the invention, DNA preferentially enriched at a target locus (e.g., a plurality of polymorphic loci) comprises obtaining a hybridization of a plurality of targeted loci (e.g., a polymorphic locus) Capture Probe; hybridizing the capture probe to DNA in a sample (eg, first sample) and physically removing some or all of the unhybridized DNA from the DNA sample (eg, first sample).

In some embodiments of any aspect of the invention, hybridization capture probes are designed to hybridize to regions flanking, but not overlapping, the polymorphic site. In some embodiments, the hybridization capture probe is designed to hybridize to a region flanking but not overlapping the polymorphic site, and wherein the length of the flanking capture probe can be selected from the group consisting of less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases. In some embodiments, the hybridization capture probes are designed to hybridize to regions of overlapping polymorphic loci, and wherein the plurality of hybridization capture probes comprises at least two hybridization capture probes for each polymorphic locus, And wherein each hybridization capture probe is designed to complement a different allele at the polymorphic locus.

In some embodiments of any aspect of the invention, DNA preferentially enriched at a plurality of polymorphic loci comprises obtaining a plurality of internal forward primers, wherein each primer targets one of the polymorphic loci, and wherein the internal The 3' end of the forward primer is designed to hybridize to a DNA region upstream of the polymorphic site and separated from the polymorphic site by a small number of bases selected from the group consisting of 1, 2, 3, 4, 5 , 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, or 31 to 60 base pairs in groups; optionally obtain multiple internal reverse primers, where each primer targets One of the polymorphic loci, and wherein the 3' end of the internal reverse primer is designed to hybridize to a DNA region upstream of the polymorphic site and separated from the polymorphic site by a small number of bases, wherein the small selected from the group consisting of 1, 2, 3, 4, 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 30, or 31 to 60 base pairs; hybridizes internal primer to DNA and amplifying the DNA using the polymerase chain reaction to form amplicons.

In some embodiments of any aspect of the invention, the method further comprises obtaining a plurality of outer forward primers, wherein each primer targets one of the targets (e.g., polymorphic loci), and wherein the outer forward primers Designed to hybridize to a DNA region upstream of an internal forward primer; optionally obtaining a plurality of external reverse primers, wherein each primer targets one of the loci of interest (e.g., polymorphic loci), and wherein The outer reverse primer is designed to hybridize to the region of DNA immediately downstream of the inner reverse primer; hybridize the first primer to the DNA; and amplify the DNA using polymerase chain reaction.

In some embodiments of any aspect of the invention, the method further comprises obtaining a plurality of outer reverse primers, wherein each primer targets one of the polymorphic loci, and wherein the outer reverse primers are designed for hybridizes to the DNA region immediately downstream of the inner reverse primer; optionally obtaining a plurality of outer forward primers, wherein each primer targets one of the loci of interest (e.g., polymorphic loci), and wherein the outer forward Primers are designed to hybridize to a region of DNA upstream of the internal forward primer; hybridize the first primer to the DNA; and amplify the DNA using polymerase chain reaction.

In some embodiments of any aspect of the invention, preparing the sample (e.g., the first sample) further comprises appending universal adapters to the DNA in the sample (e.g., the first sample) and amplifying the DNA using polymerase chain reaction. DNA in a sample (eg, first sample) is amplified. In some embodiments, at least some fraction of the amplified amplicons is less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp, and wherein said fraction Is it 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%.

In some embodiments of any aspect of the invention, amplifying the DNA is performed in one or more individual reaction volumes, and wherein each individual reaction volume contains more than 100 different pairs of forward and reverse primers , over 200 different forward and reverse primer pairs, over 500 different forward and reverse primer pairs, over 1,000 different forward and reverse primer pairs, over 2,000 different forward and reverse Primer pairs, over 5,000 different forward and reverse primer pairs, over 10,000 different forward and reverse primer pairs, over 20,000 different forward and reverse primer pairs, over 50,000 different forward and Reverse primer pairs or over 100,000 different forward and reverse primer pairs.

In some embodiments of any aspect of the invention, preparing the sample (e.g., the first sample) further comprises dividing the sample (e.g., the first sample) into a plurality of fractions, and wherein each fraction is preferentially enriched in the target DNA at a subset of loci (eg, multiple polymorphic loci). In some embodiments, internal primers are selected by identifying primer pairs that are likely to form undesired primer duplexes and removing from the plurality of primers at least one of the primer pairs that are identified as likely to form undesired primer duplexes. In some embodiments, an internal primer contains a region designed to hybridize upstream or downstream of a targeted locus (e.g., a polymorphic locus), and optionally contains a general primer designed to allow PCR amplification. trigger sequence. In some embodiments, at least some of the primers additionally contain random regions that differ from each individual primer molecule. In some embodiments, at least some of the primers additionally contain molecular barcodes.

In some embodiments of any aspect of the invention, the multiple of the average degree of allelic bias between the prepared sample and the sample (e.g., the first sample) caused by preferential enrichment is selected from the group consisting of: no more than 2 times, not more than 1.5 times, not more than 1.2 times, not more than 1.1 times, not more than 1.05 times, not more than 1.02 times, not more than 1.01 times, not more than 1.005 times, not more than 1.002 times, not more than 1.001 times and not more than 1.0001 times. In some embodiments, the plurality of polymorphic loci are SNPs. In some embodiments, measuring the DNA in the prepared sample is by sequencing.

In some embodiments of any aspect of the invention, the loci of interest are present on the same related nucleic acid (eg, the same chromosome or the same region of a chromosome). In some embodiments, at least some of the loci of interest are present on different associated nucleic acids (eg, different chromosomes). In some embodiments, nucleic acid samples include fragmented or digested nucleic acids. In some embodiments, nucleic acid samples include genomic DNA, cDNA or mRNA. In some embodiments, the nucleic acid sample includes DNA from a single cell. In some embodiments, the nucleic acid sample is a substantially cell-free blood or plasma sample. In some embodiments, the nucleic acid sample includes or is derived from blood, plasma, saliva, semen, sperm, cell culture supernatant, mucus secretion, dental plaque, gastrointestinal tissue, feces, urine, hair, bone, body fluid, Tears, tissue, skin, nails, blastomeres, embryos, amniotic fluid, villi samples, bile, lymph, cervical mucus or forensic samples. In some embodiments, the target locus is a segment of human nucleic acid. In some embodiments, the locus of interest comprises or consists of single nucleotide polymorphisms (SNPs). In some embodiments, primers are DNA molecules.

In some embodiments of any aspect of the invention, the DNA in the sample (eg, the first sample) is derived from maternal plasma. In some embodiments, preparing the sample (eg, the first sample) further comprises amplifying the DNA. In some embodiments, preparing the sample (eg, first sample) further comprises preferentially enriching DNA at loci of interest (eg, a plurality of polymorphic loci) in the sample (eg, first sample).

In various embodiments, the primer extension reaction or polymerase chain reaction includes the addition of one or more nucleotides by a polymerase. In various embodiments, the primer extension reaction or polymerase chain reaction does not include ligation-mediated PCR. In various embodiments, the primer extension reaction or polymerase chain reaction does not involve joining the two primers by a ligase. In various embodiments, the primers do not include linkage inversion probes (LIPs), which may also be referred to as precircularized probes, precircularizing probes, circularizing probes; padlock probes or molecular inversions probe (MIP).

It is to be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

definition

A single nucleotide polymorphism (SNP) refers to a single nucleotide that can differ between the genomes of two members of the same species. The use of the term should not imply any limitation on the frequency with which each variant occurs.

Sequence refers to a DNA sequence or gene sequence. It can refer to the primary physical structure of a DNA molecule or strand in an individual. It can refer to the sequence of nucleotides found in said DNA molecule, or the complementary strand of said DNA molecule. It can refer to the information contained in a DNA molecule, as used to represent the DNA molecule in computer simulations.

A locus refers to a specific associated region on an individual's DNA, which may refer to a SNP, a site of possible insertion or deletion, or some other site of associated genetic variation. A disease-linked SNP can also refer to a disease-linked locus.

Polymorphic alleles, also known as "polymorphic loci", refer to alleles or loci that differ in genotype between individuals within a given species. Some examples of polymorphic alleles include single nucleotide polymorphisms, short tandem repeats, deletions, duplications and inversions.

A polymorphic site refers to a specific nucleotide found in a polymorphic region that varies between individuals.

Alleles are genes that occupy a specific locus.

Genetic data, also known as "genotype data," refers to data describing aspects of the genome of one or more individuals. It can refer to a locus or a group of genes, part or the whole sequence, part or the whole chromosome or the whole genome. It can refer to the identity of one or more nucleotides; it can refer to a contiguous group of nucleotides or nucleotides from different locations in the genome or a combination thereof. Genotype data are usually simulated in silico, however, it is also possible to consider the actual nucleotides represented in the sequence as chemically encoded genetic data. Genotype data can be said to be "on" an individual, "of" an individual, "on" an individual, "from" an individual, or "on" an individual. Genotype data may refer to output measurements from a genotyping platform, where those measurements are made on genetic material.

Genetic material, also called "genetic sample," refers to bodily material, such as tissue or blood, from one or more individuals that contains DNA or RNA.

Noisy genetic data refers to any of the following genetic data: allelic loss, uncertain base pair measurements, incorrect base pair measurements, missing base pair measurements, uncertain insertion or deletion measurements, uncertain Measurements of copy number of chromosomal segments, spurious signals, missing measurements, other errors, or combinations thereof.

Confidence refers to the so-called statistical likelihood that a SNP, allele, set of alleles, ploidy call, or determined copy number of a chromosome segment correctly represents the true genetic state of an individual.

Ploidy calling, also known as "chromosomal copy number calling" or "copy number calling" (CNC), can refer to the act of determining the number and/or chromosome identity of one or more chromosomes present in a cell.

Aneuploidy refers to a state in which there is an incorrect number of chromosomes in a cell (eg, an incorrect number of intact chromosomes or an incorrect number of chromosome segments, eg, there is a deletion or duplication of a chromosome segment). In the case of human cells, it can refer to a situation where the cell does not contain the 22 pairs of autosomes and one pair of sex chromosomes. In the case of human gametes, it can refer to the situation where a cell does not contain one of the 23 chromosomes. In the case of a single chromosome type, it can refer to a situation where there are more or less than two homologous but non-identical copies of a chromosome, or where there are two copies of a chromosome derived from the same parent. In some embodiments, the deletion of a chromosomal segment is a microdeletion.

Ploidy state refers to the number and/or chromosome identity of one or more chromosome types in a cell.

Chromosome can refer to a single chromosomal copy, meaning a single DNA molecule of 46 in normal somatic cells; an example is 'maternally derived chromosome 18'. Chromosome can also refer to a type of chromosome of which there are 23 in normal human cells; an example is 'chromosome 18'.

Chromosomal characteristics may refer to a reference chromosome number, ie, chromosome type. Normal humans have 22 types of numbered autosomes and two types of sex chromosomes. It can also refer to the parental source of chromosomes. It can also refer to a specific chromosome inherited from a parent. It can also refer to other attribute characteristics of chromosomes.

The state of genetic material or simply "genetic state" can refer to the identity of a set of SNPs on the DNA, the phased haplotype of the genetic material, and the DNA sequence, including insertions, deletions, duplications, and mutations. It can also refer to the ploidy state of one or more chromosomes, chromosome segments, or groups of chromosome segments.

Allelic data refers to a set of genotype data for a set of one or more alleles. It can specify phase haplotype data. It can refer to SNP identity, and it can refer to sequence data of DNA, including insertions, deletions, duplications and mutations. It can include the parental origin of each allele.

Allelic state refers to the actual state of a gene in a set of one or more alleles. It can refer to the actual state of a gene described by allelic data.

The ratio of alleles or allelic ratio refers to the ratio between the amount of each allele present at a locus in a sample or in an individual. When measuring a sample by sequencing, the ratio of alleles can refer to the ratio of sequence reads that map to each allele at a locus. When a sample is measured by an intensity-based measurement method, the allele ratio may refer to the ratio of the amount of each allele present at a locus as estimated by the measurement method.

Allele count refers to the number of sequences that map to a particular locus, and if the locus is polymorphic, it refers to the number of sequences that map to each of the alleles. If each allele was counted in binary, the allele count would be an integer. If alleles are counted probabilistically, the allele count may be a fraction.

The allele count probability refers to the number and probability of mapping to a particular locus or set of alleles at a polymorphic locus. It should be noted that allele counts are equivalent to allele count probabilities, where the mapping probability for each sequence of counts is binary (zero or one). In some embodiments, allele count probabilities may be binary. In some embodiments, the allele count probability can be set equal to the DNA measurement.

Allelic distribution or 'allele count distribution' refers to the relative amount of each allele present at each locus in a set of loci. An allelic distribution can refer to an individual, a sample, or a set of measurements taken on a sample. In the context of sequencing, allelic distribution refers to the number or likely number of reads that map to a particular allele at each allele in a set of polymorphic loci. Allelic measures can be processed in a probabilistic fashion, that is, the likelihood that a given allele is present for a given sequence read is a score between 0 and 1, or they can be processed in a binary fashion, That is, any given read is considered to be exactly zero or one copy of a particular allele.

The allelic distribution pattern refers to a set of different allele distributions for different parental backgrounds. Certain allelic distribution patterns can indicate certain ploidy states.

Allelic bias refers to the degree to which the measured ratio of alleles at a heterozygous locus differs from the ratio present in the original DNA sample. The degree of allelic bias at a particular locus is equal to the ratio of alleles observed at that locus as measured divided by the ratio of alleles at that locus in the original DNA sample. The allelic bias can be defined to be greater than one, such that if the calculation of the allelic bias degree returns a value x less than 1, then the allelic bias degree can be restated as 1/x. Allelic bias may be due to amplification bias, purification bias, or some other phenomenon that affects different alleles differently.

A primer, also called a "PCR probe", refers to a single DNA molecule (DNA oligomer) or a collection of DNA molecules (DNA oligomers) in which the DNA molecules are identical, or nearly identical, and in which the primer contains A region designed to hybridize to a targeted locus (eg, targeted to a polymorphic locus or a non-polymorphic locus), and may contain a priming sequence designed to allow PCR amplification. Primers can also contain molecular barcodes. Primers may contain random regions that differ for each individual molecule. The terms "test primer" and "candidate primer" are not meant to be limiting and may refer to any of the primers disclosed herein.

A primer library refers to a population of two or more primers. In various embodiments, the library comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different primers. In various embodiments, the library comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different primer pairs, wherein each pair of primers includes a forward test primer and Reverse test primers, where each pair of test primers hybridizes to one locus of interest. In some embodiments, the library of primers comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different individual primers that each hybridize to a different target locus, wherein the Individual primers are not part of a primer pair. In some embodiments, the library has (i) primer pairs and (ii) individual primers that are not part of the primer pairs (eg, universal primers).

A hybridization capture probe refers to any nucleic acid sequence, possibly modified, produced by various methods such as PCR or direct synthesis and intended to be complementary to one strand of a specific target DNA sequence in a sample. Exogenous hybridization capture probes can be added to the prepared sample and hybridized through a denaturation-reannealing process to form exogenous-endogenous fragment duplexes. These duplexes can then be physically separated from the sample by various means.

Sequence reads refer to data representing the sequence of nucleotide bases measured using clonal sequencing methods. Clonal sequencing can generate sequence data representing a single copy or clones or clusters of an original DNA molecule. Sequence reads can also have an associated quality score at each base position in the sequence, which indicates the probability that the nucleotide was called correctly.

Mapping sequence reads is the process of determining the source location of sequence reads in the genome sequence of a particular organism. The source position of the sequence reads is based on the nucleotide sequence similarity of the reads to the genomic sequence.

Matching copy errors, also known as "matching chromosomal aneuploidy" (MCA), are aneuploidy states in which a cell contains two identical or nearly identical chromosomes. This type of aneuploidy can arise during the formation of gametes in meiosis and can be referred to as a meiotic nondisjunction error. This type of error can appear in mitosis. Matching trisomy may refer to a situation where three copies of a given chromosome are present in an individual and two of the copies are identical.

Mismatched copy errors, also known as "unique chromosomal aneuploidy" (UCA), refer to the aneuploidy state in which a cell contains two chromosomes from the same parent that can be homologous but not identical. This type of aneuploidy can arise during meiosis and can be referred to as a meiotic error. A mismatched trisomy can refer to a situation where there are three copies of a given chromosome in an individual and two of the copies are from the same parent and are homologous but not identical. It should be noted that a mismatched trisomy can refer to a situation where there are two homologous chromosomes from one parent and where some segments of the chromosomes are identical while other segments are merely homologous.

Homologous chromosomes are copies of chromosomes that contain the same set of genes that usually pair up during meiosis.

Concordant chromosomes are copies of chromosomes that contain the same set of genes and, for each gene, they have identical or nearly identical sets of alleles.

Allelic loss (ADO) refers to the condition in which at least one base pair in a set of base pairs from homologous chromosomes is not detected at a given allele.

Loss of loci (LDO) refers to the situation where two base pairs in a set of base pairs from homologous chromosomes are not detected at a given allele.

Homozygous means having similar alleles as corresponding chromosomal loci.

Heterozygous means having different alleles as corresponding chromosomal loci.

The heterozygosity rate refers to the ratio of individuals in a population that have a heterozygous allele at a given locus. Heterozygosity rate can also refer to the predicted or measured ratio of alleles at a given locus in an individual or DNA sample.

A highly informative single nucleotide polymorphism (HISNP) refers to a SNP in which the fetus has an allele that is not present in the mother's genotype.

A chromosomal region refers to a segment of a chromosome or a complete chromosome.

A segment of a chromosome refers to a portion of a chromosome that can range in size from one base pair to an entire chromosome.

Chromosome refers to a complete chromosome or a segment or part of a chromosome.

Copies refers to the number of copies of a chromosomal segment. It can refer to identical copies of a chromosomal segment or non-identical homologous copies, where different copies of the chromosomal segment contain a set of substantially similar loci, and where one or more of the alleles differ. It should be noted that in some cases of aneuploidy, such as M2 copy errors, it is possible that some copies of a given chromosome segment are identical and some copies of the same chromosome segment are not identical.

Haplotype refers to the combination of alleles at multiple loci that are usually inherited together on the same chromosome. Depending on the number of recombination events that have occurred between a given set of loci, a haplotype can refer to as few as two loci or to an entire chromosome. A haplotype can also refer to a statistically related set of single nucleotide polymorphisms (SNPs) on a single chromatid.

Haplotype data, also known as "phased data" or "ordered genetic data," refers to data from a single chromosome in a diploid or polyploid genome, that is, the segregated sequence of chromosomes in a diploid genome. copy of the maternal or paternal parent.

Phasing refers to the act of determining haplotype genetic data of an individual in view of disordered diploid (or polyploid) genetic data. It may refer to the act of determining, for a set of alleles found on a chromosome, which of the two genes at the allele is associated with each of the two homologous chromosomes in an individual.

Phased data refers to genetic data for which one or more haplotypes have been determined.

A hypothesis refers to a set of possible ploidy states at a specified set of chromosomes or a set of possible allelic states at a specified set of loci. The set of possibilities can contain one or more elements.

A copy number hypothesis, also referred to as a "ploidy state hypothesis", refers to a hypothesis about the copy number of a chromosome in an individual. It can also refer to assumptions about the identity of each of the chromosomes, including the parental origin of each chromosome and which of the two parental chromosomes is present in an individual. It can also refer to a hypothesis as to which chromosomes or chromosome segments (if any) from related individuals genetically correspond to a given chromosome of the individual.

A target individual is an individual whose genetic status is to be determined. In some embodiments, only a limited amount of DNA is available from the individual of interest. In some embodiments, the target individual is a fetus. In some embodiments, there may be more than one target individual. In some embodiments, each fetus from a pair of parents may be considered a target individual. In some embodiments, the genetic data to be determined is one or a set of allelic calls. In some embodiments, the genetic data to be determined is a ploidy call.

A related individual refers to any individual that is genetically related to, and thus shares a haplotype domain with, the individual of interest. In one instance, the related individual may be the genetic parent of the subject individual or any genetic material derived from the parent, such as sperm, polar body, embryo, fetus or child. It can also refer to siblings, parents, or grandparents.

A sibling is any individual whose genetic parents are the same as the individual in question. In some embodiments, it may refer to a born child, embryo or fetus, or one or more cells derived from a born child, embryo or fetus. Siblings can also refer to haploid individuals derived from one parent, such as sperm, polar bodies, or any other set of haplotype genetic material. Individuals can be thought of as siblings of themselves.

Fetus means "of a fetus" or "of a region of the placenta that is genetically similar to a fetus." In pregnant women, some parts of the placenta are genetically similar to the fetus, and free-floating fetal DNA found in maternal blood may have originated from parts of the placenta that match the fetal genotype. It should be noted that the genetic information of half of the chromosomes in the fetus is inherited from the mother of the fetus. In some embodiments, DNA from the chromosomes of fetal cells inherited from these mothers is considered "of fetal origin" rather than "of maternal origin."

DNA of fetal origin refers to DNA that was originally part of a cell whose genotype substantially corresponds to the fetal genotype.

DNA of maternal origin refers to DNA that was originally part of a cell whose genotype substantially corresponds to that of the mother.

A child may refer to an embryo, blastomere or fetus. It should be noted that in the presently disclosed embodiments, the concept applies equally well to an individual who is a born child, fetus, embryo, or a group of cells derived therefrom. Use of the term child may be intended to mean simply that the individual referred to as a child is the genetic descendant of the parents.

Parent refers to the genetic mother or father of an individual. Individuals usually have two parents (maternal and paternal), but this may not necessarily be the case, for example in genetic or chromosomal mosaicism. Parents can be thought of as individuals.

Parental context refers to the genetic status of a specified SNP on each of the two related chromosomes of one or both parents of the target.

Development on demand, also known as "normal development," refers to the implantation of a viable embryo into the uterus and resulting in pregnancy, and/or the continuation of pregnancy and resulting in a live birth, and/or the birth of a child without chromosomal abnormalities, and/or the birth of a child No other undesired genetic conditions, such as disease-linked genes. The term "development as needed" is intended to cover any situation that parents and health care providers may wish. "Development as needed" may refer, in some instances, to non-viable or viable embryos suitable for medical research or other purposes.

Insertion into the uterus is the process of transferring an embryo into the uterine cavity in the case of in vitro fertilization.

Maternal plasma refers to the plasma fraction of blood from a pregnant female.

A clinical decision refers to any decision to take or not to take an action that has consequences affecting the health or survival of an individual. In the case of prenatal diagnosis, the clinical decision can refer to a decision to abort or not to abort. A clinical decision can also refer to a decision to conduct further testing, to take action to alleviate an undesired phenotype, or to take action to prepare for the birth of a child with an abnormality.

A diagnostic cassette refers to a machine or combination of machines designed to perform one or more aspects of the methods disclosed herein. In one embodiment, the diagnostic cassette may be placed at the point of patient care. In one embodiment, the diagnostic cassette may perform targeted amplification followed by sequencing. In one embodiment, the diagnostic cartridge can function alone or with the aid of a technician.

An information-based approach is one that relies heavily on statistics to make sense of large amounts of data. In the context of prenatal diagnosis, it refers to the determination of the ploidy of one or more chromosomes by statistically inferring the most probable state rather than by direct physical measurement of state, given large amounts of genetic data, for example from molecular arrays or sequencing. Sexual status or the allelic status of one or more alleles. In one embodiment of the present invention, the information-based technology may be the technology disclosed in this patent. In one embodiment of the invention it may be PARENTALSUPPORT ^(TM) .

Raw genetic data refers to the simulated intensity signal output by the genotyping platform. In the case of SNP arrays, raw genetic data refers to the intensity signal before any genotype calls are made. In the context of sequencing, raw genetic data refers to the analog measurements, akin to a chromatogram, that go through a sequencer before the identity of any base pairs is determined and before the sequences have been mapped to the genome.

Secondary genetic data refers to the processed genetic data output by the genotyping platform. In the context of SNP arrays, secondary genetic data refers to allelic calls made by software associated with the SNP array reader, where the software has made a call for the presence or absence of the specified allele in the sample . In the context of sequencing, secondary genetic data means that the base pair identity of a sequence has been determined, and possibly also where in the genome that sequence has been mapped.

Non-Invasive Prenatal Diagnosis (NPD) or also known as "Non-Invasive Prenatal Screening" (NPS), refers to the method of determining the genetic status of a developing fetus in the mother's body using genetic material found in the mother's blood , wherein the genetic material is obtained by drawing intravenous blood from the mother.

Preferential enrichment of DNA corresponding to or at a locus refers to causing a higher percentage of DNA molecules corresponding to said locus in the DNA mixture after enrichment than in the DNA mixture before enrichment. Any method for the percentage of DNA molecules at the locus. The method may involve selective amplification of DNA molecules corresponding to the loci. The method may involve removing DNA molecules that do not correspond to a locus. The method may involve a combination of methods. The degree of enrichment is defined as the percentage of DNA molecules corresponding to the locus in the enriched mixture divided by the percentage of DNA molecules corresponding to the locus in the pre-enrichment mixture. Preferential enrichment can be performed at multiple loci. In some embodiments of the invention, the degree of enrichment is greater than 20. In some embodiments of the invention, the degree of enrichment is greater than 200. In some embodiments of the invention, the degree of enrichment is greater than 2,000. When performing preferential enrichment at multiple loci, the enrichment may refer to the average enrichment of all loci in the locus group.

Amplification refers to a method of increasing the number of copies of a DNA molecule.

Selective amplification may refer to a method of increasing the copy number of a specific DNA molecule or a DNA molecule corresponding to a specific region of DNA. It can also refer to methods of increasing the copy number of specific targeted DNA molecules or targeted DNA regions rather than just increasing non-targeted molecules or DNA regions. Selective amplification can be a method of preferential enrichment.

A universal priming sequence refers to a DNA sequence that can be appended to a population of DNA molecules of interest, eg, by ligation, PCR, or ligation-mediated PCR. After addition to the population of molecules of interest, primers specific to the universal priming sequence can be used to amplify the population of interest using a pair of amplification primers. The universal priming sequence is usually independent of the target sequence.

Universal adapters or 'junction adapters' or 'library tags' are DNA molecules that contain universal priming sequences that can be covalently ligated to the 5' and 3' ends of a population of double-stranded DNA molecules of interest. The addition of adapters provides a universal priming sequence for the 5' and 3' ends of the target population from which PCR amplification can occur using a pair of amplification primers to amplify all molecules from the target population.

Targeting refers to methods used to selectively amplify or preferentially enrich those DNA molecules in a DNA mixture that correspond to a set of loci.

A joint distribution model refers to a model that defines the probability of an event defined with respect to a plurality of random variables, specifying a plurality of random variables defined on the same probability space, where the probabilities of the variables are chained. In some embodiments, a degenerate case where the probabilities of the variables are not linked may be used.

Description of drawings

The disclosed embodiments of the present invention will be further explained with reference to the drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, emphasis instead primarily being placed upon illustrating the principles of the disclosed embodiments of the invention.

Figure 1: Graphical representation of the direct multiplex mini-PCR method.

Figure 2: Graphical representation of the semi-nested mini-PCR method.

Figure 3: Graphical representation of the fully nested mini-PCR method.

Figure 4: Graphical representation of the hemi-nested mini-PCR method.

Figure 5: Graphical representation of the triple hemi-nested mini-PCR method.

Figure 6: Graphical representation of the one-sided nested mini-PCR method.

Figure 7: Graphical representation of the one-sided mini-PCR method.

Figure 8: Graphical representation of the reverse semi-nested mini-PCR method.

Figure 9: Some possible workflows for a semi-nested approach.

Figure 10: Graphical representation of circular ligated adapters.

Figure 11: Graphical representation of internally labeled primers.

Figure 12: Examples of some primers with internal markers.

Figure 13: Graphical representation of the method performed using primers containing ligated adapter binding regions.

Figure 14: Simulated ploidy call accuracy of the enumeration method using two different analytical techniques.

Figure 15: Ratio of two alleles of multiple SNPs in cell lines in Experiment 4.

Figure 16: Ratios of two alleles for various chromosomally sorted SNPs in cell lines in Experiment 4.

Figures 17A-D: Ratios of two alleles for multiple chromosomally sorted SNPs in four maternal plasma samples.

Figure 18: Fraction of data that can be explained by binomial variance before and after data correction.

Figure 19: Graph showing the relative enrichment of fetal DNA in samples following the short library preparation protocol.

Figure 20: Read depth plot comparing direct PCR and semi-nested approaches.

Figure 21 : Read depth comparison of direct PCR for three genomic samples.

Figure 22: Comparison of read depth for semi-nested mini-PCR for three samples.

Figure 23: Comparison of read depth for 1,200-plex and 9,600-plex reactions.

Figure 24: Read count ratios for six cells at three chromosomes.

Figure 25: Allelic ratios of two triplet reactions at three chromosomes and one third run on 1 ng of genomic DNA.

Figure 26: Allelic ratios of two single cell responses at three chromosomes.

Figure 27: Comparison of two primer pools showing the number of loci with specific minor allele frequencies targeted by each primer pool.

Figure 28A: Electropherogram of PCR products. Figures 28B-28M are electropherograms of lanes 1-12 in Figure 28A, respectively.

Figures 29A-29E: Schematic depiction of the method of the invention for determining fetal aneuploidy (Figure 29A). Using maternal and paternal genotype data (from blood or cheek smears) and crossover frequency data from the HapMap database generated (FIG. 29B) multiple independent hypotheses for each possible fetal ploidy state simulated in silico (FIG. 29C ). Each of these hypotheses is expanded to include sub-hypotheses that take into account different possible intersections. The data model predicted what the sequencing data would look like (expected allelic distribution) given each hypothetical fetal genotype and different fetal cfDNA fractions, and compared it to the actual sequencing data; using Bayesian statistics ) measures the likelihood of each hypothesis. In this hypothetical example, the hypothesis with the highest likelihood (euploidy) was identified (Fig. 29D). The individual likelihoods of Figure 29C were summed for each copy number hypothesis family (monosomy, disomy or triploidy). Hypotheses with maximum likelihood were interpreted as ploidy status, revealed fetal fraction, and represented the accuracy of sample-specific calculations (Fig. 29E).

Figures 30A-30H: Representative graphical representations of euploidy (Figures 30A-30C), monosomy (Figure 30D) and trisomy (Figures 30E-30H). For all plots, the x-axis represents the linear position of the individual polymorphic loci along each chromosome (as indicated below the figure), and the y-axis represents A etc. as a fraction of the total (A+B) allelic reads. The read value of the bit gene. The maternal and fetal genotypes and the position of the band centers on the y-axis are indicated on the right side of the graph. To facilitate visualization if necessary, the plot can be color coded according to maternal genotype such that red represents maternal genotype AA, blue represents maternal genotype BB, and green represents maternal genotype AB. The maternal allelic contribution can be color-coded in the "Fetal Genotype" column, if desired. Allelic contributions are expressed in the maternal|fetal form, such that alleles with mother AA and fetus AB are denoted AA|AB. Figure 30A: Graph generated when two chromosomes are present and the fetal cfDNA fraction is 0%. This graph is from a non-pregnant woman, and thus represents the pattern when the genotype is exclusively maternal. The allelic clusters are thus centered around 1 (AA allele), 0.5 (AB allele) and 0 (BB allele). Figure 30B: Graph produced when two chromosomes are present and the fetal fraction is 12%. The contribution of the fetal allele to the fraction of A allele reads shifts the position of some allele points up or down along the y-axis such that the bands are scaled by 1(AA|AA allele), 0.94(AA |AB allele), 0.56(AB|AA allele), 0.50(AB|AB allele), 0.44(AB|BB allele), 0.06(BB|AB allele), and 0(BB |BB allele) as the center. Figure 30C. Graph generated when two chromosomes are present and the fetal fraction is 26%. A pattern consisting of two red and two blue fringe bands and a set of three intermediate green bands is evident (not shown in color map). Each band is represented by 1 (AA|AA allele), 0.87 (AA|AB allele), 0.63 (AB|AA allele), 0.50 (AB|AB allele), 0.37 (AB|BB allele gene), 0.13 (BB|AB allele) and 0 (BB|BB allele) as centers. Figure 30D: Graph generated when one chromosome is present and the fetal fraction is 26%. A flag pattern of one outer red and one outer blue marginal band and two intermediate green bands indicates maternally inherited monosomy (not shown in color map). Because the fetus contributes only a single allele (A or B) to the allelic readout, the red and blue bands at the inner edges are absent, and the central set of three bands is compressed into two bands (not shown in the colormap Show). Bands are centered around 1 (AA|A allele), 0.57 (AB|A allele), 0.43 (AB|B allele), and 0 (BB|B allele). Figure 30E: Graph generated when three chromosomes are present and the fetal fraction is 27%. This pattern has two red and two blue marginal bands and two intermediate green bands, indicating a maternally inherited meiotic trisomy (not shown in color map). Each band is represented by 1 (AA|AAA allele), 0.88 (AA|AAB allele), 0.56 (AB|AAB allele), 0.44 (AB|ABB allele), 0.12 (BB|ABB allele gene) and 0 (BB|BBB allele) as the center. Figure 30F: Graph generated when three chromosomes are present and the fetal fraction is 14%. This pattern has three red and three blue marginal bands and two intermediate green bands, indicating a paternally inherited meiotic trisomy (not shown in color map). Each band is represented by 1 (AA|AAA allele), 0.93 (AA|AAB allele), 0.87 (AA|ABB allele), 0.60 (AB|AAA allele), 0.53 (AB|AAB allele gene), 0.47 (AB|ABB allele), 0.40 (AB|BBB allele), 0.13 (BB|AAB allele), 0.07 (BB|ABB allele), and 0 (BB|BBB allele gene) as the center. Figure 30G: Graph generated when three chromosomes are present and the fetal fraction is 35%. This pattern has two red and two blue marginal bands and four intermediate green bands, indicating a maternally inherited mitotic trisomy (not shown in color map). Each band is represented by 1 (AA|AAA allele), 0.85 (AA|AAB allele), 0.72 (AB|AAA allele), 0.57 (AB|AAB allele), 0.43 (AB|ABB allele gene), 0.28 (AB|BBB allele), 0.15 (BB|ABB allele), and 0 (BB|BBB allele) as centers. Figure 30H: Graph generated when three chromosomes are present and the fetal fraction is 25%. This pattern has two red and two blue marginal bands and four intermediate green bands, indicating a paternally inherited mitotic trisomy (not shown in color map). This pattern can be distinguished from a maternally inherited mitotic trisomy (as in Figure 30G) by the position of the internal marginal zone. Specifically, each band is represented by 1 (AA|AAA allele), 0.78 (AA|ABB allele), 0.67 (AB|AAA allele), 0.56 (AB|AAB allele), 0.44 (AB |ABB allele), 0.33 (AB|BBB allele), 0.22 (BB|AAB allele) and 0 (BB|BBB allele).

Figure 31 : (Figure 31A) Euploid, (Figure 31B) T13, (Figure 31C) T18, (Figure 31D) T21, (Figure 31E) 45,X and (Figure 31F) 47,XXY test samples as indicated graphical representation of . Each chromosome is shown at the top of the graph, fetal and maternal genotypes are shown on the right side of the graph, the x-axis represents the linear position of the SNP along each chromosome, and the y-axis represents A as a fraction of the total reads The read value of the allele. It should be noted that the altered cluster localization is based on fetal fraction, as described in this paper. Each dot represents a single SNP locus. Fetal and maternal genotypes are shown on the right side of the graph, and chromosomal identity is shown on the top of the graph.

Figure 32: The combined birth prevalence of sex chromosome aneuploidies is greater than the combined birth prevalence of autosomal aneuploidies.

While the above figures illustrate disclosed embodiments of the invention, other embodiments are also contemplated, as noted in the discussion. The present invention presents exemplary embodiments by means of illustrations and is not limited thereto. Many other modifications and embodiments within the scope and spirit of the principles of the disclosed embodiments of the invention will occur to those skilled in the art.

Detailed ways

The present invention is based in part on the surprising discovery that generally only a relatively small number of primers in a primer library results in the formation of a large number of amplified primer-dimers during multiplex PCR reactions. Methods have been developed to select the least desirable primers for removal from the pool of candidate primers. By reducing the amount of primer-dimers to negligible amounts (approximately 0.1% of the PCR product), these methods allow the resulting primer pool to simultaneously amplify a large number of target loci in a single multiplex PCR reaction. Because a primer hybridizes to a target locus and amplifies it rather than hybridizing to other primers and forming amplified primer-dimers, the number of different target loci that can be amplified is increased. It was also found that using lower than normal primer concentrations and much longer annealing times increased the likelihood that the primers would hybridize to the locus of interest rather than each other and form primer-dimers.

During PCR amplification and sequencing of 19,488 target loci in the genomic sample, 99.4%-99.7% of the sequencing reads mapped to the genome, of which 99.99% mapped to the targeted loci. For plasma samples with 10 million sequencing reads, typically at least 19,350 (99.3%) of the 19,488 targeted loci were amplified and sequenced. Being able to simultaneously amplify such a large number of target loci at once greatly reduces the amount of time and DNA volume required to analyze thousands of target loci. For example, DNA from a single cell is sufficient for the simultaneous analysis of thousands of target loci, which is important for applications with low DNA quantities, such as genetic testing of single cells from embryos prior to in vitro fertilization or analysis of extremely DNA-less forensic samples for genetic testing. Additionally, being able to analyze the locus of interest in one reaction volume (eg, in one chamber or well) rather than splitting the sample into multiple different reactions reduces the variability that may occur between reactions. Additionally, methods have been developed to correct for amplification bias that may occur between different target loci using reference standards. For example, differences in amplification efficiency between loci of interest due to factors such as GC content can cause loci of interest that are present in virtually the same amount to produce different amounts of PCR product. The use of a reference standard similar to the target locus allows detection of such amplification bias so that it can be corrected during quantification of the target locus.

During the sequencing of PCR products, artefacts such as primer dimers are detected and thus inhibit the detection of target amplicons. Because of this limitation, microarrays containing hybridization probes are often used for detection, since microarrays are less sensitive to interference from primer-dimers. The high level of multiplexing that has now been achieved with minimal non-target amplicons allows the use of PCR followed by sequencing as an alternative to microarrays.

The multiplex PCR method of the present invention can be used in various applications, such as genotyping, detection of chromosomal abnormalities (such as fetal chromosomal aneuploidy), gene mutation and polymorphism (such as single nucleotide polymorphism, SNP ) analysis, gene deletion analysis, paternity determination, analysis of genetic differences in populations, forensic analysis, measurement of disease predisposition, quantitative analysis of mRNA, and detection and identification of infectious agents such as bacteria, parasites, and viruses. The multiplex PCR method can also be used for non-invasive prenatal testing such as paternity testing or detection of fetal chromosomal abnormalities.

Exemplary Primer Design Methods

Highly complex PCR often produces an extremely high proportion of DNA products resulting from unproductive side reactions such as primer-dimer formation. In one embodiment, specific primers most likely to cause unproductive side reactions can be removed from the primer pool, resulting in a primer pool that will generate a greater proportion of amplified DNA that maps to the genome. The step of removing problematic primers (that is, those primers that are particularly likely to form dimers) has unexpectedly achieved extremely high levels of PCR compounding for subsequent analysis by sequencing. In systems where performance is significantly degraded by primer dimers and/or other malfunctioning products, such as sequencing, greater than 10-fold, greater than 50-fold and greater than 100-fold greater complexation than otherwise described has been achieved. It should be noted that this is in contrast to probe-based detection methods such as microarray, TAQMAN, PCR, etc., where excess primer dimers will not significantly affect the results. It should also be noted that the general belief in the art is that multiplex PCR for sequencing is limited to about 100 assays in the same well. Fluidigm and Rain Dance offer platforms for performing 48 or 1000 PCR assays on one sample in parallel reactions.

There are several ways to select primers from the library that minimize the amount of non-mapped primer dimers or other primer failure products. Empirical data shows that a small number of 'bad' primers results in a large number of unmapped primer-dimer side reactions. Removing these 'bad' primers can increase the percentage of sequence reads that map to the targeted loci. One way to identify 'bad' primers is to look at the sequencing data of the DNA that was amplified by targeted amplification; those primer-dimers seen at the highest frequency can be removed, resulting in significantly less likely to produce DNA that does not map to the genome Primer library of by-product DNA. There are also publicly available programs that can calculate the binding energies of various primer combinations, and removing those with the highest binding energies will also result in a pool of primers that are significantly less likely to produce by-product DNA that does not map to the genome.

In some embodiments for selecting primers, an initial library of primer candidates is created by designing one or more primers or primer pairs to candidate target loci. A set of candidate target loci (eg, SNPs) can be selected based on publicly available information about desired parameters of the target loci, such as the frequency of the SNP or the heterozygosity rate of the SNP within the target population. In one example, PCR primers can be designed using the Primer3 program (www.primer3.sourceforge.net; libprimer3 version 2.2.3, which is hereby incorporated by reference in its entirety). Primers can be designed to anneal within a specific annealing temperature range, have a specific range of GC content, have a specific size range, produce target amplicons within a specific size range, and/or be characterized by other parameters, if desired. Starting with multiple primers or primer pairs per candidate target locus increases the likelihood that primers or primer pairs for most or all target loci will remain in the library. In one embodiment, selection criteria may require that at least one primer pair per locus of interest remain in the library. In that way, most or all loci of interest will be amplified when using the final primer pool. This is desirable for applications such as screening for deletions or duplications at large numbers of locations in the genome, or screening large numbers of sequences associated with disease or increased disease risk (eg polymorphisms or other mutations). If one primer pair from a library would produce a target amplicon that overlaps a target amplicon generated by another primer pair, one of the primer pairs can be removed from the library to prevent interference.

In some embodiments, "undesirable scores" are calculated (eg, calculated in silico) for most or all possible combinations of two primers from a pool of candidate primers (a higher score indicates minimal desirability). In various embodiments, a non-ideal score is calculated for at least 80%, 90%, 95%, 98%, 99%, or 99.5% of the possible candidate primer combinations in the library. Each undesirable score is based at least in part on the likelihood of dimer formation between two candidate primers. If desired, the undesired score can also be based on one or more other parameters selected from the group consisting of: heterozygosity rate at the target locus, disease prevalence associated with sequences (e.g., polymorphisms) at the target locus rate, disease penetrance associated with sequence (e.g., polymorphism) at the target locus, specificity of candidate primers for the target locus, size of candidate primers, melting temperature of the target amplicon, target amplification The GC content of the target amplicon, the amplification efficiency of the target amplicon, and the size of the target amplicon. If multiple factors are considered, the unfavorable score can be calculated based on a weighted average of the individual parameters. The parameters can be assigned different weights based on their importance to the particular application in which the primer will be used. In some embodiments, primers with the highest undesirable scores are removed from the library. If the removed primer is a member of a primer pair that hybridizes to one locus of interest, then the other member of the primer pair can be removed from the library. The process of removing primers can be repeated as necessary. In some embodiments, the selection method is performed until the undesired scores of the remaining candidate primer combinations in the library are all at or below a minimum threshold. In some embodiments, the selection method is performed until the number of candidate primers remaining in the library is reduced to a desired number.

In various embodiments, after calculating the undesired score, candidate primers that are the portion of the maximum number of combinations of two candidate primers with an unsatisfactory score above a first minimum threshold are removed from the library. This step ignores interactions at or below the first minimum threshold, since these interactions are less important. If the removed primer is a member of a primer pair that hybridizes to one locus of interest, then the other member of the primer pair can be removed from the library. The process of removing primers can be repeated as necessary. In some embodiments, the selection method is performed until the undesired scores of remaining candidate primer combinations in the library are all at or below a first minimum threshold. If the number of candidate primers remaining in the library is higher than desired, the number of primers can be reduced by lowering the first minimum threshold to a lower second minimum threshold and repeating the process of removing primers. If the number of candidate primers remaining in the pool is lower than desired, the method can be continued by increasing the first minimum threshold to a higher second minimum threshold and repeating the process of removing primers using the initial pool of candidate primers, thereby allowing There are more primer candidates remaining in the library. In some embodiments, the selection method is performed until the undesired scores of the remaining candidate primer combinations in the library are all at or below a second minimum threshold, or until the number of remaining candidate primer combinations in the library is reduced to a desired number until.

If desired, a primer pair that produces a target amplicon that overlaps a target amplicon produced by another primer pair can be split into separate amplification reactions. Multiple PCR amplification reactions would be required for applications requiring the analysis of all candidate target loci (rather than omitting candidate target loci from analysis due to overlap with target amplicons).

These selection methods minimize the number of candidate primers that must be removed from the library, achieving the desired reduction in primer-dimers. By removing a smaller number of candidate primers from the library, the resulting primer library can be used to amplify more (or all) of the target loci.

Complexing a large number of primers imposes a number of constraints on the assays that can be included. Detection of unintentional interactions produces spurious amplification products. Further limitations arise from the size limitations of mini-PCRs. In one embodiment, it is possible to start with a very large number of potential SNP targets (between about 500 and greater than 1 million) and attempt to design primers that amplify each SNP. When primers can be designed, it is possible to attempt to identify those likely to form spurious products by using published thermodynamic parameters for DNA duplex formation to estimate the likelihood of spurious primer duplex formation between all possible primer pairs. primer pair. Primer interactions can be ranked by a score function related to interaction and primers with the worst interaction scores are eliminated until the desired number of primers is met. It is also possible to rank the list of tests and select the most heterozygous compatible test where the SNP that is likely to be heterozygous is most applicable. Experiments have verified that primers with high mutual use fractions are most likely to form primer-dimers. At high complexes, it is not possible to eliminate all spurious interactions, but it is necessary to remove primers or primer pairs with the highest fractional interaction in silico, as they can dominate the overall reaction, greatly limiting amplification of the intended target. We have performed this procedure to create composite primer sets of up to, and in some cases, more than 10,000 primers. Thanks to this procedure, the improvement is enormous, achieving 80%, 90%, 95%, 98% and even 99% of the target product compared to 10% from reactions that did not remove the worst primers. Amplification of , as determined by sequencing of all PCR products. When combined with the partially semi-nested approach as previously described, more than 90% and even more than 95% of the amplicons could be mapped to the targeting sequence.

It should be noted that there are other methods for determining which PCR probes are likely to form dimers. In one embodiment, analyzing a pool of DNA that has been amplified using a non-optimized set of primers may be sufficient to identify problematic primers. For example, sequencing can be used for analysis and it is determined that those dimers present in the greatest number are the ones most likely to form dimers and can be removed.

This method has a variety of potential applications, such as for SNP genotyping, heterozygosity rate determination, copy number measurement, and other targeted sequencing applications. In one embodiment, the primer design method can be used in combination with the mini-PCR method described elsewhere in this document. In some embodiments, primer design methods can be used as part of a large-scale multiplex PCR method.

Use of labels on primers can reduce amplification and sequencing of primer-dimer products. In some embodiments, the primer contains an internal region that forms a loop structure with the label. In certain embodiments, the primer includes a 5' region specific for the locus of interest, an inner region that is not specific for the locus of interest and forms a loop structure, and a 3' region specific for the locus of interest. In some embodiments, the loop region may be between two binding regions designed to bind to contiguous or adjacent regions of template DNA. In various embodiments, the 3' region is at least 7 nucleotides in length. In some embodiments, the 3' region is between 7 and 20 nucleotides, such as between 7 and 15 nucleotides or 7 and 10 nucleotides in length, inclusive. In various embodiments, the primers include a 5' region that is not specific for the locus of interest (e.g., a marker or universal primer binding site), followed by a region that is specific for the locus of interest, nonspecific for the locus of interest The inner region that is homogeneous and forms a loop structure and the 3' region that is specific for the locus of interest. Tag-primers can be used to shorten the necessary target-specific sequence to less than 20, less than 15, less than 12 and even less than 10 base pairs. This can be found incidentally when fragmenting the target sequence within the primer binding site in the case of standard primer design or it can be engineered into the primer design. Advantages of this approach include: it increases the number of assays that can be engineered for a certain maximum amplicon length, and it reduces "uninformative" sequencing of primer sequences. It can also be used in combination with internal markup (see elsewhere in this document).

In one embodiment, the relative amount of non-productive products in a multiplex targeted PCR amplification can be reduced by increasing the annealing temperature. In the case of amplified pools containing the same tag as the target-specific primers, the annealing temperature can be increased compared to genomic DNA because the tag will cause the primer to bind. In some examples, we used significantly lower primer concentrations than previously reported and used longer annealing times than reported elsewhere. In some embodiments, the annealing time may be longer than 3 minutes, longer than 5 minutes, longer than 8 minutes, longer than 10 minutes, longer than 15 minutes, longer than 20 minutes, longer than 30 minutes, longer than 60 minutes, longer than 120 minutes, longer than 240 minutes, Longer than 480 minutes, and even longer than 960 minutes. In one example, longer annealing times than in previous reports were used, allowing for lower primer concentrations. In various embodiments, a longer than normal extension time is used, for example over 3, 5, 8, 10 or 15 minutes. In some embodiments, primer concentrations are as low as 50 nM, 20 nM, 10 nM, 5 nM, 1 nM and below 1 μM. This surprisingly yields robust performance for highly complex reactions, such as 1,000-fold reactions, 2,000-fold reactions, 5,000-fold reactions, 10,000-fold reactions, 20,000-fold reactions, 50,000-fold reactions, and even 100,000-fold reactions. In one embodiment, amplification uses one, two, three, four or five cycles operating with long annealing times followed by PCR cycles with labeled primers with more common annealing times.

To select target positions, one can start with a pool of candidate primer pair designs and create a thermodynamic model of potentially adverse interactions between primer pairs, and then use the model to eliminate designs that are incompatible with other designs in the pool.

After the selection process, the remaining primers in the library can be used in any of the methods of the invention.

Exemplary primer library

In one aspect, the invention features a library of primers, eg, primers selected from a library of candidate primers using any of the methods of the invention. In some embodiments, the library comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, Primers for 30,000, 40,000, 50,000, 75,000, or 100,000 different target loci. In various embodiments, the library comprises between 1,000 to 2,000, 2,000 to 5,000, 5,000 to 7,500, 7,500 to 10,000 simultaneously amplified (or capable of being simultaneously amplified) in one reaction volume , 10,000 to 20,000, 20,000 to 25,000, 25,000 to 30,000, 30,000 to 40,000, 40,000 to 50,000, 50,000 to 75,000, or 75,000 to 100,000 different target loci Primers, including endpoints. In various embodiments, the library comprises primers that simultaneously amplify (or are capable of simultaneously amplifying) between 1,000 and 100,000 different target loci in one reaction volume, such as between 1,000 and 50,000 , 1,000 to 30,000, 1,000 to 20,000, 1,000 to 10,000, 2,000 to 30,000, 2,000 to 20,000, 2,000 to 10,000, 5,000 to 30,000, 5,000 to 20,000 or 5,000 Between 10,000 and 10,000 different loci of interest, including endpoints. In some embodiments, the library comprises simultaneous amplification (or is capable of simultaneous amplification) of target loci in one reaction volume such that less than 60%, 40%, 30%, 20%, 10%, 5%, 4 %, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.5% of the amplification products are primers that are primer dimers. In various embodiments, the amount of amplification product as a primer-dimer is between 0.5% and 60%, such as between 0.1% and 40%, 0.1% and 20%, 0.25% and 20%, 0.25% to 10%, 0.5% to 20%, 0.5% to 10%, 1% to 20%, or 1% to 10%, inclusive. In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) the target loci in one reaction volume such that at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% %, 98%, 99%, or 99.5% of the amplification products are target amplicons. In various embodiments, the amount of amplified product as target amplicon is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 98%, 90% to 99.5% or 95% to 99.5%, inclusive. In some embodiments, the primers simultaneously amplify (or are capable of simultaneously amplifying) the target loci in one reaction volume such that at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% %, 98%, 99%, or 99.5% of the targeted loci were amplified. In various embodiments, the amount of amplified target loci is between 50% and 99.5%, such as between 60% and 99%, 70% and 98%, 80% and 99%, 90% and 99.5% %, 95% to 99.9%, or 98% to 99.99%, inclusive. In some embodiments, the primer library comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 primer pairs, wherein each pair of primers includes a forward test primer and a reverse test primer Primers, where each pair of test primers hybridizes to one locus of interest. In some embodiments, the library of primers comprises at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 individual primers that each hybridize to a different target locus, wherein the individual primers Not part of the primer pair.

In various embodiments, the concentration of each primer is less than 100, 75, 50, 25, 20, 10, 5, 2 or 1 nM, or less than 500, 100, 10 or 1 μM. In various embodiments, the concentration of each primer is between 1 μM to 100 nM, eg, between 1 μM to 1 nM, 1 to 75 nM, 2 to 50 nM, or 5 to 50 nM, inclusive. In various embodiments, the primers have a GC content of between 30% and 80%, such as between 40% and 70% or between 50% and 60%, inclusive. In some embodiments, the GC content of the primers ranges from less than 30%, 20%, 10%, or 5%. In some embodiments, the primers have a GC content in the range of 5% to 30%, such as 5% to 20% or 5% to 10%, inclusive. In some embodiments, the test primer has a melting temperature ( _Tm ) between 40°C to 80°C, eg, 50°C to 70°C, 55°C to 65°C, or 57°C to 60.5°C, inclusive. In some embodiments, _Tm is calculated using the Primer3 program (libprimer3 version 2.2.3) using built-in Santa Lucia parameters (www.primer3.sourceforge.net). In some embodiments, the melting temperature of the primer ranges from less than 15°C, 10°C, 5°C, 3°C, or 1°C. In some embodiments, the melting temperature of the primer ranges between 1°C to 15°C, eg, between 1°C to 10°C, 1°C to 5°C, or 1°C to 3°C, inclusive. In some embodiments, the length of the primer is between 15 and 100 nucleotides, such as between 15 and 75 nucleotides, 15 to 40 nucleotides, 17 to 35 nucleotides, 18 to 30 nucleotides, between 20 and 65 nucleotides, inclusive. In some embodiments, primers range in length from less than 50, 40, 30, 20, 10, or 5 nucleotides. In some embodiments, the primers range in length from 5 to 50 nucleotides, such as 5 to 40 nucleotides, 5 to 20 nucleotides, or 5 to 10 nucleotides inclusive. . In some embodiments, the amplicon of interest is between 50 and 100 nucleotides, such as between 60 and 80 nucleotides or 60 to 75 nucleotides in length, inclusive. In some embodiments, target amplicons range in length from less than 50, 25, 15, 10, or 5 nucleotides. In some embodiments, the target amplicon ranges in length from 5 to 50 nucleotides, such as 5 to 25 nucleotides, 5 to 15 nucleotides, or 5 to 10 nucleotides in length , including the endpoint.

These primer libraries can be used in any of the methods of the invention.

Exemplary Primer Kits

In one aspect, the invention features a kit (eg, a kit for amplifying a target locus in a nucleic acid sample) that includes any one of the primer libraries of the invention. In some embodiments, kits comprising a plurality of primers designed to carry out the methods described herein can be formulated. Primers can be outer forward and reverse primers as disclosed herein, inner forward and reverse primers, which can have been designed to have low binding to other primers in the kit as disclosed in the primer design section Affinity primers, which can be hybrid capture probes or precircularized probes, or some combination thereof, as described in the relevant section. In one embodiment, a kit designed for use with the methods disclosed herein to determine the ploidy state of a target chromosome in a gestating fetus can be formulated comprising a plurality of internal forward primers and Optionally a plurality of internal reverse primers and optionally external forward primers and external reverse primers, wherein each of said primers is designed for hybridization to immediately following the target chromosome and optionally the additional chromosome A region of DNA upstream and/or downstream of one of the target sites (eg, polymorphic site). In one embodiment, a primer kit can be used in combination with a diagnostic kit as described elsewhere in this document. In some embodiments, the kit includes instructions for using the library to amplify the locus of interest.

Exemplary multiplex PCR method

In one aspect, the invention features a method of amplifying a target locus in a nucleic acid sample that involves (i) hybridizing a nucleic acid sample to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 primer pools of different target loci are contacted to generate a reaction mixture; and (ii) subjecting the reaction mixture to primer extension reaction conditions (e.g., PCR conditions) to generate an amplicon comprising the target amplicon increase product. In some embodiments, the method further comprises determining the presence or absence of at least one target amplicon (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% %, 99%, or 99.5% of target amplicons). In some embodiments, the method further comprises determining at least one target amplicon (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of target amplicons). In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the targeted loci are amplified. In various embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, or 0.05 % of amplification products were primer dimers.

In one embodiment, the methods disclosed herein use highly efficient high multiplex targeted PCR to amplify DNA, followed by high throughput sequencing to determine the allele frequency at each locus of interest. The ability to multiplex more than about 50 or 100 PCR primers in one reaction volume in such a way that a majority of the resulting sequence reads map to targeted loci is novel and non-obvious. One technique that allows highly complex targeted PCR to be performed in a highly efficient manner involves designing primers that are less likely to hybridize to each other. By creating at least 500, at least 1,000, at least 2,000, at least 5,000, at least 7,500, at least 10,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 potentially unfavorable interactions between potential primer pairs effect, or a thermodynamic model of unwanted interactions between primers and sample DNA, and then use the model to select PCR probes, often called primers, to eliminate designs that are incompatible with other designs in the pool. Another technique that allows highly multiplex targeted PCR to be performed in a highly efficient manner is the use of a partially or fully nested approach to targeted PCR. Use of one or a combination of these methods allows for the multiplexing of at least 300, at least 800, at least 1,200, at least 4,000, or at least 10,000 primers in a single pool, wherein the resulting amplified DNA contains DNA that will map to a targeted locus when sequenced. most of the DNA molecule. Using one or a combination of these methods allows multiplexing of a large number of primers in a single pool, wherein the resulting amplified DNA comprises more than 50%, more than 60%, more than 67%, more than 80%, more than 90%, more than 95%, more than 96%, greater than 97%, greater than 98%, greater than 99%, or greater than 99.5% of the DNA molecules map to the targeted locus.

In some embodiments, detection of genetic material of interest can be performed in a multiplexed manner. The number of gene target sequences that can be manipulated in parallel can range from one to ten, ten to one hundred, one hundred to one thousand, one thousand to ten thousand, ten thousand to one hundred thousand, one hundred thousand to one million, or one million to one Variations in the range of tens of millions. Previous attempts to multiplex more than 100 primers per pool have produced significant problems with undesired side reactions, such as primer-dimer formation.

targeted PCR

In some embodiments, PCR can be used to target specific locations in the genome. In plasma samples, the initial DNA was highly fragmented (typically less than 500 bp with an average length of less than 200 bp). In PCR, forward and reverse primers anneal to the same fragment to enable amplification. Therefore, if the fragment is short, then the PCR assay must also amplify a relatively short region. Like MIP, if the polymorphic position is too close to the polymerase binding site, then it can lead to biased amplification of different alleles. Currently, PCR primers targeting polymorphic regions (such as those containing SNPs) are typically designed such that the 3' end of the primer will hybridize to the base immediately adjacent to the polymorphic base. In one embodiment of the invention, the 3' ends of the forward and reverse PCR primers are designed for hybridization to one or several positions away from the variant position (polymorphic site) of the targeted allele. base. The number of bases between the polymorphic sites (SNP or others) and the 3' end of the designed primer hybridized can be one base, it can be two bases, it can be three bases, It could be four bases, it could be five bases, it could be six bases, it could be seven to ten bases, it could be eleven to fifteen bases, or it could be Sixteen to twenty bases. Forward and reverse primers can be designed to hybridize different numbers of bases away from the polymorphic site.

Large numbers of PCR assays can be generated, however, the interaction between different PCR assays makes it difficult to multiplex them beyond about a hundred assays. Various complex molecular approaches can be used to increase the level of complexation, but it is still likely to be limited to less than 100, perhaps 200 or possibly 500 assays per reaction. Samples containing large amounts of DNA can be split into multiple sub-reactions and then reassembled prior to sequencing. Splitting the sample will introduce statistical noise with respect to the entire sample of DNA molecules or samples where some subpopulations are restricted. In one embodiment, a small or limited amount of DNA can refer to an amount below 10 pg, between 10 and 100 pg, between 100 pg and 1 ng, between 1 and 10 ng, or between 10 and 100 ng . It should be noted that while this method works especially well for small amounts of DNA, where other methods involving splitting into multiple pools can cause significant problems related to the random noise introduced, this method does not work well for samples containing any amount of DNA. It also provides the benefit of minimizing bias when operating on the fly. In these cases, a general preamplification step can be used to increase the overall sample size. Ideally, this preamplification step should not significantly alter the allelic distribution.

In one embodiment, the method of the present invention can generate a large number of targeted loci, specifically 1,000 to 5,000 loci, 5,000 to 10,000 loci, or more than 10,000 locus-specific PCR products for genotyping by sequencing or some other genotyping method. Currently, performing multiplex PCR reactions with more than 5 to 10 targets presents a significant challenge and is often hindered by primer by-products such as primer dimers and other artefacts. When microarrays are used to detect target sequences with hybridization probes, primer-dimers and other artefacts can be ignored because these species are not detected. However, when sequencing is used as a detection method, the vast majority of sequencing reads will sequence such artifacts rather than the desired target sequence in the sample. Methods described in the prior art to multiplex more than 50 or 100 reactions in one reaction volume followed by sequencing will typically yield more than 20%, and often more than 50%, in many cases more than 80% and in some cases more than 90% % of off-target sequence reads.

In general, to perform targeted sequencing of multiple targets (over 50, over 100, over 500, or over 1,000) of a sample, the sample can be split into multiple parallel reactions that amplify a single target. This has been performed in PCR multi-well plates or can be performed in commercial platforms such as FLUIDIGM ACCESS (48 reactions per sample in a microfluidic chip) or droplet PCR (DROPLET PCR) via Ray En Downs technique (hundreds to thousands of targets). Unfortunately, these split-and-merge methods are problematic for samples containing limited amounts of DNA because often not enough copies of the genome are present to ensure that one copy of each region of the genome is present in each well. This is a particularly serious problem when targeting polymorphic loci and the relative proportions of alleles at the polymorphic loci are desired, since random noise introduced by splitting and merging will result in isomorphic differences present in the initial DNA sample. The measurement of the ratio of alleles is very inaccurate. A method is described herein that can efficiently and efficiently amplify multiple PCR reactions where only limited amounts of DNA are available. In one embodiment, the method can be adapted for analysis of single cells, bodily fluids, DNA mixtures (such as free-floating DNA found in maternal plasma), biopsies, environmental and/or forensic samples.

In one embodiment, targeted sequencing may involve one, more or all of the following steps. a) Generate and amplify a library with adapter sequences on both ends of the DNA fragments. b) Divide into multiple reactions after library amplification. c) Generating and optionally amplifying a library with adapter sequences at both ends of the DNA fragments. d) Perform 1,000 to 10,000 multiplex amplifications of selected targets using one target-specific "forward" primer and one marker-specific primer per target. e) Using a "reverse" target-specific primer and one (or more) primers specific for the universal marker introduced as part of the target-specific forward primer in the first round, from this product Perform a second amplification. f) Perform 1000-plex pre-amplification of selected targets for a limited number of cycles. g) Divide the product into multiple aliquots and amplify the subpool of interest in separate reactions (eg, 50 to 500 multiplexes), but this can be used down to singleplexes. h) Combining the products of parallel subpool reactions. i) During these amplifications, primers can be sequenced to compatible markers (partial or full length) so that the products can be sequenced.

high multiplex PCR

Disclosed herein are methods that allow the targeted amplification of over a hundred to tens of thousands of target sequences (eg, SNP loci) from a nucleic acid sample (eg, genomic DNA obtained from plasma). The amplified sample can be relatively free of primer-dimer products and have low allelic bias at the locus of interest. Analysis of products can be performed by sequencing if, during or after amplification, the products are attached with sequencing compatible adapters.

Performing high multiplex PCR amplification using methods known in the art produces primer-dimer products in excess of the desired amplification product and is unsuitable for sequencing. These can be empirically reduced by eliminating primers that form these products or by performing in silico selection of primers. However, the larger the number of detections, the harder this problem becomes.

One solution is to split the 5000-plex reaction into several lower multiplex amplifications, such as one hundred 50-plex or fifty 100-plex reactions, or use microfluidics or even split the samples into separate PCRs reaction. However, if sample DNA is limited, such as in non-invasive prenatal diagnosis of pregnancy plasma, then splitting the sample between multiple reactions should be avoided as this will create a bottleneck effect.

Described herein is a method of first collectively amplifying the plasma DNA of a sample and then splitting the sample into multiple composite target enrichment reactions, each with a more modest number of target sequences. In one embodiment, the method of the present invention can be used to preferentially enrich a DNA mixture at multiple loci, the method comprising one or more of the following steps: generating and amplifying a library from the DNA mixture, wherein the Molecules have adapter sequences ligated on both ends of DNA fragments; divide amplified pool into multiple reactions; use one target-specific "forward" primer and one or more adapter-specific universal "reverse" per target Primers, which perform the first round of multiplex amplification of the selected target. In one embodiment, the method of the invention further comprises the use of a "reverse" target-specific primer and one or more markers specific for the universal marker introduced as part of the target-specific forward primer in the first round. primers, to perform a second amplification. In one embodiment, the method may involve fully nested, half-nested, half-nested, single-sided fully nested, single-sided half-nested, or single-sided half-nested PCR methods . In one embodiment, the method of the invention is used to preferentially enrich a DNA mixture at multiple loci, the method comprising performing multiplex preamplification of selected targets for a finite number of cycles, dividing the product into multiple aliquots sample and amplify target subpools in separate reactions, and combine products from parallel subpool reactions. It should be noted that this approach can be used for 50 to 500 loci, for 500 to 5,000 loci, for 5,000 to 50,000 loci, or even for 50,000 to 500,000 loci to produce low-level alleles Targeted amplification is performed in a genetically biased manner. In one embodiment, the primers carry partial or full-length sequencing compatible tags.

A workflow may require (1) extraction of DNA, such as plasma DNA; (2) preparation of a fragment library with universal adapters on both ends of the fragments; (3) amplification of the library using universal primers specific to the adapters; (4) ) divide the amplified sample "pool" into multiple aliquots; (5) perform multiplexing (e.g., about 100, 1,000, or 10,000 multiplexes on the aliquots with one target-specific primer and marker-specific (6) pool aliquots of a sample; (7) identify the barcode of the sample; (8) mix the samples and adjust the concentration; (9) sequence the sample. The workflow can contain multiple sub-steps containing one of the steps listed (e.g. step (2) library preparation step can require three enzymatic steps (end blunting, dA tailing, and adapter ligation) and three purification steps ). The steps of the workflow can be combined, split, or executed in a different order (such as identifying barcodes and merging samples).

It is important to note that library amplification can be performed in a manner that favors the more efficient amplification of short fragments. In this way, it is possible to preferentially amplify shorter sequences, such as mononucleosomal DNA fragments, such as cell-free fetal DNA (of placental origin) found in the circulation of pregnant women. It should be noted that PCR detection may have a marker, such as a sequencing marker (usually a truncated form of 15 to 25 bases). After multiplexing, the PCR multiplexing results of the samples are pooled and labeling (including identification of barcodes) is then done by marker-specific PCR (which can also be done by conjugation). Additionally, full sequencing tags can be added in the same reaction as multiplexing. In the first cycle, the target can be amplified with target-specific primers, followed by tag-specific primers taking over to complete the SQ-adaptor sequence. PCR primers may not carry a marker. A sequencing marker can be attached to the amplification product by ligation.

In one embodiment, for various applications such as the detection of fetal aneuploidy, amplified material can be estimated by clonal sequencing using high multiplex PCR sequentially. While conventional multiplex PCR estimates up to fifty loci simultaneously, the methods described herein can be used to achieve simultaneous estimation of more than 50 loci, simultaneous estimation of more than 100 loci, simultaneous estimation of more than 500 loci, More than 1,000 loci are estimated simultaneously, more than 5,000 loci are estimated simultaneously, more than 10,000 loci are estimated simultaneously, more than 50,000 loci are estimated simultaneously, and more than 100,000 loci are estimated simultaneously. Experiments have shown that up to, including 10,000, and more than 10,000 different loci can be estimated simultaneously in a single reaction with sufficient efficiency and specificity to make a non-invasive prenatal non-integrated gene with high accuracy. Ploidy diagnosis and/or copy number call. Detection can be combined in a single reaction with an entire sample, such as a cfDNA sample isolated from maternal plasma, a fraction thereof, or another processed derivative of a cfDNA sample. Samples (eg, cfDNA or derivatives) can also be split into multiple parallel multiplexing reactions. Optimal sample splitting and recombination is determined by weighing various performance specifications. Due to the limited amount of material, splitting the sample into multiple parts introduces sampling noise, processing time, and increases the possibility of error. Conversely, higher recombination produces greater amounts of spurious amplification and greater amplification inequality, both of which degrade test performance.

Two key relevant considerations in the application of the methods described herein are the finite amount of starting sample (eg, plasma) and the number of starting molecules in the material from which allele frequency or other measurements are to be obtained. If the number of initial molecules drops below a certain level, random sampling noise becomes significant and affects test accuracy. Typically, data of sufficient quality for a non-invasive prenatal diagnosis of aneuploidy can be obtained if measurements are performed on a sample containing an equal number of 500-1000 initial molecules per locus of interest. There are various ways to increase the number of different measurements, such as increasing the sample volume. Every manipulation applied to a sample also potentially creates a loss of matter. It is necessary to characterize the losses caused by various manipulations and avoid them, or improve the results of certain manipulations as necessary to avoid losses that may reduce the performance of the test.

In one embodiment, it is possible to mitigate potential losses in subsequent steps by amplifying all or a portion of the initial sample (eg, cfDNA sample). Various methods are available to amplify all genetic material in a sample, increasing the amount available for downstream procedures. In one embodiment, ligation-mediated PCR (LM-PCR) DNA fragments are amplified by PCR following ligation of one different adapter, two different adapters, or multiple different adapters. In one embodiment, multiple displacement amplification (MDA) phi-29 polymerase is used to isothermally amplify all DNA. In DOP-PCR and variants, random priming is used to amplify the starting material DNA. Each method has certain characteristics, such as uniformity of amplification across all represented regions of the genome, efficiency of capture and amplification of initial DNA, and amplification performance as a function of fragment length.

In one example, LM-PCR can be used with a single heteroduplex adapter with a 3' tyrosine. Heteroduplex adapters enable the use of a single adapter molecule that can be converted to two different sequences on the 5' and 3' ends of the original DNA fragment during the first round of PCR. In one embodiment, it is possible to fractionate the amplified library by size separation or product (eg AMPURE, TASS) or other similar methods. Prior to ligation, the sample DNA ends can be blunt-ended and then a single adenosine base added to the 3' end. Prior to ligation, the DNA can be cleaved using restriction enzymes or some other cleavage method. During ligation, the 3' adenosine of the sample fragments and the complementary 3' tyrosine overhang of the adapter can enhance ligation efficiency. From a temporal standpoint, the extension step of PCR amplification can be limited to reduce the amplification of fragments longer than about 200 bp, about 300 bp, about 400 bp, about 500 bp, or about 1,000 bp. Since the longer DNA found in maternal plasma is almost exclusively of maternal origin, this could enable 10%-50% enrichment of fetal DNA and improve test performance. Multiple reactions were run using conditions as specified by commercially available kits; resulting in successful ligation of less than 10% of the sample DNA molecules. A series of optimizations of the reaction conditions at this point increased conjugation to about 70%.

mini-PCR

The following mini-PCR method is desirable for samples containing short nucleic acids, digested nucleic acids, or fragmented nucleic acids such as cfDNA. Conventional PCR assay designs result in substantial loss of different fetal molecules, but this loss can be greatly reduced by designing extremely short PCR assays called mini-PCR assays. Fetal cfDNA in maternal serum was highly fragmented and fragment sizes were distributed roughly in a Gaussian fashion with a mean of 160 bp, a standard deviation of 15 bp, a minimum size of about 100 bp, and a maximum size of about 220 bp. The distribution of fragment start and end positions relative to targeted polymorphisms, while not necessarily random, varies widely within individual targets and across all targets and polymorphic sites at a particular target locus can occupy polymorphic sites derived from Any position from the beginning to the end of each segment of the locus. It should be noted that the term mini-PCR may equally refer to ordinary PCR without additional constraints or limitations.

During PCR, amplification will only occur from the template DNA fragment containing the forward and reverse primer sites. Because fetal cfDNA fragments are short, the likelihood that two primer sites exist, the likelihood of a fetal fragment of length L containing forward and reverse primer sites, is the ratio of amplicon length to fragment length. Under ideal conditions, assays with amplicons of 45, 50, 55, 60, 65 or 70 bp will be successfully amplified from 72%, 69%, 66%, 63%, 59% or 56% of the available template fragments, respectively molecular. Amplicon length is the distance between the 5' ends of the forward and reverse priming sites. Amplicon lengths shorter than those typically used by those skilled in the art can yield more efficient measurements of desired polymorphic loci by requiring only short sequence reads. In one embodiment, a substantial portion of the amplicon should be less than 100 bp, less than 90 bp, less than 80 bp, less than 70 bp, less than 65 bp, less than 60 bp, less than 55 bp, less than 50 bp, or less than 45 bp.

It should be noted that in methods known in the prior art, short assays such as those described herein are generally avoided, as they are not desired and they limit primer length, annealing characteristics and distance between forward and reverse primers. A number of constraints are imposed on primer design.

It should also be noted that if the 3' end of either primer is within approximately 1-6 bases of the polymorphic site, there is the potential for biased amplification. This single base difference in the initial polymerase binding site can cause preferential amplification of one allele, which can alter the observed allele frequency and reduce performance. All of these limitations make it very challenging to identify primers that will successfully amplify a particular locus and, moreover, to design a large number of primer sets that are compatible in the same multiplex reaction. In one embodiment, the 3' ends of the internal forward and reverse primers are designed to hybridize to a DNA region upstream of the polymorphic site and separated from the polymorphic site by a small number of bases. Ideally, the number of bases can be between 6 and 10 bases, but equally can be between 4 and 15 bases, between three and 20 bases, between two between 30 bases or between 1 and 60 bases, and achieve substantially the same purpose.

Multiplex PCR can involve a single round of PCR that amplifies all targets or it can involve one round of PCR followed by one or more rounds of nested PCR or some variation of nested PCR. Nested PCR consists of one or more subsequent rounds of PCR amplification using one or more new primers internally bound by at least one base pair to the primers used in the previous round. Nested PCR reduces the number of spurious amplified targets caused by amplification in subsequent reactions to only those amplified products from the previous reaction with the correct internal sequence. Reducing spurious amplification targets improves the number of useful measurements that can be obtained, especially in sequencing. Nested PCR usually requires the design of primers that are completely inside the binding site of the previous primer, necessarily increasing the minimum DNA segment size required for amplification. Regarding samples such as maternal plasma cfDNA, where the DNA is highly fragmented, larger assay sizes reduce the number of distinct cfDNA molecules from which measurements can be obtained. In one embodiment, to counteract this effect, a partially nested approach can be used, in which one or both of the second round primers overlap the first binding site and are internally extended by a certain number of bases to obtain Additional specificity while minimally increasing overall assay size.

In one embodiment, multiple pools of PCR assays are designed to potentially amplify heterozygous SNPs or other polymorphic or non-polymorphic loci on one or more chromosomes and these assays are used in a single reaction to Amplify DNA. The number of PCR tests can be between 50 and 200 PCR tests, between 200 and 1,000 PCR tests, between 1,000 and 5,000 PCR tests, or between 5,000 and 20,000 PCR tests (50 to 200 weights, 200 to 1,000 weights, 1,000 to 5,000 weights, 5,000 to 20,000 weights, and over 20,000 weights, respectively). In one embodiment, a composite pool of approximately 10,000 PCR assays (10,000 multiplexes) is designed to potentially amplify heterozygous SNP loci on chromosomes X, Y, 13, 18, and 21 and chromosomes 1 or 2 and These assays are used in a single reaction to amplify cfDNA obtained from plasma samples, chorionic villi samples, amniocentesis samples, single or small numbers of cells, other body fluids or tissues, cancer or other genetic material. The SNP frequency for each locus can be determined by cloning or some other sequencing method of the amplicon. Statistical analysis of all detected allele frequency distributions or ratios can be used to determine whether a sample contains a trisomy for one or more of the chromosomes included in the test. In another embodiment, the initial cfDNA sample was split into two samples and parallel 5,000-plex assays were performed. In another embodiment, the initial cfDNA sample is split into n samples and parallel (approximately 10,000/n) multiplexing is performed, where n is between 2 and 12, or between 12 and 24, or between Between 24 and 48, or between 48 and 96. Data were collected and analyzed in a similar manner as already described. It should be noted that this method is equally applicable to the detection of translocations, deletions, duplications and other chromosomal abnormalities.

In one embodiment, a tail having no homology to the target genome may also be added to the 3' or 5' end of any of the primers. These tails facilitate subsequent manipulations, procedures or measurements. In one example, the tail sequence can be the same for the forward and reverse target-specific primers. In one embodiment, different tails can be used for the forward and reverse target-specific primers. In one embodiment, multiple different tails can be used for different loci or groups of loci. Certain tails can be shared among all loci or among subsets of loci. For example, direct sequencing after amplification can be achieved using forward and reverse tails corresponding to the forward and reverse sequences required by any of the current sequencing platforms. In one embodiment, the tail can serve as a common priming site in all amplification targets that can be used to add other suitable sequences. In some embodiments, internal primers may contain regions designed to hybridize upstream or downstream of a targeted locus (eg, a polymorphic locus). In some embodiments, primers may contain molecular barcodes. In some embodiments, primers may contain universal priming sequences designed to allow PCR amplification.

In one example, a 10,000-plex PCR pool is created such that the forward and reverse primers have tails corresponding to the forward and reverse sequences required by a high-throughput sequencing instrument, such as commercially available HISEQ, GAIIX or MYSEQ from ILLUMINA. In addition, the 5' included in the sequencing tail is an additional sequence that can be used as a priming site in subsequent PCR to add a nucleotide barcode sequence to the amplicon, enabling multiple sequencing in a single lane of a high-throughput sequencing instrument. Composite sequencing of samples.

In one embodiment, a 10,000-plex PCR detection pool is created such that the reverse primers have tails corresponding to the reverse sequences required by high throughput sequencing instruments. After amplification with the first 10,000-plex detection, another forward primer with a partially nested (e.g., 6-base nested) targeting all targets and corresponding to the reverse sequencing included in the first round can be used. Tail the 10,000-plex pool of reverse primers to perform subsequent PCR amplification. This latter round of partially nested amplification with only one target-specific and universal primer limits the required assay size, reduces sampling noise, but greatly reduces the number of spurious amplicons. Sequencing tags can be added to the attached ligated adapters and/or as part of the PCR probes so that the tags are part of the final amplicon.

Fetal fraction affects test performance. There are various ways to enrich the fetal fraction of DNA found in maternal plasma. Fetal fraction can be increased by the already discussed LM-PCR method described previously as well as by targeted removal of long maternal fragments. In one embodiment, prior to multiplex PCR amplification of the loci of interest, additional multiplex PCR reactions can be performed to selectively remove long and largely maternally derived genes corresponding to loci targeted in subsequent multiplex PCRs. fragment of this book. Additional primers were designed for annealing sites that were more distant from the polymorphism than would be expected to be present in the cell-free fetal DNA fragment. These primers can be used in a one-cycle multiplex PCR reaction prior to multiplex PCR for the polymorphic loci of interest. These end primers are labeled with molecules or moieties that allow selective recognition of labeled DNA fragments. In one example, these DNA molecules can be covalently modified with a biotin molecule that allows removal of newly formed double-stranded DNA containing these primers after one cycle of PCR. The double-stranded DNA formed during this first round is likely to be of maternal origin. Removal of hybridized material can be achieved by using streptavidin magnetic beads. There are other labeling methods that can work equally well. In one embodiment, a size selection method can be used to enrich for shorter DNA strands in the sample; for example, those less than about 800 bp, less than about 500 bp, or less than about 300 bp. Amplification of short fragments can then be performed as usual.

The mini-PCR method described in this invention enables highly multiplexed amplification and analysis of hundreds to thousands or even millions of loci from a single sample in a single reaction. Likewise, detection of amplified DNA can be multiplexed; tens to hundreds of samples can be multiplexed in one sequencing lane by using barcoded PCR. This composite assay has been successfully tested up to 49-fold, and much higher degrees of composites are possible. In effect, this allows hundreds of samples to be genotyped at thousands of SNPs in a single sequencing operation. With respect to these samples, the method allows determination of genotype and heterozygosity rate and simultaneous determination of copy number, both of which can be used for aneuploidy detection purposes. This method is particularly useful for detecting aneuploidy in a gestating fetus from free-floating DNA found in maternal plasma. This method can be used as part of a method for identifying the sex of a fetus and/or predicting the parentage of a fetus. It can be used as part of a dosing method for mutations. This approach can be used with any amount of DNA or RNA, and the targeted regions can be SNPs, other polymorphic regions, non-polymorphic regions, and combinations thereof.

In some embodiments, ligation-mediated universal PCR amplification of fragmented DNA can be used. Ligation-mediated universal PCR amplification can be used to amplify plasma DNA, which can then be divided into multiple parallel reactions. It can also be used to preferentially amplify short fragments, thereby enriching the fetal fraction. In some embodiments, detection of shorter fragments can be achieved by adding labels to the fragments by ligation, using shorter target-sequence-specific portions of primers and/or annealing at higher temperatures that reduce non-specific reactions.

The methods described herein can be used for multiple purposes where there is a target DNA group mixed with an amount of contaminated DNA. In some embodiments, the target DNA and the contaminated DNA can be from genetically related individuals. For example, fetal (target) genetic abnormalities can be detected from maternal plasma containing fetal (target) DNA as well as maternal (contaminated) DNA; such abnormalities include whole chromosome abnormalities (such as aneuploidy), partial chromosome abnormalities Abnormalities (eg, deletions, duplications, inversions, translocations), polynucleotide polymorphisms (eg, STRs), single nucleotide polymorphisms, and/or other genetic abnormalities or differences. In some embodiments, the target and contaminating DNA may be from the same individual, but where the target and contaminating DNA differ by one or more mutations, such as in the case of cancer. (See e.g. Preferential Ampiification of Apoptotic DNA from Plasma: Potential for Enhancing Detection of Minor DNA Alterations in Circulating DNA by Preferential Amplification of Apoptotic DNA from Plasma by H. Mamon et al. Detection of Minor DNA Alterations in Circulating DNA). Clinical Chemistry 54:9 (2008). In some embodiments, DNA can be found in cell culture (apoptotic) supernatants. In some embodiments, it is possible to induce apoptosis in a biological sample (eg, blood) for subsequent library preparation, amplification, and/or sequencing. Several workflows and schemes for accomplishing this are presented elsewhere in this disclosure.

In some embodiments, the DNA of interest can be derived from a single cell, from a DNA sample consisting of less than one copy of the genome of interest, from low amounts of DNA, from DNA from mixed sources (e.g., pregnancy plasma: placental and maternal DNA; Cancer patient plasma and tumors: mixing between healthy and cancer DNA, transplantation, etc.), from other body fluids, from cell cultures, from culture supernatants, from forensic DNA samples, from ancient DNA samples ( such as insects trapped in amber), derived from other DNA samples, and combinations thereof.

In some embodiments, short amplicon sizes can be used. Short amplicon sizes are especially suitable for fragmented DNA (see e.g. A. Sikora et al. Detection of increased amounts of cell-free fetal DNA using short PCR amplicons. DNA with short PCR amplicons). Clin Chem. 2010 Jan;56(1):136-8).

The use of short amplicon sizes can yield some significant benefits. Short amplicon sizes result in optimized amplification efficiencies. Short amplicon sizes generally yield shorter products and therefore less chance of non-specific priming. Shorter products can be more densely clustered on the sequencing flow cell because the clusters will be smaller. It should be noted that the methods described herein can be equally applied to longer PCR amplicons. Amplicon length can be increased if necessary, for example when sequencing larger stretches of sequence. Positive results were obtained in single cell and genomic DNA operations with 146-fold targeted amplification experiments with a length of 100bp to 200bp as the first step in the nested PCR protocol.

In some embodiments, the methods described herein can be used to amplify and/or detect SNPs, copy number, nucleotide methylation, mRNA levels, other types of RNA expression levels, other genetic characteristics and/or expression Epigenetic features. The mini-PCR method described herein can be used in conjunction with next generation sequencing; it can be used with other downstream methods such as microarrays, counting by digital PCR, real-time PCR, mass spectrometry, etc.

In some embodiments, the mini-PCR amplification methods described herein can be used as part of a method for accurate quantification of minority populations. It can be used for absolute quantification using a spike calibrator. It can be used for mutation/minor allele quantification by very deep sequencing and can operate in a highly multiplexed fashion. It can be used for standard parentage and identity testing of relatives or ancestry in humans, animals, plants or other organisms. It can be used in forensic testing. It can be used for rapid genotyping and copy number analysis (CN) of any kind of material such as amniotic fluid and CVS, sperm, product of conception (POC). It can be used for single-cell analysis such as genotyping of biopsy samples from embryos. It can be used for rapid embryo analysis (within less than one, one or two days of biopsy) by targeted sequencing using mini-PCR.

In some embodiments, it can be used in tumor analysis: tumor biopsies are often a mixture of healthy and tumor cells. Targeted PCR allows deep sequencing of SNPs and loci without access to background sequences. It can be used for copy number and loss of heterozygosity analysis of tumor DNA. The tumor DNA can be present in many different body fluids or tissues of tumor patients. It can be used to detect tumor recurrence and/or tumor screening. It can be used for quality control testing of seeds. It can be used for breeding or fishing purposes. It should be noted that either of these methods can equally be used to target non-polymorphic loci for ploidy calling.

Some of the literature describing some of the basic methods underlying the methods disclosed herein include: (1) Wang HY, Luo M, Tereshchenko IV, Frick DM(FrikkerDM), Cui X(Cui X), Li JY(Li JY), Hu G(Hu G), Zhu Y(Chu Y), Azaro MA(AzaroMA), Lin Y(Lin Y), Shen L (Shen L), Yang Q (Yang Q), Kambouris ME (Kambouris ME), Gao R (Gao R), Shi W (Shih W), Li H (Li H). Genome Research (Genome Res.) 2005 Feb;15(2):276-83. Division of Molecular Genetics, Microbiology, and Immunology/Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903, USA. (2) High-throughput genotyping of single nucleotide polymorphisms with high sensitivity (High-throughput genotyping of single nucleotide polymorphisms with high sensitivity). Li H, Wang HY, Cui X, Luo M, Hu G, Greenawalt DM, Teresenko IV, Li JY, Zhu Y, Gao R. Methods Mol Biol. 2007; 396-PubMedPMID:18025699. (3) A method including a composite average of 9 detections for sequencing is described in: Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Varley KE (Varley KE), Mitra RD. Genome Res 2008 Nov;18(11):1844-50. Electronic version October 10, 2008. It should be noted that the methods disclosed herein allow for orders of magnitude more complexities than the above references.

Targeted PCR variant - nested

There are several possible workflows when performing PCR; some typical of the methods disclosed herein are described. The steps outlined herein are not intended to exclude other possible steps nor to imply that any of the steps described herein are required for the method to function properly. Numerous parameter changes or other modifications are known in the literature and can be made without affecting the essence of the invention. A concrete general workflow is given below, followed by many possible variants. Variants generally refer to possible second PCR reactions, such as different types of nesting that can be done (step 3). It is important to note that variants may be performed at different times or in different orders than explicitly described herein. The illustrated examples using polymorphic loci can be readily adapted for amplification of non-polymorphic loci, if necessary.

1. The DNA in the sample can have attached ligation adapters, commonly referred to as library tags or ligation adapter tags (LT), where the ligation adapters contain a universal priming sequence followed by universal amplification. In one embodiment, this can be done using standard protocols designed for creating sequencing libraries after fragmentation. In one example, the ends of the DNA sample can be blunt-ended, and an A can then be added at the 3' end. A Y adapter containing a T overhang can be added and ligated. In some embodiments, cohesive ends other than A or T overhangs may be used. In some embodiments, additional adapters may be added, such as circular ligation adapters. In some embodiments, adapters may have tags designed for PCR amplification.

2. Specific Target Amplification (STA): Pre-amplification that can multiplex hundreds to thousands to tens of thousands and even hundreds of thousands of targets in one reaction volume. A STA typically runs for 10 to 30 cycles, although it can run for 5 to 40 cycles, 2 to 50 cycles, and even 1 to 100 cycles. Primers can be tailed, e.g. for simpler workflows or to avoid sequencing large proportions of dimers. It should be noted that dimers of two primers carrying the same label will generally not be efficiently amplified or sequenced. In some embodiments, between 1 and 10 cycles of PCR may be performed; in some embodiments, between 10 and 20 cycles of PCR may be performed; in some embodiments, between 10 and 20 cycles of PCR may be performed; Between 20 and 30 cycles of PCR; in some embodiments, between 30 and 40 cycles of PCR can be performed; in some embodiments, more than 40 cycles of PCR can be performed. Amplification can be linear amplification. The number of PCR cycles can be optimized to produce an optimal depth-of-read (DOR) profile. Different purposes will require different DOR profiles. In some embodiments, a more even distribution of reads across all assays is desirable; if the DOR of some assays is too small, then the random noise may be too high for the data to be applicable; and if the read depth is too high, then each The marginal utility of the extra readings is relatively small.

Primer tails can improve detection of fragmented DNA from universal marker libraries. If the library markers and primer tails contain homologous sequences, hybridization can be improved (eg, melting temperature ( _TM ) is reduced) and primers can be extended if only a portion of the primer target sequence is in the sample DNA fragment. In some embodiments, 13 or more target-specific base pairs may be used. In some embodiments, 10 to 12 target-specific base pairs may be used. In some embodiments, 8 to 9 target-specific base pairs can be used. In some embodiments, 6 to 7 target-specific base pairs may be used. In some embodiments, STA, such as MDA, RCA, other whole-genome amplification, or adapter-mediated universal PCR, can be performed on pre-amplified DNA. In some embodiments, STA can be performed on samples enriched or depleted for certain sequences and populations, eg, by size selection, target capture, directed degradation.

3. In some embodiments, it is possible to perform a second multiplex PCR or primer extension reaction to increase specificity and reduce undesired products. For example, full nesting, half-nesting, half-side nesting, and/or parallel reactions subdivided into smaller detection pools are all techniques that can be used to increase specificity. Experiments have shown that splitting the sample into three 400-plex reactions yields product DNA with greater specificity than one 1,200-plex reaction with identical primers. Similarly, experiments have shown that splitting a sample into four 2,400-plex reactions with identical primers yields product DNA that is more specific than one 9,600-plex reaction. In one embodiment, it is possible to use target-specific and marker-specific primers with the same and opposite directionality.

4. In some embodiments, it is possible to amplify DNA samples (diluted, purified or otherwise) generated by STA reactions using marker-specific primers and "universal amplification", i.e. amplifying multiple or all preamplified And mark the target. Primers can contain additional functional sequences, such as barcodes, or full adapter sequences required for sequencing on high-throughput sequencing platforms.

These methods can be used to analyze any DNA sample, and are especially useful when the DNA sample is particularly small, or when it is a DNA sample in which the DNA originates from more than one individual, such as in the case of maternal plasma. These methods can be used with DNA samples such as single or small numbers of cells, genomic DNA, plasma DNA, amplified plasma pools, amplified apoptotic supernatant pools, or other mixed DNA samples. In one embodiment, these methods can be used in situations where cells with different genetic makeup may exist in a single individual, such as in the case of cancer or transplantation.

Scenario variants (variations and/or additions to the above workflows)

Direct multiplex mini-PCR: Specific target amplification (STA) of multiple target sequences with labeled primers is shown in Figure 1 . 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents single-stranded DNA that has been universally amplified with hybridized PCR primers. 104 represents the final PCR product. In some embodiments, STA may be performed on more than 100, more than 200, more than 500, more than 1,000, more than 2,000, more than 5,000, more than 10,000, more than 20,000, more than 50,000, more than 100,000, or more than 200,000 objects. In subsequent reactions, marker-specific primers amplify all target sequences and extend the markers to include all sequences required for sequencing, including sample indexing. In one embodiment, no primers may be labeled or only certain primers may be labeled. Sequencing adapters can be added by conventional adapter ligation. In one embodiment, an initial primer may carry a marker.

In one embodiment, primers are designed to amplify unexpectedly short lengths of DNA. The prior art indicates that one of ordinary skill in the art typically designs 100+bp amplicons. In one embodiment, the amplicon can be designed to be smaller than 80 bp. In one embodiment, the amplicon can be designed to be smaller than 70 bp. In one embodiment, the amplicon can be designed to be smaller than 60 bp. In one embodiment, the amplicon can be designed to be smaller than 50 bp. In one embodiment, the amplicon can be designed to be smaller than 45 bp. In one embodiment, the amplicon can be designed to be smaller than 40 bp. In one embodiment, the amplicon can be designed to be smaller than 35 bp. In one embodiment, the amplicon can be designed to be between 40 and 65 bp.

Experiments were performed using this protocol with 1200 multiplex amplification. Genomic DNA and pregnancy plasma were used; approximately 70% of sequence reads mapped to targeted sequences. Details are provided elsewhere in this document. Resequencing of 1042 without design and detection selection resulted in >99% of the sequences being primer dimer products.

Sequential PCR: After STA1, multiple aliquots of the product can be amplified in parallel with the same primer pool of reduced complexity. The first amplification yielded sufficient material for resolution. This method works especially well for small amounts of sample, such as about 6-100 pg, about 100 pg to 1 ng, about 1 ng to 10 ng, or about 10 ng to 100 ng of sample. Turn 1200 weights into three 400 weights to execute the scheme. Mapping of sequencing reads increased from about 60% to 70% of the 1200-plex alone to over 95%.

Semi-nested mini-PCR: (see Figure 2) After STA 1, a second STA is performed, consisting of a composite set of internally nested forward primers (103B, 105b) and one (or a small number) of marker-specific reverse primers. To the primer (103A). 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents the single-stranded DNA that has been universally amplified with hybridized forward primer B and reverse primer A. 104 represents the PCR product from 103. 105 represents the product from 104, which contains the hybridized nested forward primer b and reverse marker A, which was already part of the molecule from the PCR that occurred between 103 and 104. 106 represents the final PCR product. In the case of this workflow, typically more than 95% of the sequences map to the intended target. Nested primers can overlap the outer forward primer sequence but introduce an extra 3' base. In some embodiments, it is possible to use between one and 20 additional 3' bases. Experiments have shown that using 9 or more additional 3' bases in a 1200-plex design works equally well.

Fully nested mini-PCR: (see Figure 3) After STA step 1, it is possible to perform a second multiplex PCR (or reduced complexity parallel multiplex PCR). 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents the single-stranded DNA that has been universally amplified with hybridized forward primer B and reverse primer A. 104 represents the PCR product from 103. 105 represents the product from 104, which contains the hybridized nested forward primer b and nested reverse primer a. 106 represents the final PCR product. In some embodiments, it is possible to use two full sets of primers. Experiments using a fully nested mini-PCR protocol were used to perform 146 multiplex amplification on single and triple cells without the step 102 of attaching universal ligation adapters and amplification.

Hemi-nested mini-PCR: (see Figure 4) It is possible to use target DNA with adapters at the ends of the fragments. Perform STA with a composite set of forward primers (B) and one (or small number) of marker-specific reverse primers (A). A second STA can be performed using a universal marker-specific forward primer and a target-specific reverse primer. 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents single-stranded DNA that has been universally amplified with hybridized reverse primer A. 104 represents the PCR product from 103 amplified using reverse primer A and ligation adapter marker primer LT. 105 represents the product from 104, which contains hybridized forward primer B. 106 represents the final PCR product. In this workflow, target-specific forward and reverse primers are used in separate reactions, reducing reaction complexity and preventing dimer formation of forward and reverse primers. It should be noted that in this example, primers A and B may be considered first primers, and primers 'a' and 'b' may be considered internal primers. This method is a huge improvement over direct PCR because it works just as well, but it avoids primer-dimers. After the first round of the hemi-nested protocol, about 99% non-targeted DNA is typically seen, however after the second round there is usually a huge improvement.

Triple Hemi-Nested Mini-PCR: (see Figure 5) It is possible to use target DNA with adapters at the ends of the fragments. Perform STA with a composite set of forward primers (B) and one (or a small number) of marker-specific reverse primers (A) and (a). A second STA can be performed using a universal marker-specific forward primer and a target-specific reverse primer. 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents single-stranded DNA that has been universally amplified with hybridized reverse primer A. 104 represents the PCR product from 103 amplified using reverse primer A and ligation adapter marker primer LT. 105 represents the product from 104, which contains hybridized forward primer B. 106 represents the PCR product from 105 amplified using reverse primer A and forward primer B. 107 represents the product from 106, which contains the hybridized reverse primer 'a'. 108 represents the final PCR product. It should be noted that in this example, primers 'a' and B may be considered internal primers, and A may be considered the first primer. Optionally, both A and B may be considered the first primer, and 'a' may be considered the inner primer. The names of reverse and forward primers can be switched. In this workflow, target-specific forward and reverse primers are used in separate reactions, reducing reaction complexity and preventing dimer formation of forward and reverse primers. This method is a huge improvement over direct PCR because it works just as well, but it avoids primer-dimers. After the first round of the hemi-nested protocol, about 99% non-targeted DNA is typically seen, however after the second round there is usually a huge improvement.

Single-sided nested mini-PCR: (see Figure 6) It is possible to use target DNA with adapters at the ends of the fragments. STA can also be performed with a composite set of nested forward primers and using ligation adapter markers as reverse primers. A second STA can then be performed using a nested set of forward primers and a universal reverse primer. 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents the single-stranded DNA that has been universally amplified with hybridized forward primer A. 104 represents the PCR product from 103 amplified using forward primer A and ligation adapter marker reverse primer LT. 105 represents the product from 104, which contains the hybridized nested forward primer a. 106 represents the final PCR product. This method can detect target sequences shorter than standard PCR by using overlapping primers in the first and second STA. The method is typically performed on a DNA sample that has undergone STA step 1 above (attach universal marker and amplify); two nested primers on one side only, library marker on the other. The method was performed on apoptotic supernatants and pregnant plasma pools. In the case of this workflow, about 60% of the sequences mapped to the intended targets. Note that reads containing reverse adapter sequences were not mapped, so this number would be expected to be higher if those reads containing reverse adapter sequences were mapped.

Single-sided mini-PCR: It is possible to use target DNA with adapters at the ends of the fragments (see Figure 7). STA can be performed with a composite set of forward primers and one (or a small number) of tag-specific reverse primers. 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents the single-stranded DNA containing hybridized forward primer A. 104 represents the PCR product from 103 amplified using forward primer A and ligation adapter marker reverse primer LT and is the final PCR product. This method can detect target sequences that are shorter than standard PCR. However, it will be relatively non-specific since only one target-specific primer is used. This protocol is half as effective as single-sided nested mini-PCR.

Reverse semi-nested mini-PCR: It is possible to use target DNA with adapters at the ends of the fragments (see Figure 8). STA can be performed with a composite set of forward primers and one (or a small number) of tag-specific reverse primers. 101 represents double-stranded DNA with associated polymorphic loci at X. 102 represents double stranded DNA with ligation adapters added for universal amplification. 103 represents the single-stranded DNA containing hybridized reverse primer B. 104 represents the PCR product from 103 amplified using reverse primer B and ligation adapter marker forward primer LT. 105 represents the PCR product from 104, which contains hybridized forward primer A and internal reverse primer 'b'. 106 represents the PCR product that has been amplified from 105 using forward primer A and reverse primer 'b', and it is the final PCR product. This method can detect target sequences that are shorter than standard PCR.

There can also be more variants that are simple iterations or combinations of the above methods, such as double nested PCR, where three sets of primers are used. Another variant is a half-nested mini-PCR, where STA can also be performed with a composite set of nested forward primers and one (or a small number) of marker-specific reverse primers.

It should be noted that in all these variants the identities of the forward and reverse primers can be interchanged. It should be noted that in some embodiments, nested variants can also be manipulated without an initial library preparation comprising the attachment of adapter tags and general amplification steps. It should be noted that in some embodiments, additional rounds of PCR may be included with additional forward and/or reverse primers and amplification steps, these additional steps may be particularly useful when it is desired to further increase the percentage of DNA molecules corresponding to the loci of interest. Be applicable.

nested workflow

There are various ways to perform amplification, with different degrees of nesting and with different degrees of compositing. In Fig. 9, a flowchart with some possible workflows is given. It should be noted that the use of 10,000-plex PCRs is meant as an example only; these flow diagrams will apply equally to other multiplex degrees.

circular ligation adapter

When adding universally tagged adapters, eg, to make a library for sequencing, there are multiple ways to join the adapters. One way is to blunt the ends of the sample DNA, perform A-tailing, and ligate with adapters with T-overhangs. There are various other ways of joining adapters. There are also multiple adapters that can be ligated. For example, Y adapters can be used where the adapter consists of two strands of DNA, one of which has a double-stranded region and a region designated by the forward primer region, and where the other strand is composed of the same DNA as the first strand. The double-stranded region on the complementary double-stranded region and the region containing the reverse primer are specified. When annealed, the double stranded region may contain a T overhang for joining to double stranded DNA via an A overhang.

In one embodiment, the adapter can be a DNA loop, wherein the terminal regions are complementary, and wherein the loop region contains a forward primer tagging region (LFT), a reverse primer tagging region (LRT), and a region in between. Cleavage site (see Figure 10). 101 refers to double-stranded, blunt-ended target DNA. 102 refers to A-tailed target DNA. 103 refers to a circular ligation adapter containing a T overhang 'T' and a cleavage site 'Z'. 104 refers to the target DNA with attached circular ligation adapters. 105 refers to the target DNA with attached ligation adapter, cleaved at the cleavage site. LFT refers to ligation adapter forward tag and LRT refers to ligation adapter reverse tag. The complementary region may terminate in a T-overhang or other feature that may be used for ligation to the target DNA. The cleavage site can be a series of uracils that are cleaved by UNG, or a sequence that can be recognized and cleaved by restriction enzymes or other cleavage methods or just substantial amplification. These adapters can be used in any library preparation, for example for sequencing. These adapters can be used in combination with any of the other methods described herein, such as mini-PCR amplification methods.

internal marker primer

When sequencing is used to determine the allele present at a given polymorphic locus, sequence reads typically begin upstream of the primer binding site (a) and then arrive at the polymorphic site (X). Markers are typically configured as shown on the left in Figure 11. 101 refers to the single stranded target DNA containing the associated polymorphic locus 'X' and primer 'a' attached with marker 'b'. To avoid non-specific hybridization, the length of the primer binding site (the region complementary to 'a' in the target DNA) is usually 18 to 30 bp. The sequence marker 'b' is usually about 20 bp; in theory these could be any length longer than about 15 bp, but many people use primer sequences sold by sequencing platform companies. The distance 'd' between 'a' and 'X' may be at least 2 bp in order to avoid allelic bias. When performing multiplex PCR amplification using the methods disclosed herein or other methods where careful primer design is required to avoid excessive primer-primer interactions, the allowable distance between 'a' and 'X' The range of d' can vary widely: from 2bp to 10bp, from 2bp to 20bp, from 2bp to 30bp or even from 2bp to over 30bp. Therefore, when using the primer configuration shown on the left in Figure 11, sequence reads must be a minimum of 40bp to obtain reads long enough to measure polymorphic loci, and depending on the length of 'a' and 'd', sequence reads may Needs to be as long as 60 or 75 bp. In general, the longer the sequence reads, the more costly and time-consuming to sequence a given number of reads, so minimizing the necessary read length can save time and money. In addition, reducing the required sequence read length can also improve the accuracy of polymorphic region measurements because, on average, bases that are called earlier on the read are called more accurately than those that are called later on the read. accuracy.

In one embodiment, for internally labeled primers, the primer binding site (a) is split into segments (a', a", a"'...), and the sequence labeled (b) On the DNA segment between the two primer binding sites, as shown in Figure 11, 103. This configuration allows the sequencer to make shorter sequence reads. In one embodiment, a'+a" should be at least about 18 bp, and can be as long as 30, 40, 50, 60, 80, 100 or more than 100 bp. In one embodiment, a" should be at least about 6 bp, And in one embodiment, between about 8 and 16 bp. All other factors being equal, internal marker primers can be used to cut the length of the desired sequence read by at least 6bp, as much as 8bp, 10bp, 12bp, 15bp, and even as much as 20 or 30bp. This can yield significant monetary, time and accuracy advantages. An example of an internally labeled primer is given in FIG. 12 .

Primers containing ligated adapter binding regions

One problem with fragmented DNA is that because of its short length, the probability of polymorphisms is higher near the end of the DNA strand than in longer strands (eg 101, Figure 10). Because PCR capture of polymorphisms requires primer-binding sites of appropriate length on both sides of the polymorphism, a large number of DNA strands with targeted polymorphisms will suffer from insufficient overlap between primers and targeted binding sites. And miss. In one embodiment, the target DNA 101 may have a ligation adapter 102 attached, and the target primer 103 may have a region complementary to the ligation adapter tag (it) attached upstream of the designed binding region (a) ( cr) (see Figure 13); thus, in the case where the binding region (the region complementary to a in 101) is 18 bp shorter than normally required for hybridization, the region (cr) on the primer that is complementary to the library marker is able to increase compatibility with Binding energy at the point where PCR can be performed. It should be noted that any specificity lost due to shorter binding regions can be compensated by other PCR primers containing appropriately long target binding regions. It should be noted that this example can be used in combination with direct PCR or any of the other methods described herein, such as nested PCR, semi-nested PCR, hemi-nested PCR, single Side-nested or half- or half-side-nested PCR or other PCR schemes.

When ploidy is determined using sequencing data and analytical methods that involve comparing observed allelic data to predicted allelic distributions for individual hypotheses, each additional read from an allele with low read depth will give more informative than allelic reads with high read depth. Therefore, ideally, one would want to see a uniform depth of reads (DOR), where each locus would have a similar number of representative sequence reads. Therefore, it is desirable to minimize the DOR variance. In one embodiment, it is possible to reduce the coefficient of variance of DOR (this can be defined as standard deviation of DOR/average DOR) by increasing the annealing time. In some embodiments, the annealing temperature may be longer than 2 minutes, longer than 4 minutes, longer than ten minutes, longer than 30 minutes and longer than one hour or even longer. Since annealing is a balancing process, there is no limit to improving the DOR variance by increasing the annealing time. In one example, increasing the primer concentration can reduce the DOR variance.

Exemplary Whole Genome Amplification Methods

In some embodiments, the methods of the invention may involve amplifying DNA, for example using genome-wide applications to amplify a nucleic acid sample prior to amplifying only the loci of interest. Amplification of DNA, the process of converting a small amount of genetic material into a larger amount of genetic material containing a similar set of genetic data, can be performed by various methods including, but not limited to, the polymerase chain reaction (PCR ). One method of amplifying DNA is whole genome amplification (WGA). There are several methods available for WGA: ligation-mediated PCR (LM-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR), and multiple displacement amplification (MDA). In LM-PCR, short DNA sequences called adapters are ligated to the blunt ends of DNA. These adapters contain universal amplification sequences that are used to amplify DNA by PCR. In DOP-PCR, random primers also containing universally amplified sequences are used in the first round of annealing and PCR. Then, a second round of PCR was used to amplify additional sequences containing the universal primer sequences. MDA uses phi-29 polymerase, a highly progressive and nonspecific enzyme that replicates DNA and has been used for single-cell analysis. The major limitations to amplifying material from single cells are (1) the necessity to use very dilute DNA concentrations or very small volumes of reaction mixtures, and (2) the difficulty of reliably dissociating DNA from proteins in whole genomes. Regardless, single-cell whole-genome amplification has been successfully used in various applications for many years. Other methods exist for amplifying DNA from DNA samples. DNA amplification converts an initial DNA sample into a DNA sample that is similar in sequence set but much larger in number. In some cases, amplification may not be required.

In some embodiments, DNA can be amplified using universal amplification such as WGA or MDA. In some embodiments, the DNA can be amplified by targeted amplification, eg, using targeted PCR or circularizing probes. In some embodiments, DNA may be preferentially enriched using targeted amplification methods or methods that result in complete or partial separation of desired from undesired DNA, such as capture by hybridization methods. In some embodiments, DNA can be amplified by using a combination of general amplification methods and preferential enrichment methods. A fuller description of some of these methods can be found elsewhere in this document.

Exemplary Enrichment and Sequencing Methods

In one embodiment, the methods disclosed herein use selective enrichment techniques that preserve the presence of each gene of interest from a set of loci of interest (e.g., polymorphic loci) present in the initial DNA sample. Relative allele frequencies at loci (eg, each polymorphic locus). While enrichment is particularly advantageous for methods used to analyze polymorphic loci, these enrichment methods can easily be adapted for non-polymorphic loci, if necessary. In some embodiments, amplification and/or selective enrichment techniques may involve PCR (eg, ligation-mediated PCR), fragment capture by hybridization, molecular inversion probes, or other circularizing probes. In some embodiments, methods for amplification or selective enrichment may involve the use of probes in which, after proper hybridization to the target sequence, the 3' or 5' ends of the nucleotide probes are passed through a small number of nucleotides Separated from the polymorphic site of the allele. This spacing reduces the preferential amplification of one allele (known as allelic bias). This is an improvement over methods involving the use of probes where the 3' or 5' ends of the correctly hybridized probes are directly adjacent or very close to the polymorphic site of the allele. In one embodiment, probes where the hybridization region may or is determined to contain a polymorphic site are excluded. Polymorphic sites at hybridization sites can cause unequal hybridization of some alleles or inhibit overall hybridization, causing preferential amplification of certain alleles. An improvement of these embodiments over other methods involving targeted amplification and/or selective enrichment is that they better preserve the original allele frequency of the sample at each polymorphic locus, regardless of the sample Whether it is a pure genomic sample from a single individual or a mixture of individuals.

Using the technique of enriching a DNA sample at a set of loci of interest followed by sequencing as part of a method for non-invasive prenatal allelic or ploidy calling can lead to several unexpected advantages. In some embodiments of the invention, the methods involve measuring genetic data suitable for information-based methods, such as PARENTAL SUPPORT ^™ (PS). The end result of some of the described embodiments is actionable genetic data of an embryo or fetus. There are a variety of methods that can be used to measure an individual and/or genetic data associated with an individual as part of implementing the method. In one embodiment, disclosed herein is a method for enriching the concentration of a set of targeted alleles comprising one or more of the following steps: targeted amplification of genetic material, addition of Locus-specific oligonucleotide probes, ligation of specified DNA strands, separation of desired DNA groups, removal of unwanted reaction components, detection of certain DNA sequences by hybridization, and detection of one or more strands of DNA by DNA sequencing methods sequence of chains. In some cases, DNA strands may refer to genetic material of interest; in some cases, they may refer to primers; in some cases, they may refer to synthetic sequences, or a combination thereof. These steps can be performed in a variety of different orders.

For example, a universal amplification step of DNA prior to targeted amplification can bring several advantages, such as removing the risk of bottleneck effects and reducing allelic bias. The DNA can be mixed with oligonucleotide probes that hybridize to two adjacent regions flanking the target sequence. Following hybridization, the probe ends can be ligated by adding polymerase, building blocks for ligation, and any necessary reagents to allow circularization of the probe. Following circularization, an exonuclease can be added to digest non-circularized genetic material, followed by detection of circularized probes. The DNA can be mixed with PCR primers that hybridize to two adjacent regions flanking the target sequence. After hybridization, the probe ends can be ligated by adding polymerase, building blocks for ligation, and any necessary reagents for accomplishing PCR amplification. Amplified or unamplified DNA can be targeted by hybridization capture probes targeting a set of loci; following hybridization, the probes can be localized and separated from the mixture to provide a DNA mixture enriched for the target sequence .

Using a method of targeting certain loci followed by sequencing as part of a method for allele calling or ploidy calling can lead to several unexpected advantages. Some methods by which DNA can be targeted or preferentially enriched include the use of circularizing probes, linkage inversion probes (LIP, MIP), capture by hybridization methods such as SURESELECT, and targeted PCR or ligation mediators. Guided PCR amplification strategy.

In some embodiments, the methods of the invention involve measuring genetic data suitable for use in information-based methods further described herein, such as PARENTAL SUPPORT ^™ (PS). PARENTAL SUPPORT ^™ is an information-based method for manipulating genetic data, aspects of which are described herein. The end result of some of these embodiments is actionable genetic data for an embryo or fetus, followed by a clinical decision based on the actionable data. The algorithm behind the PS method uses the measured genetic data of the target individual, usually an embryo or fetus, and the measured genetic data of related individuals, and can improve the accuracy of knowing the genetic status of the target individual. In one embodiment, the measured genetic data is used in the context of making ploidy decisions during prenatal genetic diagnosis. In one embodiment, the measured genetic data is used in the context of ploidy determination or allelic calling of embryos during in vitro fertilization. There are a variety of methods that can be used to measure individuals and/or individual-related genetic data in the situations described above. The different methods contain multiple steps, those steps usually involve amplifying the genetic material, adding oligonucleotide probes, joining the specified DNA strands, isolating the desired DNA group, removing unwanted reaction components, detecting some DNA by hybridization Sequence, the detection of the sequence of one or more strands of DNA by DNA sequencing methods. In some cases, DNA strands may refer to genetic material of interest; in some cases, they may refer to primers; in some cases, they may refer to synthetic sequences, or a combination thereof. These steps can be performed in a variety of different orders.

It should be noted that it is theoretically possible to target any number of loci in the genome, from one locus to well over a million loci. If a DNA sample is subjected to targeting and then sequenced, the percentage of alleles read by the sequencer will be enriched relative to their natural abundance in the sample. The degree of enrichment can be anywhere from one percent (or even less) to ten, hundred, thousand or even millions of times. In the human genome, there are approximately 3 billion base pairs and nucleotides, containing approximately 75 million polymorphic loci. The more loci that are targeted, the smaller the enrichment is likely to be. The fewer the number of loci targeted, the greater the enrichment possible and, for a given number of sequence reads, the greater the depth of reads that can be obtained at those loci.

In one embodiment of the invention, targeting or prioritization can be entirely focused on SNPs. In one embodiment, targeting or preference can be focused on any polymorphic site. A variety of commercial targeting products are available to enrich for exons. Surprisingly, targeting exclusively SNPs or exclusively polymorphic loci is particularly advantageous when using methods for performing NPD that rely on allelic distribution. Methods for NPD using sequencing are also disclosed, e.g. U.S. Patent 7,888,017, which relates to read counting analysis, where read counting focuses on counting the number of reads that map to a given chromosome, where the sequence reads analyzed are not focused on polymorphisms of the genome area. Those types of methods that do not focus on polymorphic alleles will not benefit as much from targeting or preferentially enriching a set of alleles.

In one embodiment of the invention, it is possible to use a targeted approach that focuses on SNPs to enrich genetic samples in polymorphic regions of the genome. In one embodiment, it is possible to focus on a small number of SNPs, such as between 1 and 100 SNPs, or a larger number, such as between 100 and 1,000, between 1,000 and 10,000, between 10,000 and Between 100,000 or more than 100,000 SNPs. In one embodiment, it is possible to focus on one or a small number of chromosomes involved in the birth of a living trisomy, such as chromosomes 13, 18, 21, X and Y, or some combination thereof. In one embodiment, it is possible to enrich the targeted SNP by a small factor, such as between 1.01-fold and 100-fold, or by a larger factor, such as between 100-fold and 1,000,000-fold between, or even more than 1,000,000 times. In one embodiment of the invention, it is possible to use targeted methods for creating DNA samples that are preferentially enriched in polymorphic regions of the genome. In one embodiment, it is possible to use this method to create a DNA mixture having any of these characteristics, wherein the DNA mixture contains maternal DNA as well as free-floating fetal DNA. In one example, it is possible to use this method to create DNA mixtures with any combination of these factors. For example, the methods described herein can be used to create a DNA mixture comprising maternal and fetal DNA preferentially enriched for DNA corresponding to 200 SNPs, all located on chromosome 18 or 21, and on average Enriched 1000-fold. In another example, it is possible to use the method to create a DNA mixture preferentially enriched at 10,000 SNPs, all or most of which are located on chromosomes 13, 18, 21, X and Y, And each locus was enriched more than 500-fold on average. Any of the targeting methods described herein can be used to create DNA mixtures that are preferentially enriched at certain loci.

In some embodiments, the methods of the invention further comprise using a high-throughput DNA sequencer to measure fractionated DNA containing a disproportionate amount of sequence from one or more chromosomes, wherein said The one or more chromosomes are taken from the group comprising chromosome 13, chromosome 18, chromosome 21, X chromosome, Y chromosome, and combinations thereof.

Three methods are described here: multiplex PCR, targeted capture by hybridization, and linked inversion probes (LIPs), which can be used to obtain and analyze measurements from a sufficient number of polymorphic loci from maternal plasma samples, Fetal aneuploidy is thereby detected; this is not meant to exclude other methods of selectively enriching the loci of interest. Other methods can likewise be used without changing the essence of the method described. In each case, the polymorphisms analyzed may include single nucleotide polymorphisms (SNPs), small indels or STRs. A preferred method involves the use of SNPs. Each method produces allele frequency data; the allele frequency data for each locus of interest and/or the joint allele frequency distribution from these loci can be analyzed to determine the ploidy of the fetus. Each method has its own considerations due to the limited source material and the fact that maternal plasma consists of a mixture of maternal and fetal DNA. This method can be combined with other methods to provide a more accurate assay. In one embodiment, this method can be combined with sequence counting methods such as those described in US Patent 7,888,017. The method can also be used to non-invasively detect fetal parentage from maternal plasma samples. Additionally, each method can be applied to other DNA mixtures or pure DNA samples to detect the presence or absence of aneuploid chromosomes, genotype large numbers of SNPs from degraded DNA samples, detect segmental copy number variations (CNV ), detecting other relevant genotypic states or some combination thereof.

Accurately measure the distribution of alleles in a sample

Current sequencing methods can be used to estimate the distribution of alleles in a sample. One such method involves random sampling of the sequence of a pool of DNA, known as shotgun sequencing. The proportion of a particular allele in the sequencing data is usually very low and can be determined by simple statistics. The human genome contains approximately 3 billion base pairs. Thus, if the sequencing method used makes 100 bp reads, then a particular allele will be measured approximately once every 30 million sequence reads.

In one embodiment, the method of the invention is used to determine the presence or absence of two or more different haplotypes containing the same set of loci in a DNA sample from the allelic distribution of loci measured from said chromosome . Different haplotypes can represent two different homologous chromosomes from an individual, three different homologous chromosomes from a trisomy individual, and three different homologous haplotypes from the mother and fetus, where the haplotypes One of the haplotypes shared between the mother and the fetus, three or four haplotypes from the mother and the fetus, wherein one or two of the haplotypes are shared between the mother and the fetus, or other combinations. Polymorphic alleles between haplotypes tend to be more informative, however any allele where the mother and father are not homozygous for the same allele will be better informed by the measured allelic distribution than can be obtained from Useful information on the information obtained by simple read count analysis.

However, shotgun sequencing of such samples is extremely inefficient because it yields multiple sequences of non-polymorphic regions between different haplotypes in the sample or regions that are not related chromosomes and thus does not reveal information about the target haplotype ratio information. Described herein are methods for specifically targeting and/or preferentially enriching DNA segments in the genome of a sample that are more likely to be polymorphic to increase the yield of allelic information obtained by sequencing. It should be noted that, for the allelic distribution measured in the enriched sample to truly represent the actual amount present in the target individual, it is critical that at a given locus in the targeted segment, one allele is present compared to the other. Little or no preferential enrichment of one allele. Current methods known in the art for targeting polymorphic alleles are designed to ensure detection of at least some of any alleles present. However, these methods are not designed to measure the unbiased allelic distribution of the polymorphic alleles present in the initial mixture. It is not obvious that any particular method of target enrichment will be able to produce enriched samples in which the measured allelic distribution will be a better representation than any other method of the isoforms present in the unamplified initial sample. bit gene distribution. While a variety of enrichment methods can theoretically be envisioned for such purposes, those of ordinary skill in the art are well aware that there are substantial random or deterministic biases in current amplification, targeting, and other preferential enrichment methods . One embodiment of the methods described herein allows multiple alleles found in a mixture of DNA corresponding to a given locus in the genome to be amplified or preferentially amplified in such a way that each of the alleles is approximately equally enriched. Enrichment. Said another way, the method allows for an overall increase in the relative numbers of alleles present in the mixture, while the ratios between the alleles corresponding to each locus are substantially the same as they were in the initial DNA mixture. stay the same. For some of the reported methods, preferential enrichment of loci can produce allelic bias of more than 1%, more than 2%, more than 5% and even more than 10%. This preferential enrichment may be due to capture bias when using capture by hybridization methods, or amplification bias which may be small for each cycle but becomes larger when mixing exceeds 20, 30 or 40 cycles. For the purposes of the present invention, the ratio remains substantially the same meaning that the ratio of alleles in the initial mixture divided by the ratio of alleles in the resulting mixture is between 0.95 and 1.05, between 0.98 and 1.02, between 0.99 and 1.01, between 0.995 and 1.005, between 0.998 and 1.002, between 0.999 and 1.001, or between 0.9999 and 1.0001. It should be noted that the calculation of allele ratios presented here cannot be used to determine the ploidy state of an individual of interest and can only be a measure for measuring allelic bias.

In one embodiment, after the mixture has been preferentially enriched at the locus group of interest, previous, current or next generation pairs can be used to clone samples (samples generated from a single molecule; examples include Illumina GAIIx, Illumina GAIIx, Illumina It can be sequenced by any one of the sequencing instruments for HISEQ, LIFE TECHNOLOGIES SOLiD, 5500XL). Ratio can be estimated by sequencing specific alleles within the targeted region. These sequencing reads can be analyzed and counted according to the allele type and thus the determined ratio of different alleles. For variants that are one to a few bases in length, detection of alleles will be performed by sequencing and sequencing reads must cover the allele in question in order to estimate the allelic composition of the captured molecule. The total number of molecules captured for genotyping can be increased by increasing the length of the sequencing reads. Complete sequencing of all molecules will ensure collection of the largest amount of data available in the enrichment pool. However, sequencing is currently expensive, and a method that can measure allelic distribution using a smaller number of sequence reads would be of great value. In addition, there are technical limitations on the maximum possible read length and accuracy limitations as read length increases. The alleles with greatest utility will be one to a few bases in length, but in theory any allele shorter than the length of the sequencing reads could be used. Although allelic variation occurs in all types, the examples provided herein focus on SNPs or variants that contain only a few adjacent base pairs. In many cases, larger variants such as segmental copy number variants can be detected by aggregates of these smaller variants because the entire set of SNPs within the segment are replicated. Variants larger than a few bases (eg STRs) require special consideration and some targeting approaches will work while others will not.

There are a variety of targeted methods that can be used to specifically isolate and enrich for one or more variant positions in the genome. Typically, these rely on the use of constant sequences flanking the variant sequences. There are other reports related to targeting in the context of sequencing where the substrate is maternal plasma (see e.g. Liao et al., Clin. Chem. 2011; 57(1): p. 92 -101 pages). However, these methods use targeted probes that target exons and are not focused on targeting polymorphic regions of the genome. In one embodiment, the methods of the invention involve the use of targeting probes that focus exclusively or almost exclusively on polymorphic regions. In one embodiment, the methods of the invention involve the use of targeting probes that focus exclusively or almost exclusively on SNPs. In some embodiments of the invention, the targeted polymorphic loci consist of at least 10% SNPs, at least 20% SNPs, at least 30% SNPs, at least 40% SNPs, at least 50% SNPs, at least 60% SNPs, at least 70% SNPs , at least 80% SNPs, at least 90% SNPs, at least 95% SNPs, at least 98% SNPs, at least 99% SNPs, at least 99.9% SNPs or exclusively SNPs.

In one embodiment, the method of the present invention can be used to determine the genotypes (basic composition of DNA at specific loci) and the relative proportions of those genotypes of a mixture of DNA molecules, where those DNA molecules can be derived from one or more genetically distinct individuals. In one embodiment, the methods of the invention can be used to determine genotypes at a set of polymorphic loci, and the relative ratios of the amounts of the different alleles present at those loci. In one embodiment, a polymorphic locus may consist entirely of SNPs. In one embodiment, polymorphic loci can comprise SNPs, single tandem repeats, and other polymorphisms. In one embodiment, the methods of the invention can be used to determine the relative distribution of alleles at a set of polymorphic loci in a DNA mixture comprising maternally derived DNA and fetally derived DNA. In one embodiment, the joint allelic distribution of a mixture of DNA isolated from blood of a pregnant woman can be determined. In one embodiment, the distribution of alleles at a set of loci can be used to determine the ploidy state of one or more chromosomes of a gestating fetus.

In one embodiment, the mixture of DNA molecules can be derived from DNA extracted from multiple cells of an individual. In one example, the initial collection of cells from which DNA is obtained may comprise a mixture of diploid or haploid cells of the same or different genotypes, if the individual is mosaic (germline or somatic). In one embodiment, the mixture of DNA molecules can also be derived from DNA extracted from single cells. In one embodiment, the mixture of DNA molecules can also be derived from DNA extracted from a mixture of two or more cells of the same individual or different individuals. In one embodiment, the mixture of DNA molecules may be derived from DNA isolated from biological material known to contain cell-free DNA that has been released from cells such as plasma. In one embodiment, such biological material may be a mixture of DNA from one or more individuals, as is the case during pregnancy, where fetal DNA has been shown to be present in the mixture. In one embodiment, the biological material may be derived from a mixture of cells found in maternal blood, some of which are of fetal origin. In one embodiment, the biological material may be cells from the blood of a pregnant woman that have been enriched with fetal cells.

circularizing probe

Some embodiments of the invention involve the use of "Linked Inversion Probes" (LIPs), which have been previously described in the literature, to amplify a locus of interest either before or after amplification using primers other than LIPs in the multiplex PCR method of the invention . LIP is a general term intended to cover techniques involving the production of circular DNA molecules in which the probes are designed to hybridize to the targeted DNA region on either side of the targeted allele so that the appropriate polymerase The addition of and/or ligase and appropriate conditions, buffers and other reagents will complete the complementary, inverted DNA region on the targeted allele creating a circular DNA loop that captures the information found in the targeted allele. A LIP may also be referred to as a precircularizing probe, a precircularizing probe, or a circularizing probe. The LIP probe can be a linear DNA molecule between 50 and 500 nucleotides in length, and in one embodiment, between 70 and 100 nucleotides in length; in some embodiments, it can be longer or shorter than described in this article. Other embodiments of the invention involve different implementations of LIP techniques such as padlock probes and molecular inversion probes (MIP).

One approach to targeting specific locations for sequencing is to synthesize probes in which the 3' and 5' ends of the probe are positioned adjacent to and flanking the targeting region in an inverted fashion. Annealing to target DNA such that addition of DNA polymerase and DNA ligase causes extension from the 3' end, addition of bases to the single-stranded probe complementary to the target molecule (gap filling), followed by ligation of the new 3' end To the 5' end of the original probe, a circular DNA molecule is generated which can then be separated from background DNA. The probe ends are designed to flank targeting relevant regions. One aspect of this approach is commonly referred to as MIP and has been used in conjunction with array technology to determine the properties of the populated sequences. One disadvantage of using MIP in the context of measuring allele ratios is that the hybridization, circularization and amplification steps do not occur at equal rates for different alleles at the same locus. This results in the measured allele ratios not being representative of the actual allele ratios present in the initial mixture.

In one embodiment, the probes in the circularization are constructed such that a region of the probe designed to hybridize upstream of the targeted polymorphic locus and a probe designed to hybridize downstream of the targeted polymorphic locus The domains are covalently linked by a non-nucleic acid backbone. This backbone can be any biocompatible molecule or combination of biocompatible molecules. Some examples of potentially biocompatible molecules are poly(ethylene glycol), polycarbonate, polyurethane, polyethylene, polypropylene, sulfone polymers, silicone, cellulose, fluoropolymers, acrylic series compounds, styrene block copolymers and other block copolymers.

In one embodiment of the invention, this method has been modified to readily amenable to sequencing as a means of querying for fillers in sequences. In order to maintain the initial allelic ratio of the initial sample, at least one key consideration must be taken into account. The position of the variant among the different alleles in the gap-fill region must not be too close to the probe binding site, since there will be an initiation bias caused by the DNA polymerase, causing variant differences. Another consideration is that there may be other variations in the probe binding site associated with variants in the gap-fill region that would lead to unequal amplification of different alleles. In one embodiment of the invention, the 3' and 5' ends of the precircularized probes are designed to hybridize to one or more of the variant positions (polymorphic sites) away from the targeted allele. bases at positions. The number of bases between the polymorphic site (SNP or other) and the 3' end and/or 5' of the precircularized probe designed to hybridize can be one base, which can be Two bases, it could be three bases, it could be four bases, it could be five bases, it could be six bases, it could be seven to ten bases, it could be Eleven to fifteen bases, or it could be sixteen to twenty bases, twenty to thirty bases or thirty to sixty bases. Forward and reverse primers can be designed to hybridize different numbers of bases away from the polymorphic site. Large numbers of circularizing probes can be produced with current DNA synthesis techniques, allowing the generation and potentially pooling of extremely large numbers of probes, enabling simultaneous interrogation of multiple loci. Has been reported to work with over 300,000 probes. Two papers that discuss methods involving circularizing probes that can be used to measure genomic data from an individual of interest include: Porreca et al., Nature Methods, 20074(11), p. 931 - pp. 936; and Turner et al., Nature Methods, 2009, 6(5), pp. 315-316. The methods described in these papers can be used in combination with other methods described herein. Certain steps of the methods of these two papers may be used in combination with other steps of other methods described herein.

In some embodiments of the methods disclosed herein, the genetic material of the target individual is optionally amplified, followed by hybridization of the pre-circularized probes, gap filling is performed to fill bases between the two ends of the hybridized probes base, the two ends are joined to form a circularized probe, and the circularized probe is amplified using, for example, rolling circle amplification. After extracting the desired target allelic information by circularizing appropriately designed oligonucleotide probes, eg, in a LIP system, the gene sequence of the circularized probes can be measured to obtain the desired sequence data. In one example, appropriately designed oligonucleotide probes can be directly circularized on the unamplified genetic material of a target individual and subsequently amplified. It should be noted that multiple amplification procedures can be used to amplify the original genetic material or the circularized LIP, including rolling circle amplification, MDA or other amplification schemes. Different methods can be used to measure genetic information on the target genome, such as using high-throughput sequencing, Sanger sequencing, other sequencing methods, capture by hybridization, capture by circularization, multiplex PCR, other hybridization methods, and combinations thereof .

After an individual's genetic material has been measured using one or more of the above methods, the individual's ploidy of one or more chromosomes can then be determined using an informative method (such as the PARENTAL SUPPORT ^™ method) along with appropriate genetic measurements. Sexual status and/or the genetic status of one or a group of alleles, especially those alleles associated with a relevant disease or genetic status. It should be noted that the use of LIP has been reported for composite capture of gene sequences followed by genotyping with sequencing. However, sequencing data generated by LIP-based strategies for amplifying the genetic material found in single cells, small numbers of cells, or extracellular DNA have not been used to determine the ploidy state of an individual of interest.

The application of information-based methods to determine the ploidy state of individuals from genetic data as measured by hybridization arrays (e.g., Illumina Infinium arrays or AFFYMETRIX gene chips) has been described elsewhere in this document described in the documentation references for . However, the methods described herein demonstrate improvements over methods previously described in the literature. For example, a LIP-based approach followed by high-throughput sequencing unexpectedly provides better genotypic data due to better recombination capabilities, better capture specificity, better uniformity sex and low allelic bias. Larger composites allow targeting of more alleles, giving more accurate results. Better uniformity results in more targeted alleles being measured, giving more accurate results. A lower allelic bias yields a lower rate of miscalls, giving more accurate results. More accurate results yield improved clinical outcomes and lead to better medical care.

It is important to note that LIP can be used as a method for targeting specific loci in a DNA sample for genotyping by methods other than sequencing. For example, LIP can be used to target DNA for genotyping using SNP arrays or other DNA or RNA based microarrays.

conjugation-mediated PCR

Ligation-mediated PCR can be used to amplify the locus of interest either before or after PCR amplification using non-ligated primers. Ligation-mediated PCR is a PCR method for preferentially enriching a DNA sample by amplifying one or more loci in a DNA mixture, the method comprising: obtaining a set of primer pairs in which Each primer contains a target-specific sequence and a non-target sequence, wherein the target-specific sequence is preferably designed for annealing to a target region: one upstream and one downstream of the polymorphic site, and the target region can be associated with the polymorphic site. Sites are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, 21-30, 31-40, 41-50, 51-100 or more than 100 apart; from upstream The 3′ end of the primer polymerizes DNA to fill the single-stranded region between it and the 5′ end of the downstream primer with nucleotides complementary to the target molecule; the last polymerized base of the upstream primer is joined to the adjacent 5′ end of the downstream primer ' bases; and using non-target sequences contained at the 5' end of the upstream primer and the 3' end of the downstream primer to amplify only the polymerized and ligated molecule. Primer pairs for different targets can be mixed in the same reaction. The non-target sequence acts as a universal sequence so that all primer pairs that have successfully polymerized and ligated can be amplified with a single pair of amplification primers.

capture by hybridization

In some embodiments, methods of the invention may involve the use of any of the following methods of capture by hybridization in addition to the use of multiplex PCR to amplify the locus of interest. The preferential enrichment of a specific set of sequences in a target genome can be achieved in a number of ways. How to use LIP to target a specific set of sequences is described elsewhere in this document, but in all those applications other methods of targeting and/or preferential enrichment can be used for the same purpose as well. An example of another targeting method is the method of capture by hybridization. Some examples of commercially available technologies for capture by hybridization include AGILENT's SURE SELECT and Illumina's TRUSEQ. In capture by hybridization, a set of oligonucleotides that are complementary or substantially complementary to a desired targeting sequence are hybridized to a DNA mixture and then physically separated from the mixture. The act of physically removing the targeting oligonucleotide after the desired sequence has hybridized to the targeting oligonucleotide also serves to remove the targeting sequence. After the hybridized oligonucleotides are removed, they can be heated above their melting temperature and they can be amplified. Some ways of physically removing the targeting oligonucleotide are by covalently bonding the targeting oligonucleotide to a solid support, such as a magnetic bead or chip. Another way to physically remove targeting oligonucleotides is by covalently bonding them to a molecular moiety that has a strong affinity for another molecular moiety. An example of such a molecular pair is biotin and streptavidin, eg as used in SURE SELECT. Thus, the targeting sequence can be covalently attached to a biotin molecule, and after hybridization, the biotin-labeled oligonucleotide can be pulled down using a streptavidin-attached solid support, the oligonucleotide A targeting sequence is hybridized to the nucleotide.

Hybridization capture involves the hybridization of probes complementary to the target of interest to the target molecule. Hybridization capture probes were originally developed to target and enrich a large portion of the genome relatively uniformly across targets. In this application, it is important to amplify all targets with sufficient uniformity so that all regions can be detected by sequencing, but regardless of the ratio of alleles in the original sample. After capture, the alleles present in the sample can be determined by direct sequencing of the captured molecules. These sequencing reads can be analyzed and counted according to allele type. However, using current techniques, the measured allelic distribution of the captured sequence is often not representative of the original allelic distribution.

In one embodiment, detection of alleles is performed by sequencing. In order to capture the allelic identity of a polymorphic site, sequencing reads must cover the allele in question in order to estimate the allelic composition of the captured molecule. Because capture molecules are often of variable length when sequenced, overlapping of variant positions cannot be ensured unless the entire molecule is sequenced. However, cost considerations as well as technical limitations regarding the maximum possible length and accuracy of sequencing reads make sequencing the entire molecule infeasible. In one embodiment, the read length can be increased from about 30 to about 50 or about 70 bases, which can greatly increase the number of reads with overlapping variant positions within the targeted sequence.

Another way to increase the number of reads querying relevant positions is to reduce the length of the probe, as long as it does not introduce a bias towards substantially enriched alleles. The length of the synthetic probes should be long enough that two probes designed to hybridize to the two different alleles present at a locus will hybridize with nearly equal affinity to the respective alleles in the initial sample . Currently, methods known in the art describe probes that are generally longer than 120 bases. In a current embodiment, if the allele is one or a few bases, the capture probe can be less than about 110 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, and less than about 25 bases, and this is sufficient to ensure that all Equal enrichment of alleles. When the DNA mixture to be enriched using hybrid capture techniques is a mixture comprising free-floating DNA isolated from blood such as maternal blood, the average length of the DNA is very short, typically less than 200 bases. The use of shorter probes creates a greater chance that the hybrid capture probe will capture the desired DNA fragment. Larger variations may require longer probes. In one embodiment, the associated variation is one (SNP) to a few bases in length. In one embodiment, targeted regions in the genome can be preferentially enriched using hybridization capture probes, wherein the length of the hybridization capture probes is less than 90 bases, and can be less than 80 bases, less than 70 bases, Less than 60 bases, less than 50 bases, less than 40 bases, less than 30 bases, or less than 25 bases. In one embodiment, to increase the chance that the desired allele is sequenced, the length of the probes designed to hybridize to the region flanking the polymorphic allele position can be reduced from more than 90 bases to About 80 bases, or about 70 bases, or about 60 bases, or about 50 bases, or about 40 bases, or about 30 bases, or about 25 bases.

There is minimal overlap between the synthetic probe and the target molecule in order to achieve capture. The synthetic probes can be made as short as possible and still be larger than the required minimum overlap. The effect of using shorter probe lengths to target polymorphic regions is that there will be more molecules that overlap the target allelic region. The fragmentation state of the initial DNA molecule also affects the number of reads that will overlap with the targeted allele. Some DNA samples, such as plasma samples, have been fragmented due to biological processes occurring in vivo. However, samples with longer fragments will benefit from fragmentation through library preparation and enrichment prior to sequencing. Maximum specificity was achieved when both probes and fragments were short (approximately 60-80 bp), with relatively few sequence reads failing to overlap key regions of interest.

In one embodiment, hybridization conditions can be adjusted to maximize the uniformity of capture of the different alleles present in the initial sample. In one embodiment, the hybridization temperature is reduced to minimize hybridization bias between alleles. Methods known in the art avoid the use of lower temperatures for hybridization because lowering the temperature has the effect of increasing hybridization of probes to undesired targets. However, when the aim is to preserve allelic ratios with maximum fidelity, methods using lower hybridization temperatures provide optimally accurate allelic ratios, but the reality is that current technology teaches away from this method. The hybridization temperature can also be increased to require greater overlap between the target and the synthesized probe so that only targets that substantially overlap the targeted region are captured. In some embodiments of the invention, the hybridization temperature is reduced from the normal hybridization temperature to about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C.

In one embodiment, hybridization capture probes can be designed such that regions of DNA in the capture probes that are complementary to DNA found in regions flanking polymorphic alleles are not immediately adjacent to the polymorphic site. In fact, the capture probe can be designed such that the region of the capture probe designed to hybridize to the DNA flanking the polymorphic site of the target will be identical to the van der Waals polymorphic site in the capture probe. ) contacts are separated by a short distance equal in length to one or a few bases. In one embodiment, hybridization capture probes are designed to hybridize to regions that flank the polymorphic allele but do not intersect it; this may be referred to as a flanking capture probe. The flanking capture probes can be less than about 120 bases, less than about 110 bases, less than about 100 bases, less than about 90 bases, and can be less than about 80 bases, less than about 70 bases in length. bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, less than about 30 bases, or less than about 25 bases. Genomic regions targeted by flanking capture probes can be separated from polymorphic loci by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-20, or greater than 20 base pairs .

A target capture based disease screening test is described using Target Enrichment Capture. Custom target enrichment, such as those currently offered by Agilent (SURE SELECT), Roche-Nimbregen (ROCHE-NIMBLEGEN), or Illumina. Capture probes can be custom designed to ensure capture of different types of mutations. With regard to point mutations, one or more probes overlapping the point mutation should be sufficient to capture and sequence the mutation.

For small insertions or deletions, one or more probes overlapping the mutation may be sufficient to capture and sequence the fragment containing the mutation. Hybridization between probes that limit capture efficiency may be less efficient and are usually designed to work with a reference genome sequence. To ensure that fragments containing mutations are captured, two probes can be designed, one matching the normal allele and one matching the mutant allele. Longer probes can enhance hybridization. Multiple overlapping probes can enhance capture. Finally, placing probes in close proximity to, but not overlapping with, mutations may allow for relatively similar capture efficiencies of normal and mutant alleles.

With respect to single tandem repeats (STRs), probes that overlap these highly variable sites are less likely to capture fragments well. To enhance capture, probes can be placed adjacent to, but not overlapping with, the variant site. The fragments can then be sequenced in the normal manner, revealing the length and composition of the STRs.

For large deletions, a series of overlapping probes can work, which is a common approach currently used in exon capture systems. However, with this approach, it may be difficult to determine whether an individual is or is heterozygous. Targeting and estimating SNPs within a captured region can potentially reveal loss of heterozygosity within that region, indicating that the individual is a carrier. In one embodiment, it is possible to place non-overlapping probes or single probes within potentially deleted regions and use the number of fragments captured as a measure of heterozygosity. In the case of an individual carrying a large deletion, one-half the number of fragments is expected to be available for capture relative to the non-deletion (diploid) reference locus. Therefore, the number of reads obtained from deleted regions should be about half that obtained from normal diploid loci. Aggregating and averaging the sequencing read depths of multiple single probes within potentially missing regions can enhance signal and improve diagnostic confidence. It is also possible to combine the two approaches: targeting SNPs to identify loss of heterozygosity and using multiple single probes to obtain a quantitative measure of the number of essential fragments from a locus. Either or both of these strategies can be combined with other strategies to better achieve the same goal.

If during testing, cfDNA detects a male fetus (as indicated by the presence of a Y chromosome segment captured and sequenced in the same test) and an X-linked dominant mutation in which neither the mother nor the father is affected or in which the mother is unaffected A dominant mutation would indicate a high risk to the fetus. Detection of two mutant recessive alleles within the same gene in the unaffected mother would imply that the fetus inherited the mutant allele from the father and possibly a second mutant allele from the mother. In all cases, follow-up testing may be indicated by amniocentesis or chorionic villus sampling.

Targeted capture-based disease screening tests can be combined with targeted capture-based non-invasive prenatal diagnostic tests for aneuploidy.

There are various ways to reduce depth of read (DOR) variability: for example, primer concentrations can be increased, longer targeted amplification probes can be used, or more STA cycles can be run (e.g., more than 25, more than 30, more than 35 or even more than 40).

Exemplary Methods of Determining the Quantity of DNA Molecules in a Sample

Described herein is a method for determining the number of DNA molecules in a sample by generating uniquely identified molecules for each initial DNA molecule in the sample during a first round of DNA amplification. A procedure for accomplishing the above, followed by single-molecule or clone sequencing, is described here.

The method requires targeting one or more specific loci and producing tagged copies of the original molecule in such a way that most or all tagged molecules for each locus of interest will have a unique tag and when using clonal or single-molecule Sequencing such barcodes can be distinguished from one another when sequenced. Each uniquely sequenced barcode represents a unique molecule in the original sample. At the same time, the sequencing data is used to determine from which locus the molecule is derived. Using this information the number of unique molecules for each locus in the initial sample can be determined.

This method can be used in any application that requires a quantitative estimate of the number of molecules in an initial sample. In addition, the number of unique molecules of one or more targets can be correlated with the number of unique molecules of one or more other targets, thereby determining relative copy numbers, allelic distributions, or allele ratios. Alternatively, the detected copy number from each target can be modeled by distribution in order to identify the most likely copy number of the original target. Applications include (but are not limited to) detection of insertions and deletions, such as those found in carriers of Duchenne Muscular Dystrophy; quantification of deleted or duplicated segments of chromosomes, such as observed in copy number variants chromosomal copy numbers from samples from unborn individuals; chromosomal copy numbers from samples from unborn individuals (eg, embryos or fetuses).

The method can be combined with simultaneous estimation of the variation contained in the targeting sequence. This can be used to determine the number of molecules representing each allele in the initial sample. This copy number approach can be combined with: estimates of SNPs or other sequence variations to determine chromosomal copy number in born and unborn individuals; Identification and quantification of copies of regions, e.g. carrier detection in spinal muscular atrophy; determination of copy number of molecules of different origin in a sample consisting of a mixture of different individuals, e.g. in free-floating DNA assays obtained from maternal plasma In fetal aneuploidy.

In one embodiment, the method, as it involves a single locus of interest, may comprise one or more of the following steps: (1) designing a pair of standard oligos for PCR amplification of specific loci . (2) During synthesis, a sequence having designated bases having no or minimal complementarity with the target locus or genome at the 5' end of one of the target-specific oligomers is added. This sequence, called the tail, is a known sequence for subsequent amplification, followed by a random nucleotide sequence. These random nucleotides comprise random regions. Random regions contain randomly generated nucleic acid sequences that vary in probability between each probe molecule. Thus, after synthesis, the pool of tailed oligomers will consist of a collection of oligomers starting with known sequences, followed by unknown sequences that vary from molecule to molecule, followed by target specific sequences. (3) Perform one round of amplification (denaturation, annealing, extension) using tailed oligos only. (4) Adding an exonuclease to the reaction, effectively stops the PCR reaction, and incubating the reaction at an appropriate temperature to remove forward-sense single-stranded oligonucleotides that do not anneal to the template and extend to form double-stranded products. (5) Incubate the reaction at high temperature to denature and eliminate the activity of the exonuclease. (6) Adding new oligonucleotides complementary to the tail of the oligomer used in the first reaction and another target-specific oligomer to the reaction to enable PCR amplification of the product generated in the first round of PCR increase. (7) Continue to amplify to generate enough products for downstream clonal sequencing. (8) Measure the amplified PCR product by various methods, such as performing cloning and sequencing for a sufficient number of bases covering the sequence.

In one embodiment, the methods of the invention involve targeting multiple loci in parallel or otherwise. Primers for different target loci can be generated independently and mixed to create a multiplex PCR pool. In one embodiment, the initial sample can be divided into subpools and different loci in each subpool can be targeted prior to recombination and sequencing. In one example, a labeling step and multiple cycles of amplification can be performed prior to subdividing the pool to ensure that all targets are efficiently targeted prior to subdividing, and amplification continues by using smaller primer sets in the subdivided pool to improve subsequent amplification.

One example of an application where this technique would be particularly useful is non-invasive prenatal aneuploidy diagnosis, where the allele ratio at a given locus or the distribution of alleles at multiple loci can be used to help determine fetal The number of copies of chromosomes present in . In this case, it is desirable to amplify the DNA present in the original sample while maintaining the relative amounts of the individual alleles. In some cases, especially where very small amounts of DNA are present, such as less than 5,000 genome copies, less than 1,000 genome copies, less than 500 genome copies, and less than 100 genome copies, it is possible to encounter what is known as The phenomenon of bottleneck effect. This is the case where there are few copies of any given allele in the original sample, and amplification bias can cause the ratio of those alleles of the amplified pool of DNA to be significantly different than in the original DNA mixture. By applying a set of unique or nearly unique barcodes to each strand of DNA prior to standard PCR amplification, it is possible to exclude n − 1 DNA copies from a set of n consensus molecules derived from sequenced DNA of the same initial molecule.

As an example, imagine a heterozygous SNP in the genome of an individual and in a mixture of DNA from that individual, where ten molecules of each allele were present in the original DNA sample. After amplification, there may be 100,000 DNA molecules corresponding to the locus. Due to the stochastic process, the DNA ratio can be anywhere from 1:2 to 2:1, however, since each of the initial molecules is labeled with a unique label, it will be possible to determine the DNA ratio derived from each DNA in the amplified pool. DNA of exactly 10 DNA molecules for each allele. Therefore, this method will give a more accurate measure of the relative amount of each allele than a method that does not use this method. For methods where it is desired to minimize the relative amount of allelic bias, this method will provide more accurate data.

Binding of sequenced fragments to loci of interest can be obtained in a number of ways. In one embodiment, a sequence of sufficient length to cover the molecular barcode and a sufficient number of unique bases corresponding to the sequence of interest is obtained from the targeting fragment to allow unambiguous identification of the locus of interest. In another embodiment, a molecular barcode primer containing a randomly generated molecular barcode may also contain a locus-specific barcode (locus barcode) that identifies the target to which it binds. This locus barcode is for each individual target in all molecular barcode primers and thus all resulting amplicons will be identical but distinct from all other targets. In one embodiment, the labeling methods described herein can be combined with a one-sided nesting scheme.

In one embodiment, the design and generation of molecular barcode primers can become practiced as follows: A molecular barcode primer can consist of a sequence that is not complementary to a target sequence, followed by a random molecular barcode region, followed by a target-specific sequence. The sequence 5' of the molecular barcode can be used for subsequence PCR amplification and can contain sequences suitable for converting the amplicons into libraries for sequencing. Random molecular barcode sequences can be generated in a number of ways. The preferred method synthesizes the molecular marker primers in such a way that all four bases are included in the reaction during the synthesis of the barcode region. All or individual combinations of bases can be assigned using the IUPAC DNA ambiguity codes. In this way, the synthesized collection of molecules will contain a random mixture of sequences in the barcode region of the molecule. The length of the barcode region will determine how many primers will contain a unique barcode. The number of unique sequences is related to the length of the barcode region, as N ^L , where N is the number of bases, usually 4, and L is the barcode length. A five base barcode can generate up to 1024 unique sequences; an eight base barcode can generate 65536 unique barcodes. In one embodiment, DNA can be measured by sequencing methods, where sequence data represents the sequence of a single molecule. This can include methods of directly sequencing a single molecule or methods of amplifying a single molecule to form clones that are detectable by sequencing instruments but still represent a single molecule, referred to herein as clone sequencing.

Exemplary Methods and Reagents for Quantification of Amplification Products

Quantification of relevant specific nucleic acid sequences is usually performed by quantitative real-time PCR techniques, such as Tuckerman (Life Technologies), INVADER probes (THIRD WAVE TECHNOLOGIES), etc. Such techniques suffer from a number of disadvantages, such as the ability to analyze multiple sequences in parallel (multiplex) at the same time and providing for only a narrow range of possible amplification cycles (for example, when the logarithm of the PCR amplification yield versus the number of cycles is in the linear range). The ability to accurately quantify data is limited. DNA sequencing technology, especially high-throughput next-generation sequencing technology (commonly known as massively parallel sequencing technology), such as MYSEQ (Illumina), HISEQ (Illumina), ion torrent (ION TORRENT) ( Life Technologies), Genome Analyzer ILX (Illumina), GSFLEX+ (Roche 454), etc., can be used to quantitatively measure the copy number of related sequences present in the sample, thereby providing quantitative information about the starting material Information such as copy number or transcript level. High-throughput gene sequencers are capable of using barcoding (ie, sample labeling with a unique nucleic acid sequence) in order to identify specific samples from individuals, allowing simultaneous analysis of multiple samples in a single run of the DNA sequencer. The number of times (number of reads) to sequence a given region of the genome in a library artifact (or other related nuclear artifact) will be proportional to the copy number (or in the case of cDNA-containing artifacts, the expression level) of the sequence in the associated genome. Proportion. However, the preparation and sequencing of gene banks (and artifacts derived from similar genomes) can introduce many biases that interfere with obtaining accurate quantitative readouts of related nucleic acid sequences. For example, different nucleic acid sequences may be amplified with different efficiencies during the nuclear amplification step that occurs during gene bank preparation or sample preparation.

The problem of different amplification efficiencies can be alleviated by using certain embodiments of the invention. The present invention includes various methods and compositions that refer to the use of standards for inclusion in amplification processes that can be used to improve quantitative accuracy. The present invention is particularly applicable to the detection of fetal aneuploidy by analysis of free-floating fetal DNA in maternal blood, as described herein and elsewhere: US Patent No. 8,008,018; US Patent No. 7,332,277; PCT Published Application WO 2012/078792A2; and PCT Published Application WO 2011/146632A1, each of which is incorporated herein by reference in its entirety. Embodiments of the invention are also applicable to the detection of aneuploidy in embryos produced in vitro. Commercially important aneuploidies that can be tested include those of human chromosomes 13, 18, 21, X, and Y.

Embodiments of the invention can be used with human or non-human nucleic acids, and can be applied to nucleic acids of animal and plant origin. Embodiments of the invention may also be used to detect and/or quantify alleles of other genetic diseases characterized by deletions or insertions. Alleles containing deletions can be detected among suspected carriers of related alleles.

One embodiment of the invention includes standards present in known quantities (relative or absolute). As an example, consider a gene pool made from a gene source where chromosome 8 (containing locus A) is diploid and chromosome 21 (containing locus B) is triploid. A gene library can be generated from such a sample that will contain the number of sequences that are a function of the number of chromosomes present in the sample, eg 200 copies of locus A and 300 copies of locus B. However, if locus A is amplified much more efficiently than locus B, then 60,000 copies of the A amplicon and 30,000 copies of the B amplicon may be present after PCR, making it difficult to detect the presence of an amplicon by high-throughput DNA sequencing ( or other quantitative nucleic acid detection techniques) to confuse the true chromosome copy number of the initial genome sample. To alleviate this problem, a standard sequence is employed for locus A, wherein the amplification efficiency of the standard sequence is substantially the same as that of locus A. Similarly, a standard sequence for locus B is created, wherein the standard sequence amplifies at substantially the same efficiency as locus B. Prior to PCR (or other amplification technique), a standard sequence for locus A and a standard sequence for locus B are added to the mixture. These standard sequences exist in known quantities (relative or absolute). So if in the previous example a 1:1 mixture of standard sequence A and standard sequence B was added (before amplification) to the mixture, then 3000 copies of the standard A amplicon would be produced and the standard B amplicon would be produced 1000 copies of , indicating that under the same set of conditions, the amplification efficiency of locus A is 3 times that of locus B.

In various embodiments, one or more selected regions of the genome containing associated SNPs (or other polymorphisms) can be specifically amplified and subsequently sequenced. This target-specific amplification can occur during the formation of a gene library for sequencing. The library can contain multiple targeted regions for amplification. In some embodiments, at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 regions of interest. Examples of such libraries are described herein and can be found in US Patent Application No. 2012/0270212, filed November 18, 2011, which is hereby incorporated by reference in its entirety.

Many high-throughput DNA sequencing technologies require modification of genetic starting material, such as joining of universal priming sites and/or barcodes, in order to form libraries for facilitating clonal amplification of small nucleic acid fragments prior to performing subsequent sequencing reactions. In some embodiments, one or more standard sequences are added during gene bank formation or to a precursor component of the gene bank prior to bank amplification. Standard sequences can be selected to simulate (but can be differentiated based on nucleotide base sequences) target genome fragments to be prepared for sequencing by high-throughput gene sequencing technology. In one embodiment, the standard sequence can be identical to the genomic segment of interest for all but one, two, three, four to ten, or eleven to twenty nucleotides. In some embodiments, when the target gene sequence contains a SNP, the standard sequence can be consistent with the SNP except for the nucleotides at the polymorphic bases, and the nucleotides at the polymorphic bases can be selected as the nucleotides at the polymorphic bases in nature. one of the four nucleotides not observed at that position. Standard sequences can be used in highly compounded analyzes of multiple loci of interest (eg, polymorphic loci). Known quantities (relative or absolute) of standard sequences can be added during the library formation process (prior to amplification) in order to provide a standard measure with greater accuracy in determining the amount of related target sequences in an assay sample. Knowledge of the known quantity of standard sequences is used in combination with knowledge of the ploidy level formation of libraries for sequencing formed from genomes with previously characterized ploidy levels (e.g., all autosomes are known to be diploid) It can be used to calibrate the amplification properties of each standard sequence relative to its corresponding target sequence and to account for batch-to-batch variation in mixtures containing multiple standard sequences. Given that often a large number of loci must be analyzed simultaneously, it is useful to generate a mixture comprising a large set of standard sequences. Embodiments of the invention include mixtures comprising multiple standard sequences. Ideally, the amount of each standard sequence in the mixture is known with high precision. However, it is extremely difficult to achieve this ideal state because, as a practical matter, there is a large variation in the amount of each standard sequence in a mixture, especially a mixture containing a large number of different synthetic oligonucleotides. This variation has many sources, such as variations in the efficiency of in vitro oligonucleotide synthesis reactions between batches, inaccurate volume measurements, and variations in pipetting practices. Furthermore, such variations can occur between different batches that theoretically contain the exact same set of standard sequences in the exact same amounts. Therefore, it is of interest to calibrate each batch of standard sequences independently. Each batch of standard sequences can be calibrated against a reference genome of known chromosomal composition. The batch of standard sequences can be calibrated by sequencing the batch of standard sequences including minimal or no amplification steps in the sequencing protocol. Embodiments of the invention include calibrated mixtures of different standard sequences. Other embodiments of the invention include methods of calibrating mixtures of different standard sequences and calibrated mixtures of different standard sequences obtained by the methods of the invention.

Various embodiments of the standard sequence mixtures of the invention and methods of using them can comprise at least 10, 100, 500, 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 or more standard sequence, and various intermediate quantities. The number of standard sequences may be the same as the number of target sequences selected for analysis during generation of a targeted library for DNA sequencing. However, in some embodiments it may be advantageous to use a smaller number of standard sequences than the number of targeted regions in the constructed library. It may be advantageous to use lower amounts in order to avoid encountering limitations in the sequencing capabilities of the high throughput DNA sequencer used. The number of standard sequences can be 50% or less than the number of targeted regions, 40% or less than the number of targeted regions, 30% or less than the number of targeted regions, 20% or less than the number of targeted regions, 10% or less The number of targeted regions, the number of 5% or less targeted regions, the number of 1% or smaller targeted regions, and various intermediate values. For example, if a gene library was created using 15,000 primer pairs targeting loci containing a specific SNP, then a standard containing 1500 corresponding to 1500 of the 15,000 loci of interest could be added prior to the amplification step of library construction A suitable mixture of sequences.

The amount of standard sequences added during library construction can vary significantly in different embodiments. In some embodiments, the amount of each standard sequence can be about the same as the predicted amount of the target sequence present in the sample of genomic material used for library preparation. In other embodiments, the amount of each standard sequence may be greater or less than the predicted amount of the target sequence present in the sample of genomic material used for library preparation. While the initial relative amounts of target and standard sequences are not critical to the function of the invention, the amounts are preferably in the range of 100-fold greater to 100-fold less than the amount of target sequence present in the sample of genomic material used for library preparation . Excess standards use excess sequencing capacity of the DNA sequencer for a given run of the instrument. Using too low a standard sequence will result in insufficient data to aid in the analysis of changes in amplification efficiency.

A standard sequence can be selected whose nucleotide base sequence is very similar to the relevant amplified region; preferably, the standard sequence has exactly the same primer binding sites as the genomic region being analyzed, ie the "target sequence". A standard sequence must be distinguishable from the corresponding target sequence at a given locus. For convenience, such distinguishable regions of standard sequences will be referred to as "marker sequences". In some embodiments, the marker sequence region of the target sequence contains polymorphic regions, such as SNPs, and may be flanked on both sides by primer binding regions. A standard sequence can be selected that closely matches the GC content of the corresponding target sequence. In some embodiments, the primer binding region of the standard sequence is flanked by universal priming sites. These universal priming sites were chosen to match those used in the genomic libraries used for analysis. In other embodiments, the standard sequence does not have a universal priming site and the universal priming site is added during library generation. Standard sequences are usually provided as single strands. Standard sequences are defined relative to corresponding target sequences and the sequence-specific reagents used to amplify the target sequences. In some embodiments, the target sequence present in the nucleic acid sample for analysis contains associated polymorphisms, such as SNPs, deletions or insertions. A standard sequence is a synthetic polynucleotide that is similar in nucleotide base sequence to the target sequence but is still distinguishable from the target sequence by at least one nucleotide base difference, thereby providing an amplicon sequence that distinguishes the standard sequence from that of the target sequence. Mechanism of amplicon sequence derived from target sequence. A standard sequence is selected to have substantially the same amplification properties as a corresponding target sequence when amplified with the same set of amplification reagents (eg, PCR primers). In some embodiments, a standard sequence can have the same primer sequence binding site as a corresponding target sequence. In other embodiments, a standard sequence may have a different primer sequence binding site than the corresponding target sequence. In some embodiments, standard sequences can be selected to produce amplicons of the same length as amplicons generated from corresponding target sequences. In other embodiments, standard sequences may be selected to produce amplicons of slightly different lengths than amplicons generated from corresponding target sequences.

After the amplification reaction has been completed, the library is sequenced on a high-throughput DNA sequencer, in which individual molecules are clonally amplified and sequenced. The number of sequence reads for each allele of the target sequence is counted, and the number of sequence reads corresponding to the standard sequence of the target sequence is also counted. The process is also performed on at least one other pair of target sequences and corresponding standard sequences. Consider for example locus A, generate X _A1 reads for allele 1 of locus A; generate X _A2 reads for allele 2 of locus A, and generate X _AC reads for canonical sequence A. The ratio of (X _A1 plus X _A2 ) to X _AC was determined for each locus of interest. As previously discussed, the process can be performed on a reference genome, such as a genome in which all chromosomes are known to be diploid. The process can be repeated multiple times to provide a large number of reads to determine the average number of reads and the standard deviation of the number of reads. The process is performed on a mixture comprising a large number of different standard sequences corresponding to different loci. By assuming that (1) X _A1 plus X _A2 corresponds to a known number of chromosomes, e.g., 2 for a normal human female genome, and (2) the standard sequence has similar amplification (and detectability) to its corresponding natural locus properties, the relative amounts of different standard sequences in a composite standard mixture can be determined. The calibrated composite standard sequence mixture can then be used to adjust for variability in amplification efficiency between different loci in the multiplex amplification reaction.

Other embodiments of the invention include methods and compositions for measuring the copy number of specific genes of interest, including duplications and mutant genes characterized by large deletions that would interfere with quantification by sequencing. Sequencing can be problematic in detecting alleles with such deletions. Amplification procedures involving standard sequences can be used to reduce this problem.

In one embodiment of the invention, the target sequence for analysis is a gene having a wild-type (ie functional) form and a mutant form characterized by a deletion. An example of such a gene is SMN1, an allele with a deletion that causes the genetic disease spinal muscular atrophy (SMA). Of interest are the detection of individuals carrying mutant forms of the gene by means of high-throughput gene sequencing techniques. Applying such techniques to the detection of deletion mutations can be problematic, not least because the sequence is not observed in sequencing (as opposed to detection of simple point mutations or SNPs). Such embodiments employ (1) a pair of amplification primers specific for the gene of interest, wherein the amplification primers will amplify the gene of interest (or a portion thereof) and will not significantly amplify the mutant allele; ( 2) correspond to the wild-type allele of the related gene, but differ by at least one detectable nucleotide base standard sequence (i.e., target sequence); (3) have specificity for a second target sequence serving as a reference sequence a pair of amplification primers; and (4) a standard sequence corresponding to the reference sequence.

In one embodiment of the present invention, there is provided a method for measuring the copy number of a related gene having a meaningful allele comprising a deletion. The method may employ amplification reagents specific for the gene of interest, for example by amplifying at least a portion of the gene of interest or the entire gene of interest or a region adjacent to the gene of interest without amplifying deletion-containing alleles of the gene of interest. PCR primers specific to the relevant genes. In addition, the method of the present invention employs a standard sequence corresponding to a related gene, wherein the standard sequence differs by at least one nucleotide base from the related gene (so that the sequence of the standard sequence can be easily distinguished from the naturally occurring related gene open). Typically, the standard sequence will contain the same primer binding sites as the gene of interest in order to minimize any amplification discrimination between the gene of interest and the standard sequence corresponding to the gene of interest. The reaction will also contain amplification reagents specific for the reference sequence. A reference sequence is a sequence with a known (or at least assumed known) copy number in the genome to be analyzed. The reaction further comprises a standard sequence corresponding to a reference sequence. Typically, the standard sequence corresponding to the reference sequence will contain the same primer binding sites as the reference sequence in order to minimize any amplification discrimination between the reference sequence and the standard sequence corresponding to the reference sequence.

Exemplary Nucleic Acid Samples

In some embodiments, genetic samples can be prepared and/or purified. A variety of standard procedures are known in the art for this purpose. In some embodiments, the sample can be centrifuged to separate the layers. In some embodiments, filtration can be used to isolate DNA. In some embodiments, preparation of DNA may involve amplification, separation, purification by chromatography, liquid-liquid separation, isolation, preferential enrichment, preferential amplification, targeted amplification, or other methods known in the art or described herein. Any of a variety of other techniques.

In some embodiments, the methods disclosed herein may be used where, for example, in in vitro fertilization there is very little DNA or where one or a few cells (typically less than ten cells, less than twenty cells, or in 40 cells) available forensic cases. In these examples, the methods disclosed herein were used to make ploidy calls from small amounts of DNA that were not contaminated with other DNA, but where ploidy calls were very difficult. In some embodiments, the methods disclosed herein can be used in situations where the DNA of interest is contaminated with DNA from another individual, for example in maternal blood in the case of prenatal diagnosis, paternity testing, or product of conception testing. Some other situations where these methods would be particularly advantageous would be in the case of cancer testing where only one or a small number of cells are present among a larger number of normal cells. Genetic measurements used as part of these methods can be performed on any sample comprising DNA or RNA such as (but not limited to): blood, plasma, body fluids, urine, hair, tears, saliva, tissue, skin, nails , blastomeres, embryos, amniotic fluid, villi samples, stool, bile, lymph, cervical mucus, semen, or other cells or materials containing nucleic acids. In one embodiment, the methods disclosed herein can be performed using nucleic acid detection methods such as sequencing, microarrays, qPCR, digital PCR, or other methods for measuring nucleic acids. If this is found to be desirable for some reason, then the ratio of the allele count probabilities at the locus can be calculated and the allele ratio can be used to determine ploidy in combination with some of the methods described herein status, assuming the methods are compatible. In some embodiments, the methods disclosed herein involve calculating in silico allele ratios at a plurality of polymorphic loci from DNA measurements made on processed samples. In some embodiments, the methods disclosed herein involve calculating in silico alleles at multiple polymorphic loci from any combination of DNA measurements made on processed samples and other improvements described herein genetic ratio.

In some embodiments, this method can be used for single cells, few cells, two to five cells, six to ten cells, ten to twenty cells, twenty to fifty cells, fifty to one A hundred cells, a hundred to a thousand cells, or a small amount of extracellular DNA, such as one to ten picograms, ten to one hundred picograms, one hundred picograms to one nanogram, one to ten nanograms, ten to one One hundred nanograms or one hundred nanograms to one microgram for genotyping.

Exemplary RNA Expression Studies

The multiplex PCR method of the present invention can be used to increase the number of target loci that can be estimated during a gene expression profiling experiment. For example, the expression levels of thousands of genes can be monitored simultaneously to determine whether a person has sequences (eg, polymorphisms or other mutations) associated with disease (eg, cancer) or increased disease risk. These methods can be used to identify sequences (e.g., e.g. polymorphism or other mutation). Additionally, the effect of particular treatments, diseases or developmental stages on gene expression can be determined. Similarly, these methods can be used to identify genes whose expression is altered in response to pathogens or other organisms by comparing gene expression in infected and uninfected cells or tissues. In these methods, the number of sequencing reads can be adjusted based on the frequency of the polymorphism to be analyzed such that if there is a polymorphism to be detected, enough reads are performed for the polymorphism.

In some embodiments, a sample containing RNA (eg, mRNA) is amplified using reverse transcriptase (RT) and the resulting DNA (eg, cDNA) is then amplified using DNA polymerase (PCR). The RT and PCR steps can be performed sequentially or separately in the same reaction volume. Any of the primer libraries of the invention can be used in this reverse transcription polymerase chain reaction (RT-PCR) method. In various embodiments, reverse transcription is performed using oligo-dT, random primers, a mixture of oligo-dT and random primers, or primers specific for the locus of interest. To avoid amplification of contaminated genomic DNA, primers for RT-PCR can be designed such that a part of one primer hybridizes to the 3' end of one exon and the other part of the primer hybridizes to the 5' end of an adjacent exon. 'end. Such primers anneal to cDNA synthesized from spliced mRNA, but do not anneal to genomic DNA. To detect amplification of contaminated DNA, RT-PCR primer pairs can be designed to flank a region containing at least one intron. Products amplified from cDNA (without introns) were smaller than those amplified from genomic DNA (with introns). The size difference of the products is used to detect the presence of contaminated DNA. In some embodiments, primer annealing sites that are at least 300-400 base pairs apart are selected only when the mRNA sequence is known, since fragments of this size from eukaryotic DNA are likely to contain splice junctions. Alternatively, samples can be treated with DNase to degrade contaminating DNA.

Exemplary Methods of Paternity Testing

The multiplex PCR method of the present invention can be used to improve the accuracy of paternity testing because so many loci of interest can be analyzed at one time (see, e.g., U.S. Publication No. 2012/0122701, filed December 22, 2011, which is hereby incorporated herein by reference in its entirety). For example, multiplex PCR methods can allow for the analysis of thousands of polymorphic loci (eg, SNPs) suitable for use in the PARENTAL SUPPORT algorithm described herein to determine whether the putative father is the biological father of the fetus. In some embodiments, the method involves (i) simultaneously amplifying a plurality of polymorphic loci on the genetic material from the putative father, including at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000 , 50,000, 75,000, or 100,000 different polymorphic loci, thereby generating a first set of amplification products; (ii) simultaneously amplifying corresponding multiple polymorphic loci on the DNA mixture sample derived from the pregnant mother's blood sample to generating a second set of amplification products; wherein the mixed sample of DNA comprises fetal DNA and maternal DNA; (iii) determining in silico, using genotype measurements, the presumed father to be the biological father of the fetus based on the first and second sets of amplification products the probability of the father; and (iv) determining whether the hypothetical father is the biological father of the fetus using the determined probability that the hypothetical father is the biological father of the fetus. In various embodiments, the method further comprises simultaneously amplifying corresponding multiple polymorphic loci on the genetic material from the mother to generate a third set of amplification products; wherein based on the first, second, and third sets of amplification Product that uses genotype measurements to determine the probability that the putative father is the biological father of the fetus.

Exemplary Methods for Embryo Characterization and Selection

The multiplex PCR method of the present invention can be used to improve embryo selection for in vitro fertilization by allowing the analysis of thousands of loci of interest at once (see, e.g., U.S. publications filed May 27, 2008, December 22, 2011 2011/0092763, which is hereby incorporated by reference in its entirety). For example, multiplex PCR methods can allow for the analysis of thousands of polymorphic loci (eg, SNPs) suitable for use in the PARENTAL SUPPORT algorithm described herein to select embryos from a set of embryos for in vitro fertilization.

In some embodiments, the present invention provides methods of estimating the relative likelihood that each embryo in a set of embryos will develop as desired. In some embodiments, the method involves simultaneously hybridizing samples from each embryo to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different genes of interest The primer pools of the loci are contacted to generate a reaction mixture for each embryo, wherein the samples are each derived from one or more cells from the embryo. In some embodiments, each reaction mixture is subjected to primer extension reaction conditions to generate amplification products. In some embodiments, the method comprises determining in silico one or more characteristics of at least one cell from each embryo based on the amplification product; and based on the one or more characteristics of the at least one cell from each embryo, The relative likelihood that each embryo will develop as desired is estimated in silico. In some embodiments, the method comprises determining at least one characteristic using an information-based method, such as the PARENTAL SUPPORT algorithm described herein. In some embodiments, the characteristics include ploidy status. In some embodiments, the characteristic is selected from the group consisting of: aneuploidy, euploidy, mosaicism, deletion, monosomy, uniparental disomy, trisomy, tetrasomy, non- Type of euploidy, unmatched copy error trisomy, matched copy error trisomy, maternal source of aneuploidy, paternal source of aneuploidy, presence or absence of disease-linked genes, any aneuploidy Chromosomal identity of ploidy chromosomes, abnormal genetic conditions, deletions or duplications, likelihood of traits, and combinations thereof. The characteristic may be associated with a chromosome taken from the group consisting of: chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 6, chromosome 7, chromosome 8, chromosome 9 Chromosome, Chromosome 10, Chromosome 11, Chromosome 12, Chromosome 13, Chromosome 14, Chromosome 15, Chromosome 16, Chromosome 17, Chromosome 18, Chromosome 19 , chromosome 20, chromosome 21, chromosome 22, chromosome X or chromosome Y and combinations thereof.

Exemplary Prenatal Diagnostic Methods

The multiplex PCR method of the present invention can be used to improve prenatal diagnosis methods, such as determining the ploidy status of fetal chromosomes. Given that a large number of target loci can be amplified simultaneously, more accurate assays can be made.

In one embodiment, the present invention provides genotype data for measurement from a mixed sample of DNA (i.e., DNA from the mother of the fetus and DNA from the fetus) and optionally genetic data from the mother and possibly also the father. An ex vivo method for determining the ploidy state of chromosomes in a gestating fetus from genotype data measured on a sample of material, wherein said determination is performed by using a joint distribution model for different possible fetuses given the parental genotype data Ploidy status creates a set of predicted allelic distributions and compares the predicted allelic distributions to the actual allelic distributions measured in the pooled sample and selects the predicted allelic distribution pattern that most closely matches the observed allele Ploidy status of the distribution pattern. In one embodiment, the pooled sample is derived from maternal blood or maternal serum or plasma. In one embodiment, a pooled sample of DNA can be preferentially enriched at loci of interest (eg, multiple polymorphic loci). In one embodiment, preferential enrichment is performed in a manner that minimizes allelic bias. In one embodiment, the invention relates to a DNA composition that has been preferentially enriched at multiple loci such that allelic bias is low. In one embodiment, the allelic distribution is measured by sequencing DNA from a pooled sample. In one embodiment, the joint distribution model employs alleles that will be distributed in a binomial fashion. In one embodiment, sets of predicted joint allele distributions are created for genetically linked loci taking into account existing recombination frequencies from various sources, eg, using data from the International HapMap Collaboration.

In one embodiment, the present invention provides a method for non-invasive prenatal diagnosis (NPD), specifically by observing alleles at multiple polymorphic loci in genotype data measured on a mixture of DNA Measurements are used to determine the aneuploidy status of the fetus, wherein certain allelic measurements are indicative of an aneuploid fetus and other allele measurements are indicative of a euploid fetus. In one embodiment, genotype data is measured by sequencing a mixture of DNA derived from maternal plasma. In one embodiment, the DNA sample may be preferentially enriched in DNA molecules corresponding to a plurality of loci for which the allelic distribution is to be calculated. In one embodiment, a DNA sample comprising only or almost exclusively genetic material from the mother and possibly also a DNA sample comprising only or almost exclusively genetic material from the father is measured. In one embodiment, genetic measurements of one or both parents and estimated fetal fractions are used to create a plurality of predicted allelic distributions corresponding to different possible underlying genetic states of the fetus; the predicted allelic distributions can be called hypothesis. In one embodiment, the maternal genetic data is not determined by measuring genetic material that is exclusively or nearly exclusively naturally derived from the mother; rather, it is determined from the maternal This plasma is estimated from genetic measurements obtained. In some embodiments, the assumptions may include fetal ploidy at one or more chromosomes, which segments of which chromosomes in the fetus are inherited from which parents, and combinations thereof. In some embodiments, the ploidy state of the fetus is determined by comparing observed allelic measurements with different hypotheses, wherein at least some of the hypotheses correspond to different ploidy states; and given the From the observed allelic measurements, select the ploidy state that corresponds to the most likely correct hypothesis. In one embodiment, this method involves using allelic measurement data from some or all of the measured SNPs, whether the loci are homozygous or heterozygous, and thus does not involve using Alleles at the locus. This approach may not be suitable for cases where the genetic data involve only one polymorphic locus. This approach is particularly advantageous when the genetic data contains data from more than ten polymorphic loci or more than twenty polymorphic loci from the chromosome of interest. This approach is particularly advantageous when the genetic data comprises data for more than 50 polymorphic loci for the target chromosome, for more than 100 polymorphic loci, or for more than 200 polymorphic loci for the target chromosome. In some embodiments, the genetic data may comprise data for more than 500 polymorphic loci of a chromosome of interest, of more than 1,000 polymorphic loci of a chromosome of interest, of more than 2,000 polymorphic loci, or of more than 5,000 polymorphic loci.

In one embodiment, the methods disclosed herein yield a quantitative measure of the number of independent observations for each allele at a polymorphic locus. This differs from most methods such as microarray or qualitative PCR, which provide information on the ratio of two alleles, but do not quantify the number of independent observations for either allele. In the case of methods that provide quantitative information about the number of independent observations, only ratios are employed in ploidy calculations, and the quantitative information itself is not applicable. To illustrate the importance of retaining information about the number of independent observations, consider a sample locus with two alleles (A and B). In the first experiment, twenty A alleles and twenty B alleles were observed; in the second experiment, 200 A alleles and 200 B alleles were observed. In both experiments the ratio (A/(A+B)) was equal to 0.5, however the second experiment conveyed more information about the frequency certainty of the A or B allele than the first experiment. Some methods by others involve averaging or summing the allelic ratios (channel ratios) of the individual alleles (i.e. x _i /y _i ) and analyzing this ratio, comparing it to a reference chromosome or using information about the predicted The rules for how a ratio works in a particular situation. There is no allelic weighting in this type of method, where it is assumed that the same amount of PCR product is ensured for each allele and that all alleles should behave in the same way. Such methods have several disadvantages and, more importantly, prevent the use of the various improved methods described elsewhere herein.

In one embodiment, the methods disclosed herein explicitly model the expected allele frequency distribution for disomies as well as the multiple allele frequency distributions that can be expected in the case of a trisomy, the trisomy Sex arises from nondisjunction during meiosis I, nondisjunction during meiosis II, and/or nondisjunction during mitosis early in fetal development. To illustrate why this is important, imagine a situation in which there is no crossover: nondisjunction during meiosis I would produce a trisomy, in which two distinct homologues are inherited from one parent; by contrast, meiotic Nondisjunction during division II or during mitosis early in fetal development will produce two copies of the same homologue from one parent. Each scenario will yield a different predicted allele frequency at each polymorphic locus and at all loci that are considered joint due to genetic linkage. Crossover causes the exchange of genetic material between homologues, making the pattern of inheritance more complex; in one embodiment, the methods of the invention accommodate this by using recombination rate information plus the physical distance between loci. In one embodiment, in order to enable improved discrimination between meiotic I nondisjunction and meiotic II or mitotic nondisjunction, the present method incorporates an increasing crossover probability with increasing distance from the centromere into in the model. Meiotic II and mitotic nondisjunction can be distinguished by the fact that mitotic nondisjunction usually produces identical or nearly identical copies of one homologue whereas two homologues present after the meiotic II nondisjunction event are usually due to Varies by one or more crosses during gametogenesis.

In some embodiments, the methods disclosed herein involve comparing observed allelic measurements to theoretical hypotheses corresponding to possible fetal Gene ratios are quantified. When the number of loci is below about 20, a ploidy determination using a method comprising quantifying the ratio of alleles at a heterozygous locus and using a method comprising comparing the observed allelic measurements with those corresponding to Ploidy determinations based on assumptions about the allelic distribution of likely fetal genetic status may yield similar results. However, when the number of loci exceeds 50, the two methods may give significantly different results; result. These differences are due to the fact that methods involving quantification of the ratio of alleles at a heterozygous locus without independently measuring the magnitude of each allele and summing or averaging the ratios prevent techniques including the use of joint distribution models, performing linkage analysis, using binomial distribution models, and/or the use of other advanced statistical techniques; Methods that state theoretical allele distribution assumptions can use these techniques, which can substantially improve the accuracy of the assay.

In one embodiment, the methods disclosed herein involve using a joint distribution model to determine whether the observed distribution of allelic measurements is indicative of a euploid or aneuploid fetus. The use of a joint distribution model differs from and significantly improves upon methods for determining heterozygosity rates by independently addressing polymorphic loci; the difference being that the resulting decisions are significantly more accurate. Without being bound by any particular theory, it is believed that one reason for their greater accuracy is that the joint distribution model takes into account the linkage between SNPs and the likelihood of crossovers that have occurred during meiosis, which Gametes are produced, the gametes form embryos, and the embryos grow into fetuses. The purpose of using the linkage concept when creating predicted distributions of allelic measurements against one or more assumptions is that it allows the creation of predicted distributions of allelic measurements that correspond significantly better to reality than if linkage were not used. As an example, imagine that there are two SNPs: 1 and 2 positioned next to each other, and that on one homologue of the mother is A at SNP 1 and A at SNP 2, and on the second homologue is A at SNP 1 is B and is B at SNP 2. If both SNPs on both congeners of the father are A, and B is measured against fetal SNP 1, then this indicates that the second congener has been inherited by the fetus, and therefore B is present at the fetal SNP The likelihood on 2 is much higher. Models that account for linkage will predict this, while models that do not account for linkage will not. Alternatively, if the mother has AB at SNP 1 and AB at nearby SNP2, then two hypotheses corresponding to maternal trisomy at said position can be used: one involving matching copy errors (either meiosis II or nondisjunction in mitosis early in fetal development), and a copy error involving a mismatch (nondisjunction in meiosis I). In the case of a matched-copy-error trisomy, if the fetus inherited AA from the mother's SNP 1, the fetus was more likely to inherit AA or BB from the mother's SNP 2, but not AB. In the case of a mismatched copy error, the fetus will inherit AB from both SNPs from the mother. Allelic distribution assumptions derived by ploidy calling methods that account for linkage will make these predictions and thus correspond to actual allelic measurements to a significantly greater extent than ploidy calling methods that do not account for linkage. It should be noted that linkage methods are not possible when using methods that rely on calculating allele ratios and summing those allele ratios.

One reason for the greater accuracy of ploidy determination using methods that involve comparing observed allelic measurements to theoretical hypotheses corresponding to probable genetic states of the fetus is believed to be that when sequencing is used to measure alleles, this This method can glean more information from data from alleles where the total number of reads is lower than other methods; for example, methods that rely on calculating and summing allele ratios will generate disproportionately weighted random noise. As an example, imagine a situation involving the use of sequencing to measure alleles, and where there is a set of loci where only five sequence reads are detected for each locus. In one embodiment, for each of the alleles, the data can be compared to a hypothetical allelic distribution and weighted according to the number of sequence reads; thus the data from these measurements will be appropriately weighted and into the entire measurement. This is in contrast to methods that involve quantifying the ratio of alleles at a heterozygous locus, since such methods can only calculate 0%, 20%, 40%, 60%, 80%, or 100% ratio; none of these came close to the predicted allelic ratio. In this latter case, the calculated allele ratios would have to be discarded due to insufficient reads, or would have disproportionate weighting and introduce random noise into the assay, reducing the accuracy of the assay. In one embodiment, individual allele measurements may be processed into independent measurements, where the relationship between measurements obtained for alleles at the same locus is the same as measurements obtained for alleles at different loci The relationship between the results is not different.

In one embodiment, the methods disclosed herein involve determining the observed alleles without comparing any metric to allelic measurements observed on a reference chromosome predicted to be disomic (referred to as the RC method). The distribution of allelic measurements obtained indicates whether the fetus is euploid or aneuploid. This significantly improves methods, such as those using shotgun sequencing, that detect aneuploidy by estimating the proportion of randomly sequenced fragments of a suspected chromosome relative to one or more putative disomic reference chromosomes. This RC method gives incorrect results if the assumed disomic reference chromosome is not actually disomic. This may occur in cases where aneuploidy is more true than trisomy of a single chromosome or where the fetus is triploid and all autosomes are trisomy. In the case of a female triploid (69,XXX) fetus, disomic chromosomes are practically completely absent. The methods described herein do not require a reference chromosome and will correctly identify trisomy in female triploid fetuses. With respect to each chromosome, hypothesis, child score, and noise level; the joint distribution model can be fitted in the absence of either: reference chromosome data, overall child score estimates, or a fixed reference hypothesis.

In one example, the methods disclosed herein demonstrate how observing the distribution of alleles at polymorphic loci can be used to determine the ploidy state of a fetus with greater accuracy than methods in the prior art. In one embodiment, the method uses targeted sequencing to obtain mixed maternal-fetal genotypes and optionally maternal and/or paternal genotypes at multiple SNPs to first determine the individual predicted genotypes under different assumptions. Allele frequency distribution, and then look at the quantitative allelic information obtained on the maternal-fetal mixture and estimate which hypothesis fits the data best, where the genetic state corresponding to the hypothesis with the best fit to the data is called for the correct genetic status. In one embodiment, the methods disclosed herein also use the degree of fit to generate a confidence that the called genetic state is the correct genetic state. In one embodiment, the methods disclosed herein involve using an algorithm that analyzes the distribution of alleles found at loci with different parental backgrounds, and comparing the different ploidy states of the different parental backgrounds (different parental genotype patterns) The observed and predicted allelic distributions of . This differs from and is an improvement over methods that do not use the ability to estimate the number of independent instances of each allele at each locus in a mixed maternal-fetal sample. In one embodiment, the methods disclosed herein involve using the observed distribution of alleles measured at a locus to determine whether the distribution of observed allelic measurements indicates euploidy or aneuploidy Fetus in which the mother is heterozygous. This differs from and is an improvement over methods that do not use the allelic distribution observed at the locus if the mother is heterozygous, as in it the DNA targets genes that are known not to be highly informative for that particular target individual The absence of preferential enrichment or preferential enrichment of loci allows for the use of approximately twice as many gene measurements from one set of sequence data in ploidy determinations, resulting in more accurate determinations.

In one embodiment, the methods disclosed herein use a joint distribution model that assumes that the allele frequency at each locus is multinomial in nature (and thus biallelic when the SNP is biallelic). item). In some embodiments, the joint distribution model uses a B-binomial distribution. When a measurement technique such as sequencing is used to provide a quantitative measure of each allele present at each locus, a binomial model can be applied to each locus and the fundamental degree of allele frequency and the Confidence in frequency. The certainty of the observed ratios cannot be determined by means of methods known in the art to generate ploidy calls from allele ratios or methods in which quantitative allelic information is discarded. The method of the present invention differs from and is an improvement over methods of calculating allele ratios and summing those ratios to make a ploidy call, since any method involving calculating allele ratios at a particular locus and then summing those ratios necessarily assumes that from The measured intensities or counts indicative of the amount of DNA for any given allele or locus will be distributed in a Gaussian manner. The methods disclosed herein do not involve calculation of allele ratios. In some embodiments, the methods disclosed herein may involve incorporating in the model the observed quantities of each allele at multiple loci. In some embodiments, the methods disclosed herein may involve calculating the predicted distribution itself, allowing the use of a joint binomial distribution model that may be more accurate than any model that assumes a Gaussian distribution of allele measurements. The likelihood that the binomial distribution model is significantly more accurate than the Gaussian distribution increases with the number of loci. For example, when querying fewer than 20 loci, the likelihood that the binomial distribution model is significantly better is low. However, when using more than 100, or especially more than 400, or especially more than 1,000, or especially more than 2,000 loci, the binomial distribution model is significantly more accurate than the Gaussian distribution model, resulting in a more accurate likelihood of ploidy determination will be very high. The likelihood that the binomial distribution model is significantly more accurate than the Gaussian distribution also increases with the number of observations at each locus. For example, when fewer than 10 distinct sequences at each locus are observed, the likelihood that the binomial distribution model is significantly better is low. However, the binomial distribution model was significantly more accurate than the Gaussian distribution model when using more than 50 sequence reads per locus, or especially more than 100 sequence reads, or especially more than 200 sequence reads, or especially more than 300 sequence reads , resulting in a more accurate ploidy determination with a very high likelihood.

In one embodiment, the methods disclosed herein use sequencing to measure the number of instances of each allele at each locus in a DNA sample. Each sequencing read can be mapped to a specific locus and processed into a binary sequence read; alternatively, read identities and/or probabilities of mapping can be incorporated as part of the sequence reads, resulting in probabilistic sequence reads, i.e., probabilistic sequence reads that map to a given locus Possible integer or fraction of sequence reads. Using binary counts or count probabilities, it is possible to use a binomial distribution for each set of measurements, allowing the calculation of confidence intervals about the number of counts. This ability to use the binomial distribution allows for more accurate ploidy estimates and the calculation of more precise confidence intervals. This is different from and an improvement over methods that use intensity to measure the amount of alleles present, such as methods using microarrays or measurements using fluorescent readers to measure the intensity of fluorescently labeled DNA in electrophoretic bands method.

In one embodiment, the methods disclosed herein use aspects of the data sets of the invention to determine parameters of estimated allele frequency distributions for that set of data. This improves the method of using the training set of data or previous sets of data to set the parameters of the present invention's predicted allele frequency distribution or possibly predicted allele ratios. This is because the conditions involved in the collection and measurement of each genetic sample are set differently, and therefore the method of using data from the dataset of the present invention to determine the parameters of the joint distribution model to be used in the ploidy determination of said samples will be tends to be more accurate.

In one embodiment, the methods disclosed herein involve determining whether an observed distribution of allelic measurements is indicative of a euploid or aneuploid fetus using maximum likelihood techniques. The use of the maximum likelihood technique differs from and significantly improves the method using the single hypothesis rejection technique, with the difference that the resulting assay will have significantly higher accuracy. One reason is that the single hypothesis rejection technique sets the cutoff threshold based on only one measurement distribution rather than two, meaning that the threshold is often not optimal. Another reason is that the maximum likelihood technique allows optimization of the cut-off threshold for each individual sample rather than determining a cut-off threshold for all samples regardless of the specific characteristics of each individual sample. Another reason is that the use of maximum likelihood techniques allows the calculation of a confidence level for each ploidy call. The ability to make a confidence calculation for each reading allows the practitioner to know which readings are accurate and which are more likely to be wrong. In some embodiments, various methods can be combined with maximum likelihood estimation techniques to enhance the accuracy of ploidy calls. In one embodiment, the maximum likelihood technique may be used in combination with the method described in US Patent 7,888,017. In one embodiment, the maximum likelihood technique can be used in combination with methods that use targeted PCR amplification to amplify DNA in mixed samples, followed by sequencing and analysis using read counting methods such as those described in 2011 TANDEMDIAGNOSTICS presented at the 2011 International Congress of Human Genetics in Montreal in October 2011. In one embodiment, the methods disclosed herein involve estimating the fetal fraction of DNA in a pooled sample and using the estimate to calculate a ploidy call and a confidence in the ploidy call. It should be noted that this is distinct and distinct from the approach of using estimated fetal fraction as a screen for sufficient fetal fraction followed by ploidy calls using a single hypothesis exclusion technique that does not take fetal fraction into account and does not generate Confidence calculations for calls.

In one embodiment, the methods disclosed herein take into account the tendency of data to be noisy and contain errors by attaching a probability to each measurement. The use of maximum likelihood techniques to select correct hypotheses from a set of hypotheses derived using measurements appended with probabilistic estimates will result in fewer incorrect measurements and fewer correct measurements will be used in the calculations used to generate ploidy calls. possible. To be more precise, this approach systematically reduces the influence of incorrectly measured data on ploidy determinations. This improves upon methods in which all data are assumed to be equally correct or in which outlying data are arbitrarily excluded from calculation results to obtain ploidy calls. Existing methods using channel ratio measurements require extending the method to multiple SNPs by averaging the individual SNP channel ratios. Not weighting individual SNPs based on SNP quality and observed read depth by expected measurement variance reduces the accuracy of the resulting statistics, leading to significantly less accurate ploidy calls, especially in borderline cases.

In one embodiment, the methods disclosed herein do not presuppose knowledge of which SNPs or other polymorphic loci are heterozygous on the fetus. This approach allows for ploidy calls in situations where paternal genotype information is not available. This improves methods where knowledge of which SNPs are heterozygous must be known in advance in order to properly select loci for a target or to interpret genetic measurements made on mixed fetal/maternal DNA samples.

The methods described herein are particularly advantageous when used on samples where small amounts of DNA are available or where the percentage of fetal DNA is low. This is due to a correspondingly higher rate of allelic loss that occurs when only small amounts of DNA are available and/or when the percentage of fetal DNA in a mixed sample of fetal and maternal DNA is low. A high allele loss rate means that the target individual has a large percentage of unmeasured alleles, leading to inaccurate fetal fraction calculations and inaccurate ploidy determinations. Because the methods disclosed herein can use a joint distribution model that takes into account the linkage of inheritance patterns between SNPs, significantly more accurate ploidy determinations can be made. The methods described herein allow accurate ploidy determination to be made when the percentage of fetal DNA molecules in the mixture is less than 40%, less than 30%, less than 20%, less than 10%, less than 8%, and even less than 6%.

In one embodiment, it is possible to determine the ploidy state of an individual based on the measurements when the individual's DNA is mixed with the DNA of a related individual. In one embodiment, the DNA mixture is the free-floating DNA found in maternal plasma, which can include DNA from the mother with a known karyotype and a known genotype, and which can be compared with DNA with an unknown karyotype. mixed with fetal DNA of unknown genotype. It is possible to use known genotype information from one or both parents to predict multiple potential genetic states of DNA in a mixed sample of different ploidy states, different chromosomal contributions from each parent to the fetus, and optionally different fetuses in the mixture DNA score. Each potential component can be called a hypothesis. The ploidy state of the fetus can then be determined by looking at the actual measurements and determining which underlying components are most likely given the observed data.

Further discussion of the above points can be found elsewhere in this document.

Non-Invasive Prenatal Diagnosis (NPD)

The process of non-invasive prenatal diagnosis involves several steps. Some of the steps may include: (1) obtaining genetic material from the fetus; (2) ex vivo enrichment of genetic material from the fetus that may be in a mixed sample; (3) ex vivo amplification of genetic material; (4) ex vivo (5) measuring genetic material ex vivo; and (6) analyzing genotype data in silico and ex vivo. Methods for practicing these six and other related steps are described herein. At least some of the method steps are not applied directly to the body. In one embodiment, the present invention relates to therapeutic and diagnostic methods applied to tissues and other biological materials that have been isolated and separated from the body. At least some of the method steps are performed on a computer.

Some embodiments of the present invention allow a clinician to determine the genetic status of a fetus being conceived in the mother in a non-invasive manner so that the health of the baby is not at risk by collecting the genetic material of the fetus and the mother does not need to go through invasive procedure. Furthermore, in certain aspects, the present invention allows determination of fetal genetic status with high accuracy, significantly greater than, for example, non-invasive maternal serum analyte-based screening (eg, triple testing widely used in prenatal care).

The high accuracy of the methods disclosed herein is a result of the informative methods used to analyze genotype data as described herein. Modern technological advances have yielded the ability to measure large amounts of genetic information from genetic samples using such methods as high-throughput sequencing and genotyping arrays. The methods disclosed herein allow clinicians to make better use of the vast amount of data available and to make more accurate diagnoses of the genetic status of the fetus. Details of various embodiments are given below. Different embodiments may involve different combinations of the steps described above. Various combinations of different embodiments of different steps may be used interchangeably.

In one embodiment, a blood sample is taken from a pregnant mother, and free floating DNA in the plasma of the mother's blood, containing a mixture of DNA of maternal and fetal origin, is separated and used to determine the ploidy state of the fetus. In one embodiment, the methods disclosed herein involve preferential enrichment of those DNAs in a mixture of DNA that correspond to polymorphic alleles in such a way that the allele ratios and/or allele distributions remain approximately the same after enrichment sequence. In one embodiment, the methods disclosed herein involve highly efficient targeted PCR-based amplification such that a very high percentage of the resulting molecules correspond to the loci of interest. In one embodiment, the methods disclosed herein involve sequencing a DNA mixture comprising DNA of maternal origin and DNA of fetal origin. In one embodiment, the methods disclosed herein involve the use of measured allelic distributions to determine the ploidy state of a gestating fetus in a mother. In one embodiment, the methods disclosed herein involve reporting the determined ploidy status to a clinician. In one embodiment, the methods disclosed herein involve taking clinical action, such as performing subsequent invasive tests, such as chorionic villus sampling or amniocentesis, preparing for the birth of a trisomy individual or selectively aborting a trisomy fetus.

This application refers to U.S. Utility Application No. 11/603,406 (U.S. Publication No. 20070184467), filed November 28, 2006; U.S. Utility Application No. 12/076,348, filed March 17, 2008 (U.S. Publication No. 20080243398) ; PCT Application No. PCT/US09/52730, filed August 4, 2009 (PCT Publication No. WO/2010/017214); PCT Application No. PCT/US10/050824, filed September 30, 2010 (PCT Publication No. WO/2011/041485); U.S. Utility Application No. 13/110,685, filed May 18, 2011, and PCT Application No. PCT/12/58578, filed October 3, 2012, each in its entirety Incorporated herein by reference. Some of the words used in this archive may have their antecedents in these references. Some of the concepts described herein may be better understood from concepts found in these references.

Screening of maternal blood for free-floating fetal DNA

The methods described herein can be used to aid in genotyping a child, fetus, or other individual of interest in which genetic material of interest is found to be present in amounts of other genetic material. In some embodiments, a genotype can refer to the ploidy state of one or more chromosomes, it can refer to one or more disease-linked alleles, or some combination thereof. In the present invention, the discussion focuses on determining the genetic status of a fetus where fetal DNA is found in maternal blood, but this example is not intended to limit the possible situations in which this method can be applied. In addition, the method may be applicable in cases where the amount of target DNA is in any proportion to non-target DNA; for example, target DNA may comprise any value between 0.000001% and 99.999999% of the DNA present. Additionally, the non-target DNA need not necessarily be from one individual, or even from related individuals, so long as genetic data from some or all of the related non-target individuals is known. In one embodiment, the methods disclosed herein can be used to determine fetal genotype data from maternal blood containing fetal DNA. It can also be used in cases where there are multiple fetuses in the uterus of a pregnant woman or where there may be other contaminated DNA in the sample, eg from other already born siblings.

This technique can take advantage of the close proximity of fetal blood cells to the maternal circulation through the placental villi. Typically, only a very small number of fetal cells enter the maternal circulation in this way (not enough to produce a positive Kleihauer-Betke test for fetal-maternal hemorrhage). Fetal cells can be sorted and analyzed for specific DNA sequences by various techniques, but without the risks inherent in invasive procedures. This technique can also take advantage of the phenomenon of free-floating fetal DNA approaching the maternal circulation through DNA release following apoptosis of placental tissue containing DNA of the same genotype as the fetus. Free-floating DNA found in maternal plasma has been shown to contain fetal DNA in proportions as high as 30%-40% fetal DNA.

In one embodiment, blood may be drawn from a pregnant woman. Studies have shown that maternal blood can contain small amounts of free-floating DNA from the fetus in addition to free-floating DNA of maternal origin. In addition, there may be enucleated fetal blood cells containing DNA of fetal origin in addition to a plurality of blood cells of maternal origin which generally do not contain nuclear DNA. Various methods are known in the art to isolate fetal DNA or create fetal DNA-enriched fractions. For example, chromatography has been shown to create enriched fractions of fetal DNA.

Once a sample of maternal blood, plasma, or other bodily fluid is obtained that is drawn in a relatively non-invasive manner and contains a certain amount of fetal DNA, the DNA found in the sample can be genotyped, the fetal DNA or the cells Either free-floating, or enriched in proportion to maternal DNA or at its initial ratio. In some embodiments, blood may be drawn using a needle to draw blood from a vein, such as the basilica vein. The methods described herein can be used to determine fetal genotype data. For example, it can be used to determine the ploidy state at one or more chromosomes, it can be used to determine the identity of a SNP or a group of SNPs, including insertions, deletions and translocations. It can be used to determine the parental origin of one or more haplotypes, including one or more genotypic traits.

It should be noted that this method will work on any nucleic acid that can be used in any genotyping and/or sequencing method, such as the Illumina Infinium array platform, Affiliate Microarray, Illumina Genome Analyzer or Life Technologies Solid Systems. This includes free-floating DNA extracted from plasma or amplification thereof (eg whole genome amplification, PCR); genomic DNA or amplification thereof from other cell types (eg human lymphocytes from whole blood). With regard to DNA preparation, any extraction or purification method that produces genomic DNA suitable for one of these platforms will equally apply. This method can work equally well for RNA samples. In one embodiment, storage of the sample may be performed in a manner that will minimize degradation (eg, subzero, at about -20° C., or at lower temperatures).

Parental Support

Some embodiments may be used in combination with the PARENTAL SUPPORT ^™ (PS) method, examples of which are described in U.S. Application No. 11/603,406 (U.S. Publication No. 20070184467), U.S. Application No. 12/076,348 (U.S. Publication No. 20080243398), U.S. Application No. 13/110,685, PCT Application No. PCT/US09/52730 (PCT Publication No. WO/2010/017214), and PCT Application No. PCT/US10/050824 (PCT Publication No. WO/2011/041485) , said application is incorporated herein by reference in its entirety. PARENTAL SUPPORT ^TM is an information-based approach that can be used to analyze genetic data. In some embodiments, the methods disclosed herein may be considered part of the PARENTAL SUPPORT ^™ method. In some embodiments, the PARENTAL SUPPORT ^™ method can be used to determine with high accuracy a target individual, one or a small number of cells from said individual, or DNA from a target individual and from one or more of other individuals. A collection of genetic data for a constituent DNA mixture, specifically methods for determining disease-associated alleles, other associated alleles and/or the ploidy state of one or more chromosomes in an individual of interest. PARENTALSUPPORT ^(TM) may refer to any of these methods. PARENTAL SUPPORT ^™ is an example of an information-based approach. An exemplary embodiment of the PARENTAL SUPPORT ^™ method is shown in FIGS. 29-31G and described in Experiment 19.

The PARENTAL SUPPORT ^TM method utilizes known parental genetic data, i.e. haplotype and/or diploid genetic data of the mother and/or father, together with different knowledge of meiotic machinery and target DNA and possibly one or more related individuals. Refine measured knowledge, and population-based crossover frequencies, to reconstruct in silico with high confidence genotypes at multiple alleles and/or ploidy states of embryos containing any cell of interest and at key genes locus of target DNA at the location. The PARENTAL SUPPORT ^TM method can reconstruct not only poorly measured single nucleotide polymorphisms (SNPs), but also insertions and deletions as well as completely unmeasured SNPs or entire regions of DNA. In addition, the PARENTAL SUPPORT ^™ method can measure multiple disease-linked loci as well as screen single cells for aneuploidy. In some embodiments, the PARENTAL SUPPORT ^™ method can be used to characterize one or more cells from an embryo biopsy during an IVF cycle to determine the genetic condition of the one or more cells.

The PARENTAL SUPPORT ^TM method allows cleaning of noisy genetic data. This can be done by inferring the correct gene alleles in the target genome (embryo) using the genotypes of the related individuals (parents) as a reference. PARENTAL SUPPORT ^™ would be especially appropriate when only a small amount of genetic material is available (eg PGD) and when direct measurements of genotype are inherently noisy due to the limited amount of genetic material. PARENTAL SUPPORT ^™ would be especially appropriate when only a small portion of genetic material (eg NPD) is available from the individual of interest and when direct measurements of genotype are inherently noisy due to contaminated DNA signals from another individual. The PARENTAL SUPPORT ^TM method enables highly accurate reconstruction of ordered diploid allelic sequences and copy numbers of chromosomal segments on embryos, although routine disordered diploid measurements may be characterized by high rates of allelic loss, Insertions, variable amplification bias, and other errors. The method can employ a basic genetic model and a basic measurement error model. The genetic model can determine the allelic probability at each SNP and the probability of crossover between SNPs. Allele probabilities can be modeled at each SNP based on data obtained from the parents and crossover probabilities between SNPs can be modeled based on data obtained from the HapMap database as developed by the International HapMap Project. Given an appropriate underlying genetic model and measurement error model, maximum a posteriori probability (MAP) estimation can be used, modified for computational efficiency, to estimate the correct ordered allelic value at each SNP in an embryo.

In some cases, the techniques outlined above are capable of determining the genotype of an individual given very small amounts of DNA derived from the individual. This can be DNA from one or a small number of cells, or it can come from the small amount of fetal DNA found in the mother's blood.

suppose

In the context of the present invention, hypotheses refer to possible genetic states. It can refer to possible ploidy states. It can refer to probable allelic states. A set of hypotheses can refer to a set of possible genetic states, a set of possible allelic states, a set of possible ploidy states, or a combination thereof. In some embodiments, a set of hypotheses can be designed such that one hypothesis from the set will correspond to the actual genetic state of any given individual. In some embodiments, a set of hypotheses can be designed such that every possible genetic state can be described by at least one hypothesis from the set. In some embodiments of the invention, an aspect of the method is determining which hypothesis corresponds to the actual genetic state of the individual in question.

In another embodiment of the invention, one step involves creating a hypothesis. In some embodiments it may be a copy number hypothesis. In some embodiments, it may involve assumptions about which segments of chromosomes from each of the related individuals genetically correspond to which segments, if any, of other related individuals. Creating a hypothesis can refer to the act of setting the bounds on variables such that the entire set of possible gene states being studied is encompassed by those variables.

A "copy number hypothesis" also known as a "ploidy hypothesis" or "ploidy state hypothesis" can refer to a hypothesis about the likely ploidy state in a target individual for a given chromosome copy, chromosome type, or chromosome segment. It can also refer to the ploidy state at more than one chromosome type in an individual. A set of copy number hypotheses can refer to a set of hypotheses where each hypothesis corresponds to a different possible ploidy state in an individual. A set of hypotheses can relate to a set of possible ploidy states, a set of possible parental haplotype contributions, a set of possible fetal DNA percentages in a pooled sample, or a combination thereof.

A normal individual contains one copy of each chromosome from each parent. However, due to meiotic and mitotic errors, it is possible for an individual to have 0, 1, 2 or more of a given chromosome type from each parent. In practice, it is rare to see more than two specified chromosomes from one parent. In the present invention, some embodiments only consider possible hypotheses where 0, 1 or 2 copies of a given chromosome came from one parent; microextensions consider more or fewer possible copies from one parent. In some embodiments, there are nine possible hypotheses for a given chromosome: three possible hypotheses for 0, 1 or 2 chromosomes of maternal origin, multiplied by three possible hypotheses for 0, 1 or 2 chromosomes of paternal origin possible hypothesis. (m, f) refers to the hypothesis, where m is the number of a given chromosome inherited from the mother, and f is the number of a given chromosome inherited from the father. Therefore, the nine hypotheses are (0, 0), (0, 1), (0, 2), (1, 0), (1, 1), (1, 2), (2, 0), (2 , 1) and (2, 2). These can also be written as H ₀₀ , H ₀₁ , H ₀₂ , H ₁₀ , H ₁₂ , H ₂₀ , H ₂₁ and H ₂₂ . Different hypotheses correspond to different ploidy states. For example, (1,1) refers to a normal disomic chromosome; (2,1) refers to a maternal trisomy, and (0,1) refers to a paternal monosomy. In some embodiments, the situation where two chromosomes are inherited from one parent and one chromosome is inherited from the other parent can be further divided into two situations: one in which the two chromosomes are identical (match copy error), and one in which is where two chromosomes are homologous but not identical (mismatched copy error). In these examples, there are sixteen possible hypotheses. It should be appreciated that it is possible to use other sets of assumptions and a different number of assumptions.

In some embodiments of the invention, a ploidy hypothesis refers to a hypothesis as to which chromosomes from other related individuals correspond to the chromosomes found in the genome of the target individual. In some embodiments, key to the method is the fact that related individuals can be expected to share haplotype domains, and using measured genetic data from related individuals, and the knowledge of which haplodomains are between the target individual and the related individual With knowledge of the match, it is possible to infer the correct genetic data of the target individual with a higher degree of confidence than using only the genetic measurements of the target individual. Thus, in some embodiments, the ploidy hypothesis may relate not only to the number of chromosomes, but also to which chromosomes in the related individual are identical or nearly identical to one or more chromosomes in the target individual.

After the set of hypotheses have been defined, when the algorithms operate on the input genetic data, they can output a determined statistical probability for each of the hypotheses under study. The probabilities of the respective hypotheses can be determined by mathematically calculating values of equal probabilities for each of the respective hypotheses, using appropriate genetic data as input, as in techniques, algorithms, and/or methods described elsewhere herein. one or more of the above.

After the probabilities of different hypotheses have been estimated, as determined by various techniques, they can be combined. For each hypothesis, this may entail multiplying the probabilities determined by each technique. The product of hypothesis probabilities can be normalized. It should be noted that a ploidy hypothesis refers to one possible ploidy state of a chromosome.

"Combining probabilities" also known as "combining hypotheses" or the process of combining outcomes of expertise is a concept that should be familiar to those skilled in the field of linear algebra. One possible way to combine probabilities is as follows: When a specialized technique is used to estimate a set of hypotheses given a set of genetic data, the output of the method is a set of associated probabilities. When a set of probabilities determined by a first technique (each associated with one of the hypotheses in the set) is combined with a set of probabilities determined by a second technique (each associated with the same set of hypotheses), Multiply the two sets of probabilities together. This means that for each hypothesis in the set, the two probabilities associated with that hypothesis, as determined by two specialized methods, are multiplied together, and the corresponding product is the output probability. This process can be extended to any number of specialized techniques. If only one specialized technique is used, the output probabilities are the same as the input probabilities. If more than two specialized techniques are used, the associated probabilities can be multiplied simultaneously. The products can be normalized so that the probabilities of the hypotheses in the hypothesis group add up to 100%.

In some embodiments, a hypothesis determination may be considered most likely if the combined probability of a given hypothesis is greater than the combined probability of any of the other hypotheses. In some embodiments, if the normalized probability is greater than a threshold, then the hypothesis can be determined to be most likely and the ploidy state or other genetic state can be called. In one embodiment, this may mean that the number and identity of the chromosomes associated with the hypothesis may be referred to as the ploidy state. In one embodiment, this may mean that the identity of the allele associated with the hypothesis may be referred to as the allelic state. In some embodiments, the threshold may be between about 50% and about 80%. In some embodiments, the threshold may be between about 80% and about 90%. In some embodiments, the threshold may be between about 90% and about 95%. In some embodiments, the threshold may be between about 95% and about 99%. In some embodiments, the threshold may be between about 99% and about 99.9%. In some embodiments, the threshold may exceed about 99.9%.

parental background

Parental background refers to the genetic status of a given allele on each of the two related chromosomes in one or both parents of a target. It should be noted that in one embodiment, the parental context does not refer to the allelic state of the target, it actually refers to the allelic state of the parents. The parental context of a given SNP can consist of four base pairs (two paternal and two maternal); they can be the same or different from each other. It is usually written "m ₁ m ₂ | f ₁ f ₂ ", where m ₁ and m ₂ are the genetic status of the specified SNP on the two maternal chromosomes, and f ₁ and f ₂ are the specified SNPs on the two paternal chromosomes Genetic status of SNPs. In some embodiments, the parental context can be written as "f ₁ f ₂ |m ₁ m ₂ ". Note that the subscripts "1" and "2" refer to the genotypes at the indicated alleles of the first and second chromosomes; note also which chromosome is labeled "1" and which is labeled " The choice of 2" is arbitrary.

It should be noted that in the present invention, A and B are generally used to denote base pair identity generically; A or B equally well denote C (cytosine), G (guanine), A (adenine), or T (Thymine). For example, if at an allele based on a given SNP, the mother's genotype at that SNP on one chromosome is T, and the homologous chromosome is G at that SNP, and the father's two homologous The genotype at the allele at the SNP on the source chromosome is G, then it can be said that the allele of the target individual has the parental background AB|BB; it can also be said that the allele has the parental background AB|AA. It should be noted that, theoretically, any of the four possible nucleotides could occur at a given allele, and thus it is possible that at a given allele, for example, the mother's genotype is AT, and the father's gene Type is GC. However, empirical data show that in most cases only two of the four possible base pairs are observed at a given allele. It is possible, eg when using a single tandem repeat, to have more than two, more than four and even more than ten parental contexts. In the present invention, the discussion assumes that only two possible base pairs will be observed at a given allele, but the examples disclosed herein can be modified to account for cases where this assumption does not hold.

"Parental context" can refer to a group or subgroup of SNPs of interest that share the same parental context. For example, if 1000 alleles on a given chromosome of a target individual are to be measured, the background AA|BB can refer to the group of 1,000 alleles for which the genotype of the target's mother is homozygous, and the set of all alleles for which the genotype of the target's father is homozygous, but where the maternal and paternal genotypes are not similar at that locus. If the parental data are unphased, and thus AB=BA, then there are nine possible parental contexts: AA|AA, AA|AB, AA|BB, AB|AA, AB|AB, AB|BB, BB|AA, BB |AB and BB|BB. If the parental data are phased, and thus AB≠BA, then there are sixteen different possible parental contexts: AA|AA, AA|AB, AA|BA, AA|BB, AB|AA, AB|AB, AB| BA, AB|BB, BA|AA, BA|AB, BA|BA, BA|BB, BB|AA, BB|AB, BB|BA, and BB|BB. Every SNP allele on a chromosome (excluding some SNPs on the sex chromosomes) has one of these parental backgrounds. A set of SNPs in which the parental background of one parent is heterozygous may be referred to as a heterozygous background.

Use of Parental Background in NPD

Non-invasive prenatal diagnosis is an important technique that can be used to determine the genetic status of a fetus from non-invasively obtained genetic material, such as blood drawn on a pregnant mother. The blood can be separated and the plasma separated, followed by the plasma DNA. DNA of the appropriate length can be isolated using size selection. The DNA may be preferentially enriched at a set of loci. This DNA can then be measured by various means, such as by hybridization to a genotyping array and measuring fluorescence, or by sequencing on a high throughput sequencer.

In the context of non-invasive prenatal diagnosis, when sequencing is used for ploidy calling of fetuses, there are various ways to use the sequence data. Most commonly, sequence data can be used to simply count the number of reads that map to a given chromosome. For example, imagine that you are trying to determine the ploidy state of chromosome 21 of a fetus. It is further envisioned that the DNA in the sample consists of 10% DNA of fetal origin and 90% DNA of maternal origin. In this case, you can look at the average number of reads on a chromosome that might be expected to be disomic (eg chromosome 3) and compare the average number of reads to the number of reads on chromosome 21 for which Reads are adjusted on the number of base pairs that are part of the unique sequence. If the fetus is euploid, then one would expect the amount of DNA per unit of genome to be approximately equal at all positions (subject to random variation). On the other hand, if the fetus is trisomy on chromosome 21, then one would expect slightly more DNA per gene unit on chromosome 21 than elsewhere on the genome. Specifically, about 5 percent more DNA on chromosome 21 could be expected in the mixture. When using sequencing to measure DNA, one can expect approximately 5% more uniquely mappable reads per unique segment on chromosome 21 than on other chromosomes. An aneuploidy diagnosis can be based on the observation that an amount of DNA from a particular chromosome is above a certain threshold when adjusted for the number of sequences that can be uniquely mapped to that chromosome. Another method that can be used to detect aneuploidy is similar to the above method, except that parental background can be considered.

When considering which alleles to target, one can consider the likelihood that certain parental backgrounds may be more informative than others. For example, AA|BB and the symmetric background BB|AA are the most informative backgrounds because the fetus is known to carry different alleles than the mother. For symmetry reasons, the AA|BB and BB|AA backgrounds may be referred to as AA|BB. Another set of informative parental backgrounds are AA|AB and BB|AB because in these cases the fetus has a 50% chance of carrying an allele that the mother does not have. For symmetry reasons, the AA|AB and BB|AB backgrounds may be referred to as AA|AB. A third set of informative parental contexts is AB|AA and AB|BB, since in these cases the fetus carries a known paternal allele that is also present in the maternal genome. For symmetry reasons, the AB|AA and AB|BB backgrounds may be referred to as AB|AA. The fourth parental context is AB|AB, where the fetus has an unknown allelic state and it is the parental context in which the mother has the same allele regardless of the allelic state. A fifth parental background is AA|AA, where the mother and father are heterozygous.

Different implementations of the disclosed embodiments of the present invention

Disclosed herein are methods for determining the ploidy state of an individual of interest. Target individuals can be blastomeres, embryos or fetuses. In some embodiments of the present invention, the method for determining the ploidy state of one or more chromosomes in a target individual may include any one and a combination of the steps described in this document.

In some embodiments, the source of genetic material to be used in determining the genetic status of the fetus may be fetal cells isolated from maternal blood, such as nucleated fetal red blood cells. The method can involve obtaining a blood sample from a pregnant mother. The method may involve using visualization techniques to separate fetal red blood cells based on the idea that certain color combinations are uniquely associated with nucleated red blood cells and similar color combinations are not associated with any other cells present in maternal blood. The color combination associated with nucleated red blood cells may include the red color of the hemoglobin surrounding the nucleus, which may be made more pronounced by staining, and the color of the nuclear material may, for example, be stained blue. By isolating the cells from maternal blood and spreading them on a glass slide, and then identifying at which points red (from hemoglobin) and blue (from nuclear material) are seen, it may be possible to identify the location of the nucleated red blood cells. Those nucleated red blood cells can then be extracted using a micromanipulator, and genotyped aspects of the genetic material in those cells can be measured using genotyping and/or sequencing techniques.

In one embodiment, nucleated red blood cells can be stained with a mold that only fluoresces in the presence of fetal and not maternal hemoglobin, and thus removes uncertainty as to whether the nucleated red blood cells are of maternal or fetal origin. Some embodiments of the invention may involve staining or otherwise marking nuclear material. Some embodiments of the invention may specifically relate to labeling fetal nuclear material using fetal cell-specific antibodies.

There are various other ways of isolating fetal cells from maternal blood, or isolating fetal DNA from maternal blood, or enriching a sample of fetal genetic material in the presence of maternal genetic material. Some of these methods are listed here, but this is not intended to be an exhaustive list. For convenience, some appropriate techniques are listed here: use of fluorescently or otherwise labeled antibodies, size exclusion chromatography, magnetically or otherwise labeled affinity tags, epigenetic differences (e.g. in specific sex alleles, differential methylation between maternal and fetal cells), density gradient centrifugation followed by CD45/14 depletion and CD71 positive selection from CD45/14 negative cells, single or Double Percoll gradient or galactose-specific lectin method.

In one embodiment of the invention, the target individual is a fetus, and different genotype measurements are made on multiple DNA samples from the fetus. In some embodiments of the invention, the fetal DNA sample is from isolated fetal cells, wherein the fetal cells can be mixed with maternal cells. In some embodiments of the invention, the fetal DNA sample is from free-floating fetal DNA, wherein the fetal DNA can be mixed with free-floating maternal DNA. In some embodiments, the fetal DNA sample can be derived from maternal plasma or maternal blood containing a mixture of maternal DNA and fetal DNA. In some embodiments, fetal DNA may be mixed with maternal DNA at a maternal:fetal ratio ranging from 99.9%:0.1% to 99%:1%, 99%:1% to 90%:10% , 90%: 10% to 80%: 20%, 80%: 20% to 70%: 30%, 70%: 30% to 50%: 50%, 50%: 50% to 10%: 90% or 10 %: 90% to 1%: 99%, 1%: 99% to 0.1%: 99.9%.

Genetic data of a target individual and/or related individuals can be converted from a molecular state to an electronic state by measuring the appropriate genetic material using tools and/or techniques drawn from the group including (but not limited to): microarrays and high-throughput sequencing. Some high-throughput sequencing methods include Sanger DNA sequencing, pyrosequencing, Illumina Solexa (SOLEXA) platform, Illumina Genome Analyzer or Applied Biosystems (APPLIEDBIOSYSTEM) 454 sequencing platform, Helik HELICOS' true single-molecule sequencing platform, HALCYON MOLECULAR's electron microscope sequencing method, or any other sequencing method. All of these methods physically convert the genetic data stored in the DNA sample into a set of genetic data that is usually stored in a memory device en route to being processed.

Genetic data of related individuals can be measured by analyzing material taken from groups including (but not limited to): bulk diploid tissue from an individual, one or more diploid cells from an individual, One or more haploid cells from an individual, one or more blastomeres from an individual of interest, extracellular genetic material found on an individual, extracellular genetic material from an individual found in maternal blood, from Cells of an individual found in maternal blood, one or more embryos produced from gametes from a related individual, one or more blastomeres obtained from such embryos, extracellular genetic material found on a related individual, known Genetic material derived from related individuals and combinations thereof.

In some embodiments, a set of at least one ploidy state hypothesis may be created for each of the relevant chromosome types of the target individual. Each of the ploidy state hypotheses can refer to one possible ploidy state of a chromosome or chromosome segment of a target individual. The set of hypotheses can include some or all of the possible ploidy states that the target individual's chromosomes are expected to have. Some of the possible ploidy states may include absence, monosomy, disomy, uniparental disomy, euploidy, trisomy, matched trisomy, unmatched trisomy, maternal trisomy, Paternal trisomy, tetrasomy, balanced (2:2) tetrasomy, unbalanced (3:1) tetrasomy, pentasomy, hexasomy, other aneuploidy, and combinations thereof. Any of these aneuploidy states can be mixed or partial aneuploidy, such as unbalanced translocations, balanced translocations, Robertsonian translocations, recombinations, deletions, insertions, crossovers, and combinations thereof.

In some embodiments, knowledge of the determined ploidy state can be used to make clinical decisions. This understanding is typically stored in a memory device as a physical arrangement of content, which can then be converted into reports. Actions can then be taken based on the report. For example, the clinical decision may be to terminate the pregnancy; alternatively, the clinical decision may be to continue the pregnancy. In some embodiments, a clinical decision may involve an intervention designed to reduce the severity of the phenotypic expression of a genetic disorder, or a decision to take related steps to prepare a child with special needs.

In one embodiment of the invention, any of the methods described herein can be modified to allow multiple targets from the same target individual, eg, multiple blood draws from the same pregnant mother. This improves the accuracy of the model because multiple genetic measurements provide more data that can be used to determine the genotype of interest. In one embodiment, one set of target genetic data serves as the primary data reported and the other serves as data for review of the primary target genetic data. In one embodiment, multiple sets of genetic data, each measured from genetic material taken from a target individual, are considered to be parallel, and thus both sets of target genetic data are used to help determine which of the parental genetic data are measured with high accuracy. part of the fetal genome.

In one embodiment, the method can be used for paternity testing purposes. For example, given the SNP-based genotype information from the mother and from the man who may or may not be the genetic father, as well as genotype information measured from a pooled sample, it is possible to determine whether the man's genotype information is indeed representative of gestation The true genetic father of the fetus in . A simple way to do this is to simply look at the context where the mother is AA and possibly the father is AB or BB. In these cases, expect to see the father contributing half (AA|AB) or all (AA|BB) at this point, respectively. Taking into account predicted ADO, it is straightforward to determine whether observed fetal SNPs are related to those of the likely father.

An embodiment of the invention may be as follows: a pregnant woman wants to know whether her fetus has Down syndrome and/or whether it will develop cystic fibrosis, and she does not wish to give birth to a baby with any of these conditions child. Doctors take her blood and stain the hemoglobin with one marker so that it is clearly red, and the nuclear material with another marker so that it is clearly blue. Knowing that maternal red blood cells are usually non-nucleated, while a high proportion of fetal cells contain nuclei, doctors can visually separate multiple nucleated red blood cells by identifying those cells that show both red and blue colors. Doctors use a micromanipulator to pick up the cells from the slide and send them to a lab where ten individual cells are expanded and genotyped. By using genetic measurements, the PARENTALSUPPORT ^™ method was able to determine that six out of ten cells were maternal blood cells and four out of ten cells were fetal cells. If the pregnant mother has given birth to a child, PARENTAL SUPPORT ^™ can also be used to determine that fetal cells differ from those of the already born child by making reliable allelic calls of the fetal cells and showing that they are not similar to those of the already born child. It should be noted that this approach is conceptually similar to the parental test embodiment of the present invention. The quality of genetic data measured from fetal cells can be very poor due to the difficulty of genotyping single cells, containing multiple allelic losses. Using PARENTAL SUPPORT ^TM , clinicians are able to infer with high accuracy aspects of the fetal genome using measured fetal DNA as well as reliable DNA measurements of the parents, thereby converting the genetic data contained on the genetic material from the fetus into stored in Predicted genetic status of the fetus on a computer. Clinicians are able to determine the ploidy status of the fetus and the presence or absence of multiple associated disease-linked genes. It turned out that the fetus was euploid and not a carrier of cystic fibrosis, and the mother decided to continue the pregnancy.

In one embodiment of the invention, a pregnant mother wishes to determine whether her fetus has any global chromosomal abnormalities. She went to her doctor and gave a sample of her blood, and she and her husband gave samples of their own DNA from cheek swabs. Laboratory researchers amplified the parental DNA using the MDA protocol and genotyped the parental DNA using the Illumina Infinium array to measure the parental genetic data at a large number of SNPs. The researchers then spun down the blood, removed the plasma, and used size-exclusion chromatography to separate the free-floating DNA samples. Alternatively, researchers use one or more fluorescent antibodies, such as antibodies specific for fetal hemoglobin, to isolate nucleated fetal red blood cells. Researchers then take the isolated or enriched fetal genetic material and use 70-mer oligonucleotides that are appropriately designed so that the two ends of each oligonucleotide correspond to the flanking sequences flanking the allele of interest The acid library amplifies it. After the addition of polymerase, ligase, and appropriate reagents, the oligonucleotides undergo gap-filling circularization, capturing the desired allele. Exonuclease is added, heat inactivated, and the product used directly as template for PCR amplification. PCR products were sequenced on an Illumina Genome Analyzer. The sequence reads are used as input to the PARENTALSUPPORT ^™ method, which then predicts the ploidy state of the fetus.

In another embodiment, a couple in which the mother is pregnant and is of advanced maternal age would like to know whether the gestating fetus has Down syndrome, Turner syndrome, Prader-Willi syndrome, or some other full chromosome abnormal. The obstetrician draws blood from the mother and father. The blood is sent to a laboratory where a technician centrifuges the maternal sample to separate the plasma and buffy coat. The DNA in the buffy coat and paternal blood samples is transformed by amplification and the genetic data encoded in the amplified genetic material is converted from molecularly stored DNA by running the genetic material on a high-throughput sequencer to measure the parental genotype The genetic data is further converted into genetic data stored electronically. Plasma samples were preferentially enriched at a panel of loci using a 5,000-plex hemi-nested targeted PCR approach. The DNA fragment mixture is made into a DNA library suitable for sequencing. The DNA is then sequenced using a high-throughput sequencing method, such as the Illumina GAIIx Genome Analyzer. The sequencing converts information molecularly encoded in DNA into information electronically encoded in computer hardware. Information-based techniques, including embodiments disclosed herein, such as PARENTAL SUPPORT ^(TM) , can be used to determine the ploidy status of a fetus. This may involve calculating in silico allele count probabilities at multiple polymorphic loci from DNA measurements taken on the prepared sample; assumptions; for each ploidy hypothesis, a joint distribution model is constructed in silico for the expected allele counts at multiple polymorphic loci on the chromosome; using the joint distribution model and the allele counts measured on the prepared samples, determining on a computer the relative probability of each of the ploidy hypotheses; and deciphering the ploidy state of the fetus by selecting the ploidy state corresponding to the hypothesis with the greatest probability. It was determined that the fetus had Down syndrome. The report is printed or sent electronically to the pregnant woman's obstetrician, who transmits the diagnosis to the woman. The woman, her husband, and the doctor sat down to discuss their options. Based on knowledge that the fetus had a trisomy, the couple decided to terminate the pregnancy.

In one example, a company may decide to offer a diagnostic technology designed to detect aneuploidy in a gestating fetus from drawn maternal blood. Their product could involve a mother showing up to her obstetrician who can draw her blood. The obstetrician may also collect a genetic sample from the father of the fetus. Clinicians can separate plasma from maternal blood and purify DNA from plasma. Clinicians can also isolate the buffy coat from maternal blood and prepare DNA from the buffy coat. Clinicians can also prepare DNA from paternal genetic samples. Clinicians can use the molecular biology techniques described in this invention to attach universal amplification markers to DNA in DNA derived from plasma samples. Clinicians can amplify universally labeled DNA. Clinicians can preferentially enrich DNA through a variety of techniques, including capture by hybridization and targeted PCR. Targeted PCR may involve nesting, hemi-nesting or hemi-nesting or any other method for causing efficient enrichment of plasma-derived DNA. Targeted PCR can be multiplexed on a large scale, e.g. 10,000 primers targeting 13, 18, 21, chromosome X and those loci common to both X and Y as well as any Optionally target SNPs on other chromosomes. Selective enrichment and/or amplification may involve labeling each individual molecule with a different marker, molecular barcode, marker for amplification, and/or marker for sequencing. The clinician can then sequence the plasma sample, and possibly also the prepared maternal and/or paternal DNA. The molecular biological steps can be carried out completely or partially by the diagnostic cartridge. Sequence data can be fed into a single computer or another type of computing platform such as can be found in the "cloud". The computing platform can calculate allele counts at targeted polymorphic loci from measurements made by the sequencer. Computing platform can create multiple ploidy hypotheses for absence, monosomy, disomy, matched trisomies, and unmatched trisomies for each of chromosomes 13, 18, 21, X, and Y . The computing platform can build a joint distribution model for predicted allele counts at the target locus on the chromosome for each ploidy hypothesis for each of the five chromosomes to be queried. The computing platform can determine the probability that each of the ploidy hypotheses are correct using the joint distribution model and the allele counts measured for the preferentially enriched DNA derived from the plasma sample. The computing platform can interpret the ploidy state of the fetus by selecting the ploidy state corresponding to the closely related hypothesis with the highest probability for each of chromosomes 13, 18, 21, X and Y. A report containing the called ploidy status can be generated and can be sent electronically to the obstetrician, displayed on an output device, or a printed hard copy of the report can be submitted to the obstetrician. The obstetrician can inform the patient and optionally the father of the fetus, and they can decide which clinical options they are willing to accept and which are most desirable.

In another embodiment, a pregnant woman (hereinafter "mother") may decide that she wants to know whether her fetus carries any genetic abnormalities or other conditions. She may want to make sure there are no gross abnormalities before she is sure to continue the pregnancy. She can go to her obstetrician, and her obstetrician can get a sample of her blood. He can also obtain genetic samples, such as a cheek smear from her cheek. He may also obtain a genetic sample, such as a cheek smear, sperm sample, or blood sample, from the fetus' father. He can send the sample to the clinician. Clinicians can enrich the fraction of free-floating fetal DNA in maternal blood samples. Clinicians can enrich the fraction of enucleated fetal blood cells in a maternal blood sample. A clinician can use aspects of the methods described herein to determine genetic data for a fetus. The genetic data may include the ploidy state of the fetus and/or the identity of one or more disease-linked alleles in the fetus. A report can be produced summarizing the results of the prenatal diagnosis. The report can be transmitted or mailed to a doctor who can inform the mother of the genetic status of the fetus. The mother may decide to interrupt the pregnancy based on the fact that the fetus has one or more chromosomal or genetic abnormalities or undesired conditions. She may also decide to continue the pregnancy based on the fact that the fetus does not have any gross chromosomal or genetic abnormalities or any related genetic conditions.

Another example could involve a pregnant woman who has been artificially inseminated by a sperm donor and has become pregnant. She wants to minimize the risk of genetic disease in the fetus she is carrying. She has drawn blood at a phlebotomist and used the techniques described in this invention to isolate three nucleated fetal red blood cells and also collected tissue samples from the mother and the genetic father. Genetic material from the fetus and from the mother and father was amplified as appropriate and genotyped using the Illumina Infinium Bead Array, and the methods described herein segregate parental and fetal genes with high accuracy. Type cleaning and phasing, and fetal ploidy calls. The fetus is found to be euploid, and phenotypic sensitivity is predicted from the reconstructed fetal genotype, and a report is produced and sent to the mother's physician so they can decide what clinical decision might be best.

In one embodiment, the original genetic material of the mother and father is converted by means of amplification into a quantity of DNA that is similar in sequence but greater in quantity. Then, by means of the genotyping method, the genotype data encoded by the nucleic acid is converted into genetic measurements, such as those described above, that can be stored physically and/or electronically on a memory device. Using a programming language, the relevant algorithms making up the PARENTAL SUPPORT ^™ algorithm (the relevant parts of which are discussed in detail herein) are translated into computer programs. Then, by executing a computer program on computer hardware, rather than physically encoding bits and bytes, arranged in patterns representing raw measurement data, they are converted into patterns representing high-confidence determinations of fetal ploidy status. The details of such conversion will depend on the data itself and the computer language and hardware system used to perform the methods described herein. The data physically configured to represent the high quality ploidy determination of the fetus is then converted into a report that can be sent to a healthcare practitioner. This conversion can be performed using a printer or computer monitor. The report may be a printed copy on paper or other suitable medium, or it may be electronic. In the case of an electronic report, it can be transmitted, it can be physically stored on a memory device in a location accessible by the healthcare practitioner's computer; it can also be presented on a screen so that it can be read. In the case of a screen display, the data can be converted into a readable format by causing a physical transformation of pixels on the display device. Switching can be achieved by physically emitting electrons to the phosphor screen, by altering the electrical charge, by physically changing the transparency of a specific set of pixels on the screen that may be located in front of the substrate that emits or absorbs the photons. This switch can be achieved by changing the nanoscale orientation of the molecules in the liquid crystal, for example from a nematic phase to a cholesteric or smectic phase at a specific set of pixels. This switching can be achieved with the aid of an electrical current that causes photons to be emitted from a specific set of pixels made from a number of light-emitting diodes arranged in a meaningful pattern. This conversion may be accomplished by any other means for displaying information, such as a computer screen or some other output device or means of transmission of information. A healthcare practitioner can then act on the report such that the data in the report is translated into action. The action may be continuation or interruption of pregnancy, in which case a gestating fetus with a genetic abnormality is converted into a non-living fetus. The transformations outlined herein can be aggregated such that, for example, the genetic material of the pregnant mother and father can be transformed through the multiple steps outlined in this invention into a medical decision to consist of abortion of a genetically abnormal fetus or to continue a pregnancy. Alternatively, a set of genotype measurements can be converted into a report that helps a physician treat his pregnant patient.

In one embodiment of the invention, the methods described herein can be used to determine the ploidy state of a fetus even when the host mother (ie, pregnant woman) is not the biological mother of the fetus she is carrying. In one embodiment of the invention, the methods described herein can be used to determine the ploidy state of a fetus using only a maternal blood sample and without the need for a paternal genetic sample.

Several mathematical methods in the presently disclosed embodiments are used to establish hypotheses about a finite number of aneuploidy states. In some cases, it is expected that, for example, only zero, one or two chromosomes are derived from each parent. In some embodiments of the invention, the mathematical derivation can be extended to account for other forms of aneuploidy, such as tetrasomy (where three chromosomes are derived from one parent), pentasomy, hexasomy, etc., without changing the invention basic concept. At the same time, it is possible to focus on a smaller number of ploidy states, such as only trisomies and disomies. It should be noted that a ploidy determination result indicating a non-integer number of chromosomes may indicate mosaicism in a sample of genetic material.

In some embodiments, the genetic abnormality is a type of aneuploidy, such as Down syndrome (or trisomy 21), Edwards syndrome (trisomy 18), Patau's syndrome Patau syndrome (13-trisomy), Turner syndrome (45X), Klinefelter syndrome (male with 2 X chromosomes), Prader-Willi syndrome, and DiGeorge syndrome (DiGeorge syndrome) syndrome) (UPD 15). Congenital anomalies, such as those listed in the previous sentence, are usually undesired, and knowledge that the fetus has one or more phenotypic abnormalities can inform the decision to: terminate the pregnancy, take necessary precautions as special needs Preparing for the birth of a child in your child or taking some treatments to lessen the severity of the chromosomal abnormality.

In some embodiments, the methods described herein can be used at very early gestational ages, such as as early as four weeks, as early as five weeks, as early as six weeks, as early as seven weeks, as early as eight weeks, as early as nine weeks , as early as ten weeks, as early as eleven weeks and as early as twelve weeks.

In some embodiments, the methods disclosed herein are used for embryo selection during in vitro fertilization in the context of preimplantation genetic diagnosis (PGD), where the target individual is an embryo, and parental genotype data can be used to derive from Ploidy determinations on embryos were made from sequencing data from single or two cell biopsies of day 3 embryos or from day 5 or 6 embryo trophoblast biopsies. In the case of PGD, only the child's DNA is measured, and only a small number of cells are tested, typically one to five, but as many as ten, twenty or fifty. The total number of starting copies of the A and B alleles (at the SNP) is then simply determined from the child genotype and cell number. In NPD, the starting copy number is extremely high and therefore the allele ratio after PCR is expected to accurately reflect the starting ratio. However, the low number of starting copies in PGD means that contamination and imperfect PCR efficiency have a significant impact on the allele ratio after PCR. This effect may be more important than read depth in predicting bias in allele ratios measured after sequencing. A distribution of allele ratios measured for known child genotypes can be created by Monte Carlosimulation of the PCR process based on PCR probe efficiencies and contamination probabilities. Given the allele ratio distribution for each possible child genotype, the likelihood of each hypothesis can be calculated as described for NIPD.

maximum likelihood estimation

Most methods known in the art for detecting the presence or absence of a biological phenomenon or medical condition involve the use of a single hypothesis rejection test in which a metric related to the condition is measured and if the metric is on one side of a specified threshold, then the presence or absence condition; and if the metric falls to the other side of the threshold, the condition does not exist. A single hypothesis rejection test looks only at the null distribution when deciding between the null hypothesis and the alternative hypothesis. Without taking into account the alternative distributions, the likelihood of each hypothesis cannot be estimated given the observed data and thus the confidence in the call cannot be calculated. Thus, in the case of a single-hypothesis rejection test, a yes or no answer is obtained without knowing the degree of confidence associated with a particular situation.

In some embodiments, the methods disclosed herein are capable of detecting the presence or absence of a biological phenomenon or medical condition using maximum likelihood methods. This substantially improves methods using single hypothesis rejection techniques, as the thresholds for calls of absence or presence of the condition can be adjusted for each case as appropriate. This is useful for diagnostics aimed at determining the presence or absence of aneuploidy in a gestating fetus from genetic data obtainable from a mixture of fetal and maternal DNA present in free-floating DNA found in maternal plasma Technology is especially appropriate. This is because the optimal threshold for calling aneuploidy versus euploidy changes as the fraction of fetal DNA in the plasma-derived fraction changes. The distribution of data associated with aneuploidy became more and more similar to that of data associated with euploidy as fetal fraction decreased.

Maximum likelihood estimation uses the distributions associated with each hypothesis to estimate the likelihood of the data conditioned on each hypothesis. These conditional probabilities can then be translated into hypothesis calls and confidence levels. Similarly, maximum a posteriori probability estimation uses the same conditional probabilities as maximum likelihood estimation, and incorporates a population prior when selecting the best hypothesis and determining confidence.

Thus, the use of the Maximum Likelihood Estimation (MLE) technique or the closely related Maximum A Posteriori Probability (MAP) technique yields two advantages, first it increases the chance of a correct interpretation, and it also allows the calculation of a confidence level for each interpretation . In one embodiment, selecting the ploidy state corresponding to the hypothesis with the greatest probability is performed using maximum likelihood estimation or maximum a posteriori probability estimation. In one embodiment, a method for determining the ploidy state of a gestating fetus is disclosed that involves taking any method currently known in the art that uses a single hypothesis rejection technique and reformulating it as makes it use MLE or MAP techniques. Some examples of methods that can be significantly improved by applying these techniques can be found in US Patent 8,008,018, US Patent 7,888,017 or US Patent 7,332,277.

In one embodiment, a method for determining the presence or absence of fetal aneuploidy in a maternal plasma sample comprising fetal and maternal genomic DNA is described, the method comprising: obtaining a maternal plasma sample; measuring DNA fragments found in a plasma sample with a high-throughput sequencer; mapping the sequences to chromosomes and determining the number of sequence reads mapped to each chromosome; calculating the fraction of fetal DNA in the plasma sample; using the fetal fraction and a number of sequence reads that map to one or more reference chromosomes predicted to be euploid, calculating a predicted distribution of the amount of target chromosomes that would be predicted to be present if the second target chromosome were euploid; and if said If the chromosome is aneuploid, one or more predicted distributions are predicted; and MLE or MAP is used to determine which of said distributions is most likely to be correct, thereby indicating the presence or absence of fetal aneuploidy. In one embodiment, measuring DNA from plasma may involve performing massively parallel shotgun sequencing. In one embodiment, measuring DNA from a plasma sample may involve sequencing DNA that has been preferentially enriched at multiple polymorphic or non-polymorphic loci, eg, by targeted amplification. Multiple loci can be designed to target one or a small number of suspected aneuploid chromosomes and one or a small number of reference chromosomes. The purpose of preferential enrichment is to increase the number of sequence reads that inform ploidy determination.

ploidy reading information method

Described herein is a method for determining the ploidy status of a fetus in view of sequence data. In some embodiments, this sequence data can be measured on a high throughput sequencer. In some embodiments, sequence data may be measured on DNA derived from free-floating DNA isolated from maternal blood, wherein the free-floating DNA comprises some maternally derived DNA and some fetal/placental derived DNA. This section will describe an embodiment of the invention in which the ploidy state of the fetus is determined under the assumption that the fraction of fetal DNA in the mixture that has been analyzed is not known and will be estimated from the data. It will also describe an embodiment wherein the fraction of fetal DNA in the mixture ("fetal fraction") or percentage of fetal DNA can be measured by another method and assumed to be known when determining the ploidy status of the fetus. In some embodiments, the fetal fraction can be calculated using only genotyping measurements taken on the maternal blood sample itself, which is a mixture of fetal and maternal DNA. In some embodiments, the score may also be calculated using the measured or otherwise known maternal genotype and/or the measured or otherwise known paternal genotype. In another embodiment, the ploidy state of the fetus may be determined solely based on the fraction of fetal DNA calculated for the chromosome in question compared to the fraction of fetal DNA calculated for a presumed disomy reference chromosome.

In a preferred embodiment, given that we observe and analyze N SNPs on a particular chromosome, for which we:

• Set NR free-floating DNA sequence measurements S = (s ₁ , . . . , s _NR ). Because this method utilizes SNP measurements, all sequence data corresponding to non-polymorphic loci can be ignored. In a simplified version, where we have (A, B) counts at each SNP, where A and B correspond to the two alleles present at a given locus, S can be written as S=((a ₁ , b ₁ ),..., (a _N , b _N )), where a _i is the A count on SNP i, b _i is the B count on SNP i, and

● Parental data consisting of

○ Genotypes from SNP microarrays or other intensity-based genotyping platforms: mother M=(m ₁ , . . . , m _N ), father F=(f ₁ , . . . , f _N ), where m _i , f _i ∈ (AA, AB, BB).

○ and/or sequence data measurements: NRM mother measurements SM = (sm ₁ , . . . , sm _nrm ), NRF father measurements SF = (sf ₁ , . . . , sf _nrf ). Similar to the above simplification, if we have (A, B) counts at each SNP, then SM = ((am ₁ , bm ₁ ), . . . , (am _N , bm _N )), SF = (( af ₁ , bf ₁ ), . . . , (af _N , bf _N )).

In general, mother, father and child data are expressed as D=(M, F, SM, SF, S). It should be noted that parental data is desirable and improves the accuracy of the algorithm, but not required, especially paternal data. This means that very accurate copy number results are still possible even in the absence of maternal and/or paternal data.

It is possible to derive an optimal copy number estimate by maximizing the log-likelihood data for all hypotheses (H) considered. Specifically, it is possible to determine the relative probability of each of the ploidy hypotheses using the joint distribution model and allele counts measured on the prepared samples, and use those relative probabilities to determine the hypothesis most likely to be correct as follows:

Similarly, given the data, the posterior hypothesis likelihood can be written as:

where the prior probability (H) is the prior probability assigned to each hypothesis H based on the model design and prior knowledge.

It is also possible to find the maximum a posteriori probability estimate using the prior:

In one embodiment, copy number hypotheses that may be considered are:

●Singleness:

○Maternal H10 (one copy from mother)

○Sire parent H01 (one copy from father)

●Disomy: H11 (one copy each for mother and father)

●Simple trisomy without crossover:

○Maternal: H21_match (two identical copies from mother, one copy from father), H21_mismatch (two copies from mother, one copy from father)

○Paternal: H12_match (one copy from mother, two identical copies from father), H12_mismatch (one copy from mother, two copies from father)

Compound trisomy, allowing crossover (using joint distribution model):

○ female parent H21 (two copies from mother, one from father),

○ Male parent H12 (one copy from mother, two copies from father)

In other embodiments, other ploidy states may be considered, such as absence (H00), uniparental disomy (H20 and H02), and tetrasomy (H04, H13, H22, H31 and H40).

If there were no crossover, then every trisomy, whether of mitotic, meiotic I, or meiotic II origin, would be either a matched or mismatched trisomy. A true trisomy is usually a combination of two due to crossover. First, a method for deriving hypothesis likelihoods about simple hypotheses is described. Then, a method for deriving hypothesis likelihoods about composite hypotheses is described, combining individual SNP likelihoods with crossovers.

LIK(D|H) on Simple Hypotheses

In one embodiment, for simple assumptions, LIK(D|H) can be determined as follows. For a simple hypothesis H, the log-likelihood LIK(H) of hypothesis H over the entire chromosome can be calculated as the sum of the log-likelihoods of the individual SNPs, assuming the known or derived child fraction is cf. In one embodiment, it is possible to derive cf from the data.

This assumption does not assume any linkage between SNPs, and thus does not exploit joint distribution models.

In some embodiments, log-likelihoods can be determined on a per-SNP basis. On a particular SNP i, assuming fetal ploidy hypothesis H and percent fetal DNA cf, the log-likelihood of the observed data D is defined as:

where m is the likely true maternal genotype and f is the likely true paternal genotype given assumption H, where m, f{AA,AB,BB}, and c are the likely child genotypes. Specifically, about monosomy, c; about disomy, c; about trisomy, c.

Genotype prior frequency: p(m|i) is the general prior probability of maternal genotype m at SNP i based on the known population frequency denoted pA _i at SNP i. Specifically:

;;

The paternal genotype probability p(f|i) can be determined in a similar manner.

True child probability: is the probability of obtaining true child genotype = c, given the parents m, f, and assuming assumptions H (these can be easily calculated). For example, p(c|m, f, H) is given below for H11, H21 match and H21 mismatch.

Data Likelihood: is the probability of assigning data D on SNP i given the true mother genotype m, true father genotype f, true child genotype c, hypothesis H, and child fraction cf. It can be broken down into probabilities for mother, father and child data as follows:

Maternal SNP array data likelihood: Assuming that the SNP array genotype is correct, at SNP i, the probability of maternal SNP array genotype data compared to the true genotype m is simply

Maternal sequence data likelihood: Given counts S _i = (am _i , bmi ₎ , the probability of maternal sequence data at SNP i is the binomial probability, defined by is P(SM|m, i)=P _X|m (am _i ), where X|m～Binom(p _m (A), am _i +bm _i ), which is defined as

m AAA AB BB A B No interpretation p(A) 1 0.5 0 1 0 0.5

Paternal Data Likelihood: A similar equation applies to paternal data likelihood.

It should be noted that it is possible to determine the genotype of a child without parental data, especially paternal data. For example, paternal genotype data F may only be used if not available. If the parent sequence data SF is not available, then P(SF|f,i)=1 can only be used.

In some embodiments, the method involves constructing a joint distribution model for predicted allele counts at multiple polymorphic loci on a chromosome for each ploidy hypothesis; a method for doing so is described herein. method. Free fetal DNA data likelihood: is the probability of free fetal DNA sequence data at SNP i given the true maternal genotype m, true child genotype c, child copy number hypothesis H, and assumed child fraction cf. It is actually the probability of sequence data S at SNP I given the true probability of A content at SNP i

Regarding counts, where S _i = (a _i , _bi ), no additional data noise or bias is involved,

where X~Binom(p(A), a _i + _bi ), where p(A)=. In more complex cases, where the exact alignment and (A,B) counts for each SNP are unknown, is a combination of synthetic binomials.

True Probability of A Content: , assuming true maternal genotype = m, true child genotype = c, and overall child fraction = cf, the true probability of A content at SNP i in this mother/child mixture is defined for

where #A(g) = number of A's in genotype g, is the chromosomal sex of the mother and is the ploidy of the child under assumption H (monosomy is 1, disomy is 2, trisomy is 3).

Using Joint Distribution Models: LIK(D|H) for Composite Hypotheses

In some embodiments, the method involves constructing a joint distribution model for predicted allele counts at multiple polymorphic loci on a chromosome for each ploidy hypothesis; a method for doing so is described herein. method. In many cases, trisomy is often not just a match or mismatch due to crossover, so in this section, the results of the composite hypotheses H21 (maternal trisomy) and H12 (paternal trisomy) are derived, so The above hypotheses combine matching and mismatching trisomies, explaining the possible crossover.

In the case of a trisomy, if there is no crossover, then the trisomy will simply be a matching or mismatching trisomy. Matched trisomy is a condition in which a child inherits two copies of an identical chromosomal segment from one parent. Unmatched trisomy is a condition in which a child inherits one copy of each homologous chromosome segment from a parent. Due to crossing over, some segments of a chromosome may have a matching trisomy and other parts may have an unmatched trisomy. This section describes how to construct a joint distribution model for heterozygosity rates for a set of alleles; that is, for one or more assumptions, to construct a joint distribution model for predicted allele counts at multiple loci.

Assume that on SNP _i , is the fit of the matching hypothesis H _m and is the fit of the mismatching hypothesis Hu, and pc(i)=crossover probability between SNP i−1 and i. Then the full likelihood can be calculated as follows:

where is the likelihood for SNP 1:N with hypothesis E as the outcome. E = Hypothesis for the last SNP, E. Recursively, one can compute:

where ~E is a hypothesis other than E (not E), where the hypotheses considered are H _m and _Hu . Specifically, the likelihood of the 1:i SNP can be calculated based on the likelihood of the 1:(i-1) SNP under the same assumption and no crossover or the opposite assumption and crossover, multiplied by the likelihood of SNP i sex

For SNP 1, i = 1, .

For SNP 2, i = 2,

And about i=3:N etc.

In some embodiments, child scores may be determined. A child fraction can refer to the proportion of sequences in a DNA mixture that are derived from a child. In the context of non-invasive prenatal diagnosis, the child fraction can refer to the proportion of sequence in maternal plasma that is derived from the fetus or contains the placental portion of the fetal genotype. It can refer to the child's fraction in a DNA sample that has been prepared from maternal plasma and can be enriched for fetal DNA. One purpose of determining the child's fraction in a DNA sample applies to algorithms that can make ploidy calls to fetuses, thus, child's fraction can refer to any DNA sample that is sequenced for non-invasive prenatal diagnosis.

Some of the algorithms proposed in this invention as part of the non-invasive prenatal aneuploidy diagnosis method assume that the child's score is known, which may not always be the case. In one embodiment, it is possible to find the most probable child score by maximizing the likelihood of disomy on selected chromosomes, in the presence or absence of parental data

Specifically, regarding the disomy hypothesis and regarding the child fraction cf on chromosome chr, given LIK(D|H11, cf, chr) = log-likelihood as above, regarding the selected chromosome in Cset (usually is 1:16), assuming euploidy, the full likelihood is:

The most probable child score (derived as .

It is possible to use either set of chromosomes. It is also possible to derive child scores without assuming euploidy on the reference chromosome. Using this approach, it is possible to determine child scores for either: (1) with array data on the parents and shotgun sequencing data on the maternal plasma; (2) with array data on the parents and the case of targeted sequencing data on maternal plasma; (3) the case with targeted sequencing data on parental and maternal plasma; (4) the case with targeted sequencing data on maternal and maternal plasma fractions; (5) Cases with targeted sequencing data on maternal plasma fractions; (6) Other combinations of parental and child fractional measurements.

In some embodiments, the informatics method can incorporate data loss; this can result in a ploidy determination with greater accuracy. Elsewhere in the present invention, the probability of obtaining an A has been assumed to be a direct function of the true mother's genotype, the true child's genotype, the fraction of children in the pool, and the child's copy number. It is also possible to miss the mother or child allele, for example instead of measuring the true child AB in the mixture, it might be the case that only the sequence mapped to allele A is measured. The parental loss rate for genomic illumina data can be denoted as d _pg , the parental loss rate for sequence data as d _ps and the child loss rate for sequence data as d _cs . In some embodiments, the mother loss rate may be assumed to be zero, and the child loss rate relatively low; in this case, the results would not be significantly affected by loss. In some embodiments, the probabilities of allelic loss can be so great that they have a significant impact on predicting ploidy calls. For this case, allelic dropout is incorporated into the algorithm here:

The parental SNP array data is missing: Regarding the maternal genome data M, if the genotype after the loss is m _d , then

where, as before; and is the likelihood of genotype m _d after a possible loss, given the true genotype m, against the dropout rate d, defined as

Similar equations apply to paternal SNP array data.

Loss of parental sequence data: About maternal sequence data SM

where as defined in the previous section and probabilities from the binomial distribution as previously defined in the Parental Data Likelihood section. Similar equations apply to paternal sequence data.

Free-floating DNA sequence data is missing:

where as defined in the free-floating data possibilities section.

In one embodiment, given the true maternal genotype, the assumed dropout rate d _ps , is the observed probability of the maternal genotype; and given the true child genotype, the assumed dropout rate d _cs , is the observed probability of the child Probability of genotype. If _nAT = number of A alleles in true genotype c, nA _D = number of A alleles in observed genotypes, where _nAT ≥ _nAD , and similarly nB _T = true genotype c The number of B alleles in , nB _D = the number of B alleles in the observed genotype, where nB _T ≥ nB _D and d = dropout rate, then

In one embodiment, the information method can incorporate random and uniform bias. Ideally, there would be no one SNP consistent sampling bias or random noise (other than binomial distribution variation) in the number of sequence counts. Specifically, on SNPi, with respect to mother genotype m, true child genotype c, and child fraction cf, and X = number of A in the set of (A+B) reads on SNP i, X works as if X∼ Binomial(p,A+B), where p==true probability of A content.

In one embodiment, the information method may incorporate random bias. It is often the case that, given that the measurements are biased, the probability of getting an A at this SNP is therefore equal to q, which is slightly different from p as defined above. How different p is from q depends on the accuracy of the measurement process and a number of other factors and can be quantified by the standard deviation of q from p. In one embodiment, it is possible to model q as having a beta distribution, with parameters depending on the mean of said distribution centered at p, and some specified standard deviation s. Specifically, this gives, where . If we let , then the parameters can be derived as, where.

This is the definition of the beta-binomial distribution, where sampling is done from a binomial distribution with variable parameter q, where q follows a beta distribution with mean p. Thus, without bias, at SNP i, assuming the true maternal genotype (m), given maternal sequence A counts at SNP i (am _i ) and maternal sequence B counts at SNP i (bm _i ), the parental sequence data (SM) probability can be calculated as follows:

P(SM|m, i)＝P _X|m (am _i ), where X|m～Binom(p _m (A), am _i +bm _i )

Now, including the random deviation and standard deviation s, this becomes:

X|m～BetaBinom(p _m (A), am _i +bm _i , s)

With no bias, given the true maternal genotype (m), true child genotype (c), child fraction (cf), assumed child hypothesis H, given a free-floating DNA sequence A at SNP i Counts (a _i ) and free-floating sequence B counts (bi ) on SNP _i , the maternal plasma DNA sequence data probabilities can be calculated as follows:

where X~Binom(p(A), a _i + _bi ), where p(A)=.

In one embodiment, including random deviation and standard deviation s, this becomes X ~ BetaBinom(p(A), a _i + _bi , s), where the amount of additional variation is specified by the bias parameter s or equivalently in N. The smaller the value of s (or the larger the value of N), the closer this distribution is to the regular binomial distribution. It is possible to estimate the amount of bias from the explicit background AA|AA, BB|BB, AA|BB, BB|AA, i.e. estimate N above, and use the estimated Depending on the characteristics of the data, N can be made a constant, independent of the reading depth a _i + b _i ; or a function of a _i + b _i , so that the greater the reading depth, the smaller the deviation.

In one embodiment, informational methods can incorporate concordant per-SNP biases. Due to artifacts of the sequencing process, some SNPs can have consistently lower or higher counts, independent of the true amount of A content. Suppose SNP i consistently adds w _i % bias to the A count population. In some embodiments, this bias can be estimated from the derived set of training data under the same conditions and added back to the parental sequence data estimate as:

P(SM|m, i)＝P _X|m (am _i ), where X|m～BetaBinom(p _m (A)+w _i , am _i +bm _i , s)

and in the case of free-floating DNA sequence data probability estimates, is:

, where X～BetaBinom(p(A)+w _i , a _i +b _i , s),

In some embodiments, the method can be written to account for, inter alia, additional noise, differential sample quality, differential SNP quality, and random sampling bias. An example of it is given here. This approach has been shown to be particularly applicable in the context of data generated using a large-scale multiplex mini-PCR protocol and was used in experiments 7 to 13. The described method involves several steps, each of which introduces different kinds of noise and/or bias to the final model:

(1) If the first sample comprising a mixture of maternal and fetal DNA contains an initial amount of DNA molecules of size = N ₀ , typically in the range of 1,000-40,000, where p = true % reference

(2) In amplification using universal ligated adapters, N1 molecules are assumed to be sampled; typically N1 is _{approximately N0} / ₂ molecules and a random sampling bias is introduced due to sampling. The amplified sample may contain N ₂ molecules, where N ₂ >>N ₁ . Let X ₁ denote the amount of reference locus (on a per SNP basis) among the N ₁ molecules sampled, where variation of p ₁ =X ₁ /N ₁ introduces random sampling bias in the rest of the protocol. This sampling bias was included in the model by using the beta-binomial (BB) distribution instead of using the simple binomial distribution model. The parameter N of the beta-binomial distribution can later be estimated from the training data on a SNP, 0<p<1, after adjusting for leakage and amplification bias on a per-sample basis. Leakage is the tendency to read SNPs incorrectly.

(3) The amplification step will amplify any allelic bias, thereby amplifying the bias introduced by possible non-uniform amplification. If one allele at a locus is amplified by a factor of f, the other allele at that locus is amplified by a factor of g, where f=ge ^b , where b=0 means no bias. The bias parameter b is centered around 0 and represents how much more or less the A allele is amplified compared to the B allele at a particular SNP. The parameter b can vary from SNP to SNP. The bias parameter b can be estimated on a per SNP basis, eg from training data.

(4) The sequencing step involves sequencing the amplified molecular sample. During this step, there can be leaks, where leaks are instances of SNPs being incorrectly read. Leakage can be caused by any number of issues, and can result in a SNP read that is not the true allele A, but another allele B found at that locus or not normally found at that locus Allele C or D. Suppose the sequencing measures sequence data for a plurality _of DNA molecules from an amplified sample _of size N3, where N3< _N2 . _In some embodiments, N3 may range from 20,000 to 100,000, 100,000 to 500,000, 500,000 to 4,000,000, 4,000,000 to 20,000,000, or 20,000,000 to 100,000,000. The probability that each sampled molecule is correctly read is p _g , in which case it will correctly represent allele A. The probability that a sample will be incorrectly read as an allele unrelated to the initial molecule is 1- _pg , and the probability that it looks like allele A will be _pr , _allele B will be pm or etc. The probability of allele C or allele D is p _o , where p _r +p _m +p _o =1. The parameters p _g , p _r , p _m , p _o are estimated from the training data on a per SNP basis.

Different protocols may involve similar steps, with variations in molecular biology steps resulting in different amounts of random sampling, different levels of amplification, and different leakage biases. The following models work equally well for each of these situations. On a per SNP basis, the model for the amount of DNA sampled is given by:

X ₃ ～BetaBinomial(L(F(p, b), p _r , p _g ), N*H(p, b))

where p = true amount of reference DNA, b = bias per SNP, and as above, _pg is the probability of a correct read and _pr is the probability of an incorrect read but by chance a read that looks like the correct allele , in the case of bad readings as above, then:

F(p,b)=pe ^b /(pe ^b +(1-p)), H(p,b)=(e ^b p+(1-p)) ² /e ^b , L(p,p _r , p _g )=p*p _g +p _r *(1−p _g ).

In some embodiments, the method uses a beta-binomial distribution instead of a simple binomial distribution; this takes care of random sampling bias. The parameter N of the β-binomial distribution was estimated on a per-sample basis as needed. Using bias correction F(p,b), H(p,b) instead of just p takes care of amplification bias. The bias parameter b is estimated in advance from the training data on a per-SNP basis.

In some embodiments, the method uses a leak correction L(p, pr, p _g ), not just p; this takes into account leak bias, ie different SNPs and sample quality. In some embodiments, the parameters p _g , p _r , p _o are estimated in advance from the training data on a per SNP basis. In some embodiments, the parameters p _g , p _r , p _o may be updated with the current sample to account for different sample masses.

The model described herein is very general and can account for differential sample mass and differential SNP mass. Different samples and SNPs are treated differently, as exemplified by the fact that some embodiments use a beta-binomial distribution whose mean and variance are functions of the initial amount of DNA and the quality of the sample and SNP.

platform modeling

Consider a single SNP where the predicted allele ratio present in plasma is r (based on maternal and fetal genotypes). The predicted allele ratio is defined as the predicted fraction of the A allele in the combined maternal and fetal DNA. With respect to the maternal genotype _gm and the child _gc , the predicted allele ratio is given by Equation 1, assuming that the genotypes are also presented in the form of allele ratios.

r＝fg _c +(1-f)g _m (1)

The observations at a SNP consist of the number _{n a} and n _b of mapped reads for each allele present, which together is the read depth _d . It is assumed that thresholds have been applied to the mapping probabilities and phred scores so that mapping and allele observations can be considered correct. A phred score is a numerical measure that relates to the probability that a particular measurement at a particular base is wrong. In one embodiment, where a base has been measured by sequencing, a phred score can be calculated from the ratio of the dye intensity corresponding to the called base to the dye intensity of other bases. The simplest model for the likelihood of observation is the binomial distribution, which assumes that each of d reads is independently taken from a large pool with allele ratio r. Equation 2 describes this model.

P(n _a , n _b |r) = p _bino (n _a ; n _a +n _b , r) = (2)

The binomial model can be extended in several ways. When the maternal and fetal genotypes are all A or all B, the expected allele ratios in plasma will be 0 or 1, and the binomial probabilities will not be well defined. In fact, unexpected alleles were sometimes observed in practice. In one embodiment, it is possible to use corrected allele ratios to allow for a small number of unexpected alleles. In one embodiment, it is possible to use the training data to model the unexpected allele ratios that occur at each SNP, and use this model to correct the expected allele ratios. When the predicted allele ratio is not 0 or 1, the observed allele ratio cannot converge to the predicted allele ratio at a sufficiently high read depth due to amplification bias or other phenomena. The allele ratios can then be modeled as a beta distribution centered on the predicted allele ratios, yielding a beta-binomial distribution for P(n _a , n _b |r), which has higher variance than the binomial .

The platform model for the response at a single SNP would be defined as F(a, b, g _c , g _m , f) (3), or given the maternal and fetal genotypes, observe n _a = a and n _b = b The probability of , which also depends on the fetal fraction via Equation 1. The functional form of F may be a binomial distribution, a beta-binomial distribution, or a similar function as discussed above.

F(a,b,g _c ,g _m ,f)=P(n _a =a,n _b =b|g _c ,g _m ,f)=P(n _a =a,n _b =b|r( g _c ，g _m ，f)) (3)

In one embodiment, child scores may be determined as follows. Maximum likelihood estimates of fetal fraction f for prenatal testing can be derived without using paternal information. This may be appropriate where paternal genetic data is not available, for example where the recorded father is not actually the genetic father of the fetus. The fetal fraction is estimated from the set of SNPs whose maternal genotype is 0 or 1, resulting in a set of only two possible fetal genotypes. Define S ₀ as the set of SNPs with maternal genotype 0 and define S ₁ as the set of SNPs with maternal genotype 1 . The possible fetal genotypes on S ₀ are 0 and 0.5, resulting in a set of possible allele ratios R ₀ (f)={0,f/2}. Similarly, R ₁ (f)={1-f/2,1}. This approach could be slightly extended to include SNPs whose maternal genotype is 0.5, but these SNPs would be less informative due to the larger set of possible allele ratios.

Define N _a0 and N _{b0 as} the vectors formed by s in S ₀ with respect to nas and n _bs of the SNP, and N _a1 and N _b1 similarly for S ₁ . Maximum Likelihood Estimation of f Defined by Equation 4.

arg max _f P(N _a0 ，N _b0 |f)P(N _a1 ，N _b1 |f) (4)

Assuming that the allele count at each SNP is independently conditioned on the SNP's plasma allele ratio, the probability can be expressed as the outcome over the SNPs in each pool (5).

P(N _a0 , N _b0 |f)=P(n _as , n _bs |f) (5)

P(N _a1 , N _b1 |f)=P(n _as , n _bs |f)

The correlation with f is through the set of possible allelic ratios R ₀ (f) and R ₁ (f). The SNP probability P(n _as , n _bs |f) can be approximated by assuming a maximum likelihood genotype conditional on f. At suitably high fetal fractions and read depths, maximum likelihood genotypes will be selected with high confidence. For example, at a fetal fraction of 10% and a read depth of 1000, consider that the mother has a SNP with genotype zero. The expected allelic ratios were 0 and 5%, which would be easily distinguishable at sufficiently high read depths. Substituting the estimated child genotype into Equation 5 yields the complete Equation (6) for fetal fraction estimation.

arg max _f (6)

The fetal fraction must be in the range [0,1] and thus optimization can be easily implemented by a constrained one-dimensional search.

In the presence of low read depth or high noise levels, it may be preferable not to assume a maximum likelihood genotype, which would result in an artificially high confidence. Another approach would be to sum the possible genotypes at each SNP, yielding the following expression (7) for P(n _a , n _b |f) for the SNP in S ₀ . The prior probability P(r) may be assumed to be uniform over R ₀ (f), or may be based on population frequencies. The expansion _of the S1 group is trivial.

P(n _a , n _b |f) = (7)

In some embodiments, the probability can be derived as follows. Confidence can be calculated from the likelihood of the data for the two hypotheses _Ht and _Hf . The likelihood of each hypothesis was derived based on the response model, estimated fetal fraction, maternal genotype, allele population frequencies, and plasma allele counts.

Define the following symbols:

The likelihood of the parentage hypothesis is a consequence of the likelihood at the SNP, assuming that the observation at each SNP is independently conditioned on the plasma allele ratio. The following equations derive the likelihood of a single SNP. Equation 8 is a general expression for the likelihood of any hypothesis h, which will then be decomposed into the specific cases of _Ht and _Hf .

P(n _a , n _b |h, G _m , G _tf , f) =

=

= (8)

In the case of _Ht , according to Equation 9, the hypothetical father is the true father and the fetal genotype is inherited from the maternal genotype and the hypothetical paternal genotype.

P(n _a , n _b |, G _m , G _tf , f) = (9)

=

In the case of H _f the hypothetical father is not the real father. The best estimate of the true paternal genotype is given by the population frequency at each SNP. Thus, the probability of a child's genotype is determined from the known maternal genotype and population frequency, as in Equation 10.

P(n _a , n _b |, G _m , G _tf , f) =

=

=

=

The confidence _Cp of correct parentage is calculated from the results for SNPs with two likelihoods using Bayes rule (11).

Cp= (11)

Maximum Likelihood Model Using Percent Fetal Fraction

Determination of fetal ploidy status by measuring free-floating DNA contained in maternal serum or by measuring genotypic material in any pooled sample is of interest. There are various approaches, such as performing a read count analysis, where it is assumed that if the fetus is trisomy for a particular chromosome, then the total amount of DNA from that chromosome found in the maternal blood will be elevated relative to a reference chromosome. One way to detect trisomies in such fetuses is to normalize the amount of DNA expected for each chromosome, e.g., according to the number of SNPs in the analysis set that correspond to a given chromosome, or according to the number of SNPs that are uniquely available for the chromosome. The number of mapped sections. After the measurements have been normalized, any chromosome whose measured amount of DNA exceeds a certain threshold is judged to be trisomy. This approach is described in Fan et al. PNAS, 2008; 105(42); pp. 16266-16271; and Chiu et al. BMJ 2011; 342:c7401. In the paper by Chiu et al., via Compute Z-scores for normalization:

Z-score for the percentage of chromosome 21 in the test case = ((percent of chromosome 21 in the test case) - (average percentage of chromosome 21 in the reference control))/(standard deviation of the percentage of chromosome 21 in the reference control).

These methods determine fetal ploidy status using a single hypothesis exclusion method. However, they have some significant disadvantages. Because these methods for determining fetal ploidy do not vary according to the percentage of fetal DNA in the sample, they use a cut-off value; the consequence of this is that the accuracy of the assay is not optimal and the percentage of fetal DNA in the mixture is relatively low. Those cases that are low will suffer from the worst accuracy.

In one embodiment, the method of the invention is used to determine the ploidy state of a fetus, said method involving taking into account the fraction of fetal DNA in a sample. In another embodiment of the invention, the method involves the use of maximum likelihood estimation. In one embodiment, the methods of the invention involve calculating the percentage of DNA of fetal or placental origin in a sample. In one embodiment, the threshold for calling aneuploidy is adaptively adjusted based on the calculated percent fetal DNA. In some embodiments, the method for estimating the percentage of fetal-derived DNA in a DNA mixture comprises: obtaining a mixed sample comprising genetic material from the mother and genetic material from the fetus; obtaining a genetic sample from the father of the fetus; measuring the mixed sample DNA in the pooled sample; measuring the DNA in the paternal sample; and using the DNA measurements in the pooled sample and the paternal sample, calculating the percentage of fetal-derived DNA in the pooled sample.

In one embodiment of the invention, the fraction of fetal DNA or the percentage of fetal DNA in the mixture can be measured. In some embodiments, the fraction may be calculated using only genotyping measurements made on the maternal plasma sample itself, which is a mixture of fetal and maternal DNA. In some embodiments, the score may also be calculated using the measured or otherwise known maternal genotype and/or the measured or otherwise known paternal genotype. In some embodiments, the percent fetal DNA can be calculated using measurements made on a mixture of maternal and fetal DNA and knowledge of the parental background. In one embodiment, fractions of fetal DNA can be calculated using population frequencies to adjust the model based on the probability of a particular allele measurement.

In one embodiment of the present invention, the confidence level may be calculated based on the accuracy of the determination of the ploidy status of the fetus. In one embodiment, the confidence for the hypothesis with maximum likelihood (H _dominant ) can be calculated as follows: (1-H _dominant )/Σ(all H). If the distributions of all hypotheses are known, then it is possible to determine the confidence of the hypotheses. If parental genotype information is known, it is possible to determine all hypothesized distributions. If the knowledge about the expected distribution of data for euploid fetuses and the expected distribution of data for aneuploid fetuses is known, then it is possible to calculate the confidence level of the ploidy determination. If the parental genotype data are known, then it is possible to calculate these expected distributions. In one embodiment, knowledge of the distribution of test statistics on the normal hypothesis and on the abnormal hypothesis can be used to determine the reliability of the call and optimize thresholds to make more reliable calls. This is especially true when the amount and/or percentage of fetal DNA in the mixture is low. It will help to avoid situations where a fetus that is actually aneuploid is found to be euploid by test statistics such as Z-statistics not exceeding thresholds based on thresholds optimized for cases with higher fetal DNA percentages.

In one embodiment, the methods disclosed herein can be used to determine fetal aneuploidy by determining the copy number of a maternal and fetal chromosome of interest in a mixture of maternal and fetal genetic material. Such methods would entail obtaining maternal tissue containing both maternal and fetal genetic material; in some embodiments, such maternal tissue may be maternal plasma or tissue isolated from maternal blood. Such a method would also entail obtaining a mixture of maternal and fetal genetic material from said maternal tissue by processing said maternal tissue. This approach would require partitioning of the resulting genetic material into multiple reaction samples; randomly providing a separate reaction sample containing the target sequence of the target chromosome and a separate reaction sample not containing the target sequence of the target chromosome, e.g. performing high-throughput sequencing on the samples . Such an approach would entail analyzing the presence or absence of a target sequence of genetic material in said individual reaction samples to provide a binary result representing the presence or absence of a first number of possibly euploid fetal chromosomes in the reaction sample and expressed in Responding to the binary outcome of the presence or absence of a second number of possibly aneuploid fetal chromosomes in the sample. Any number of binary outcomes can be calculated, for example, by means of information technology that counts sequence reads that map to a particular chromosome, a particular region of a chromosome, a particular locus, or a set of loci. Such methods may involve normalizing the number of binary events based on chromosome length, the length of a chromosome region, or the number of loci in the set. Such an approach would entail using the first quantity to calculate a predicted distribution for the number of binary outcomes of possible euploid fetal chromosomes in the reaction sample. Such an approach would entail using the first quantity and estimated fraction of fetal DNA found in the mixture, e.g. by multiplying the expected read count distribution for the number of binary outcomes of likely euploid fetal chromosomes by (1+n/2) , where n is the estimated fetal fraction, to calculate the expected distribution of the number of binary outcomes for possible aneuploid fetal chromosomes in the reaction sample. In some embodiments, sequence reads can be processed as a probabilistic map rather than a binary outcome; this approach will yield higher accuracy, but requires greater computing power. Fetal fraction can be estimated by a variety of methods, some of which are described elsewhere herein. Such a method may involve determining whether the second number corresponds to a probable aneuploid fetal chromosome that is euploid or aneuploid using a maximum likelihood method. Such a method may involve interpreting the ploidy state of the fetus as corresponding to the most likely hypothesized ploidy state of the correct given the measured data.

It should be noted that the use of maximum likelihood models can be used to improve the accuracy of any method of determining the ploidy status of a fetus. Similarly, confidence levels for any method of determining fetal ploidy status can be calculated. The use of maximum likelihood models will improve the accuracy of any method in which ploidy determination is performed using single hypothesis exclusion techniques. Maximum likelihood models can be used with any method that can compute likelihood distributions for normal and abnormal conditions. The use of maximum likelihood models implies the ability to calculate confidence levels for ploidy calls.

Further discussion of the method

In one embodiment, the methods disclosed herein utilize a quantitative measure of the number of independent observations of each allele at a polymorphic locus, where this does not involve calculating ratios of alleles. This differs from methods such as microarray-based methods that provide information about the ratio of two alleles at a locus, but do not quantify the number of independent views of either allele. Some methods known in the art can provide quantitative information on the number of independent observations, but the calculations used to determine ploidy only use allele ratios, not quantitative information. To illustrate the importance of retaining information about the number of independent observations, consider a sample locus with two alleles (A and B). In the first experiment, twenty A alleles and twenty B alleles were observed; in the second experiment, 200 A alleles and 200 B alleles were observed. In both experiments the ratio (A/(A+B)) was equal to 0.5, however the second experiment conveyed more information about the frequency certainty of the A or B allele than the first experiment. Rather than utilizing allele ratios, the methods of the present invention use quantitative data to more accurately model the most likely allele frequency at each polymorphic locus.

In one embodiment, the present method constructs a genetic model for summing measurements of multiple polymorphic loci to better distinguish trisomies from disomies and determine trisomy types. In addition, the method of the present invention incorporates genetic linkage information to enhance method accuracy. This is in contrast to some methods known in the art in which the allele ratios for all polymorphic loci on a chromosome are averaged. The methods disclosed herein explicitly predict disomies and trisomies resulting from nondisjunction during meiosis I, nondisjunction during meiosis II, and nondisjunction during mitosis in early fetal development. Allele frequency distributions were modeled. To illustrate why this is important, nondisjunction during meiosis I would produce a trisomy, in which two distinct homologues are inherited from one parent, if there were no crossover; either during meiosis II or at Nondisjunction during mitosis early in fetal development will produce two copies of the same homologue from one parent. Each case yields a different predicted allele frequency at each polymorphic locus and at all physically linked loci (ie, loci on the same chromosome) that are considered jointly. Crossover causes the exchange of genetic material between homologues, making the pattern of inheritance more complex; but the method of the present invention accommodates this by using genetic linkage information, ie recombination rate information and the physical distance between loci. To better distinguish meiotic I nondisjunction from meiotic II or mitotic nondisjunction, the method of the present invention incorporates in the model an increasing probability of crossover with increasing distance from the centromere. Meiotic II and mitotic nondisjunction can be distinguished by the fact that mitotic nondisjunction usually produces identical or nearly identical copies of one homologue whereas two homologues present after the meiotic II nondisjunction event are usually due to Varies by one or more crosses during gametogenesis.

In one example, the methods of the invention may fail to determine the haplotype of the parents if disomy is assumed. In one embodiment, in the case of trisomy, the method of the invention can make a determination about the haplotype of one or both parents by using the fact that plasma was obtained from both copies of a parent, and the parental phase information This can be determined by noting which two copies were inherited from the parent in question. Specifically, a child can inherit two identical copies of a parent (matching trisomy) or two copies of a parent (unmatching trisomy). At each SNP, the likelihood of matching trisomies and mismatching trisomies can be calculated. Ploidy calling methods that do not use a linkage model to account for crossover will calculate the total likelihood of a trisomy as a simple weighted average of matching and mismatching trisomies on all chromosomes. However, due to the biological mechanisms that produce segregation errors and crossovers, a trisomy on a chromosome can change from matching to mismatching (and vice versa) only when crossing over occurs. The method of the present invention takes into account the likelihood of crossover in a probabilistic manner, resulting in greater accuracy of ploidy calls than those methods that do not consider the likelihood of crossover.

In one embodiment, a reference chromosome is used to determine child scores and noise level quantities or probability distributions. In one embodiment, child scores, noise levels and/or probability distributions are determined using only genetic information available from the chromosomes whose ploidy status is to be determined. The inventive method works without a reference chromosome and without fixing a specific child score or noise level. This is a significant improvement and distinguishing point over methods known in the art where genetic data from a reference chromosome must be used to calibrate child scores and chromosome behaviour.

In one embodiment where a reference chromosome is not required to determine fetal fraction, the following determination assumptions are made:

* Prior probability (H)

By means of an algorithm with a reference chromosome, it is usually assumed that the reference chromosome is disomy, and then one can (a) fix the most probable child score and the random noise level N based on this assumption and the reference chromosome data:

and then simplifies to

Or (b) estimate child score and noise level distributions based on this assumption and reference chromosome data. Specifically, for cfr and N, instead of fixing just one value, the probability p(cfr, N) is assigned for a wider range of possible cfr, N values:

where the prior probability (cfr, N) is the prior probability of a specific child's score and noise level, determined by prior knowledge and experiments. Uniform only in cfr, N range if necessary. Then one can write:

The above two methods have obtained good results.

It should be noted that in some cases it may be undesirable, impossible or infeasible to use a reference chromosome. In this case, it is possible to deduce the optimal ploidy call for each chromosome individually. Specifically:

As described above, each chromosome can be determined individually, assuming hypothesis H, not only postulated disomy for the reference chromosome. Using this approach, to keep the two parameters noise and child score fixed, it is possible to fix either of the parameters, or keep both parameters in probabilistic form, for each chromosome and each hypothesis.

Measurements of DNA are prone to noise and/or errors, especially when the amount of DNA is small or where the DNA is mixed with contaminated DNA. This noise results in less accurate genotype data and less accurate ploidy calls. In some embodiments, platform modeling or some other method of noise modeling can be used to counteract the detrimental effect of noise on ploidy determinations. The method of the present invention uses a joint model of two channels, which accounts for random noise due to the amount of input DNA, DNA quality and/or protocol quality.

This is in contrast to some methods known in the art in which ploidy determinations are made using ratios of allelic intensities at loci. This approach prevents accurate SNP noise modeling. Specifically, the errors of the measurements are usually not particularly dependent on the measured channel strength ratios, which reduces the model to use one-dimensional information. Accurate modeling of noise, channel quality, and channel interaction requires a two-dimensional joint model, which cannot be modeled using allele ratios.

In particular, projecting two channel information to a ratio r (where f(x,y) is r=x/y) is not suitable for accurate channel noise and bias modeling. The noise at a particular SNP is not a function of the ratio, ie noise(x,y)≠f(x,y), but is actually a joint function of the two channels. For example, in a binomial model, the variance of the noise of the measured ratio is r(1-r)/(x+y), which is not a pure function of r. In such a model, which includes any channel bias or noise, if at SNP i, the observed channel X value is x=a _i X+ _bi , where X is the true channel value and _bi is the extra channel Bias and random noise. Similarly, suppose y=c _i Y+d _i . The observed ratio r=x/y cannot accurately predict the true ratio X/Y or model residual noise because (aiX+bi)/(ciY+di) is not a function of X/Y.

The methods disclosed herein describe an efficient way to model noise and bias using the joint binomial distribution of all channels measured individually. Relevant equations can be found elsewhere in this document in the sections talking about each SNP Consensus Bias, P(good) and P(Ref|Bad), P(Mutation|Bad), which effectively tune the behavior of the SNP. In one embodiment, the inventive method uses a beta-binomial distribution, which avoids the practice of being restricted to relying solely on allele ratios, but actually models behavior based on two channel counts.

In one embodiment, the methods disclosed herein can call the ploidy of a gestating fetus from genetic data found in maternal plasma by using all available measurements. In one embodiment, the methods disclosed herein can call the ploidy of a gestating fetus from genetic data found in maternal plasma by using measurements from only a subset of parental backgrounds. Some methods known in the art use only measured genetic data where the parental background is from an AA|BB background, that is, where both parents are homozygous at a given locus, but for different alleles. A problem with this approach is that the proportion of polymorphic loci from the AA|BB background is small, typically less than 10%. In one embodiment of the methods disclosed herein, the method does not use genetic measurements of maternal plasma at loci where the parental background is AA|BB. In one embodiment, the methods of the invention use only plasma measurements for those polymorphic loci whose parental backgrounds are AA|AB, AB|AA and AB|AB.

Some methods known in the art involve averaging the allele ratios from SNPs in an AA|BB background where the genotypes of both parents are present, and requiring the determination of ploidy calls from the average allele ratios at these SNPs . This approach suffers from significant inaccuracy due to differential SNP behavior. It should be noted that this method assumes that the genotypes of both parents are known. In contrast, in some embodiments, the present methods use a joint channel distribution model that does not assume the presence of either parent and does not assume uniform SNP behavior. In some embodiments, the methods of the invention account for different SNP behaviors/weightings. In some embodiments, the methods of the invention do not require knowledge of the genotype of one or both parents. The following is an example of how the inventive method can accomplish this:

In some embodiments, the log-likelihood of a hypothesis can be determined on a per SNP basis. The log-likelihood of the observed data D at a particular SNP i is defined as:

where m is the likely true maternal genotype, f is the likely true paternal genotype given assumption H, where m, f{AA,AB,BB}, and where c is the likely child genotype. Specifically, about monosomy, c; about disomy, c; about trisomy, c. It should be noted that including parental genotype data generally results in more accurate ploidy determinations, however, parental genotype data is not required for the method of the invention to work well.

Some methods known in the art involve averaging allele ratios from SNPs where the mother is homozygous but measure different alleles in plasma (AA|AB or AA|BB background), and require The average allele ratio at the SNP determines the ploidy call. This method is intended for use in situations where paternal genotypes are not available. It should be noted that it is questionable how accurate it is to declare that the plasma is heterozygous at a particular SNP and that no homozygous and opposite paternal BB is present: since in the case of low child fractions it appears that the presence of the B allele may simply be Noise is present; otherwise, the apparent absence of B may simply be an allelic loss of fetal measurements. Even where plasma heterozygosity could actually be determined, this approach would not be able to distinguish paternal trisomies. Specifically, for SNPs where the mother is AA, and where some B is measured in the plasma, if the father is GG, then the resulting child genotype is AGG such that the mean rate of A is 33% (child fraction = 100%) . But in cases where the father is AG, the resulting child genotype could be AGG for a matched trisomy, contributing 33% of the A rate, or AAG for an unmatched trisomy, skewing the average rate towards 66% A. Given multiple trisomies on crossed chromosomes, the overall chromosome can have any mismatched trisomies between no mismatched trisomies and all mismatched trisomies, this rate can range from 33% to 66% change between. For ordinary disomy, the ratio should be about 50%. Without using linkage models or accurate error models for the mean, this approach would miss multiple cases of paternal trisomy. In contrast, the methods disclosed herein assign a parental genotype probability to each candidate parental genotype based on available genotype information and population frequencies, and do not explicitly require parental genotypes. In addition, the methods disclosed herein are capable of detecting trisomies even in the absence or presence of parental genotype data, and can use linkage models to compensate by identifying possible crossover points from matched to unmatched trisomies .

Some methods known in the art claim one for averaging the allelic ratios for SNPs where neither the maternal or paternal genotypes are known, and for determining ploidy calls from the average ratios over these SNPs Methods. However, methods for achieving these objects are not disclosed. The methods disclosed herein enable accurate ploidy calls to be made in this situation, and are disclosed elsewhere in this document in practice, using joint probabilistic maximum likelihood and optionally exploiting SNP noise and bias model and chain model.

Some methods known in the art involve averaging the allele ratios and require determination of ploidy calls from the average allele ratios at one or a few SNPs. However, such methods do not exploit the chain concept. The methods disclosed herein do not have these disadvantages.

Using sequence length as a prior to determine DNA origin

It has been reported that the sequence length distribution of maternal and fetal DNA differs, with the fetus generally being shorter. In one embodiment of the invention, it is possible to use prior knowledge in the form of empirical data and construct prior distributions for the expected lengths of maternal (P(X|maternal)) and fetal DNA (P(X|fetal)) . Given a new unidentified DNA sequence of length x, it is possible to assign a probability that a given DNA sequence is maternal or fetal DNA based on the prior likelihood that x is maternal or fetal. Specifically, if P(x|maternal)>P(x|fetal), then the DNA sequence can be classified as maternal, where P(x|maternal)=P(x|maternal)/[(P( x|maternal)+P(x|fetal)]; and if p(x|maternal)<p(x|fetal), then the DNA sequence can be classified as fetal, P(x|fetal)=P(x| Fetus)/[(P(x|maternal)+P(x|fetus)]. In one embodiment of the present invention, it can be determined specifically for the The distribution of maternal and fetal sequence lengths for a sample, and then the sample-specific distribution can be used as the predicted size distribution for the sample.

Variable read depth for minimizing sequencing costs

In several clinical trials on diagnosis, for example in Yau et al. BMJ 2011;342:c7401, a protocol with multiple parameters is set up and then the same protocol is performed with the same parameters for each patient in the trial. In cases where sequencing is used as a method of measuring genetic material to determine the ploidy state of a fetus in a mother's gestation, a relevant parameter is the number of reads. The number of reads can refer to the number of actual reads, the number of scheduled reads, a fraction of lanes on a sequencer, all lanes, or a full flow cell. In these studies, the number of reads is typically set at a level that will ensure the desired level of accuracy for all or nearly all samples. Sequencing is currently an expensive technology, costing approximately $200 per 5 million mappable reads, and while prices are decreasing, any technology that allows sequencing-based diagnostics to operate at a similar level of accuracy but with fewer reads The method is sure to save a lot of money.

The accuracy of ploidy determination often depends on several factors, including the number of reads and the fraction of fetal DNA in the mixture. Accuracy is generally higher when the fraction of fetal DNA in the mixture is higher. Also, the accuracy is usually higher if the number of readings is greater. There may be situations where ploidy status is determined with comparable accuracy in two cases, wherein the first case has a lower fraction of fetal DNA in the mixture than the second case; There are more readings than in the second case. It is possible to use the estimated fraction of fetal DNA in the mixture as a guide to the number of reads that must be determined to achieve a specified level of accuracy.

In one embodiment of the invention, a set of samples may be run where different samples in the set are sequenced at different depths of reads, where the number of reads run per sample is determined by the calculated fraction of fetal DNA in each mixture. Select the specified level of accuracy that can be achieved. In one embodiment of the invention, this would require measurement of the pooled sample to determine the fraction of fetal DNA in the pool; such estimation of the fetal fraction can be done with sequencing, sequencing can be done with Tuckerman, sequencing can be done with qPCR Sequencing can be performed using SNP arrays, and sequencing can be performed by any method that can distinguish between different alleles at a given locus. The need to perform fetal fraction estimation can be eliminated by including assumptions covering all or a selected set of fetal parts in the set of hypotheses considered when compared to actual measured data. After the fraction of fetal DNA in the mixture has been determined, the number of sequences to be read for each sample can be determined.

In one example of the present invention, 100 pregnant women visit their respective OBs, and their blood is drawn into blood collection tubes containing anti-lysing agents and/or reagents for inactivating DNase. They each took home a kit for the father of the fetus to give a saliva sample. Both sets of genetic material from all 100 couples were sent back to the laboratory, where the mother's blood was spun down and the buffy coat and plasma were separated. Plasma contains a mixture of maternal DNA as well as DNA of placental origin. Maternal buffy coat and paternal blood were genotyped using SNP arrays, and Hull Select hybridization probes were used to target DNA in maternal plasma samples. The DNA developed with the probes was used to generate a marker library of 100 for each of the maternal samples, where each sample was labeled with a different marker. A fraction was taken from each library, and each of those fractions were pooled together and added in a multiplexed fashion to two lanes of the Illumina HISEQ DNA sequencer, where each lane yielded approximately 50 million mappable reads, Generates ~100 million mappable reads on 100 complex mixtures, or ~1 million reads per sample. Sequence reads were used to determine the fraction of fetal DNA in each mixture. The pool of 50 of the samples contained more than 15% fetal DNA, and 1 million reads were sufficient to determine the ploidy state of the fetus with 99.9% confidence.

Of the remaining mixtures, 25 had between 10% and 15% fetal DNA; a portion of each of the relevant libraries prepared from these mixtures was multiplexed and run on one lane of HISEQ, yielding an additional 200 per sample. million readings. These two sets of sequence data for each of the mixtures between 10% and 15% fetal DNA were added together, and the resulting 3 million reads per sample were sufficient to determine the ploidy status of those fetuses with 99.9% confidence .

Of the remaining mixtures, 13 had between 6% and 10% fetal DNA; a portion of each of the relevant libraries prepared from these mixtures was multiplexed and run on one lane of HISEQ, yielding 400 additional samples per sample. million readings. These two sets of sequence data for each of the pools with fetal DNA between 6% and 10% were added together and the resulting total of 5 million reads per pool was sufficient to determine the ploidy of those fetuses with 99.9% confidence state.

Eight of the remaining mixtures had between 4% and 6% fetal DNA; a portion of each of the relevant libraries prepared from these mixtures was multiplexed and run on one lane of HISEQ, yielding 600 additional samples per sample. million readings. These two sets of sequence data for each of the pools with fetal DNA between 4% and 6% were added together, and the resulting 7 million total reads per pool were sufficient to determine the ploidy of those fetuses with 99.9% confidence state.

In the remaining four mixtures, all of them had between 2% and 4% fetal DNA; a portion of each of the relevant libraries prepared from these mixtures was multiplexed and run on one lane of HISEQ, and each sample was reassembled. Generates 12 million readings. These two sets of sequence data for each of the pools with fetal DNA between 2% and 4% were added together, and the resulting total of 13 million reads per pool was sufficient to determine the ploidy of those fetuses with 99.9% confidence state.

This method requires six sequencing lanes on the HISEQ machine to achieve 99.9% accuracy over 100 samples. If each sample required the same number of runs to ensure that each ploidy determination was 99.9% accurate, then 25 sequencing lanes would be required; and if a 4% no-call or error rate was tolerated, then 14 sequencing Swimlanes are already available.

Using raw genotyping data

There are a number of ways in which NPD can be accomplished using fetal genetic information measured on fetal DNA found in maternal blood. Some of these methods involve the use of SNP arrays to measure fetal DNA, some methods involve non-targeted sequencing, and some methods involve targeted sequencing. Targeted sequencing can target SNPs, can target STRs, can target other polymorphic loci, can target non-polymorphic loci, or some combination thereof. Some of these methods may involve reading the identity of the allele from the intensity data from the sensor of the machine making the measurement using a commercial or dedicated allele reader. For example, the Illumina Infinium system or the Affilix gene chip microarray system involve beads or microchips attached with DNA sequences that can hybridize to complementary segments of DNA; after hybridization, sensors that can detect The fluorescent properties of the molecule change. There are also sequencing methods, such as the Illumina Solesa Genome Sequencer or the ABI Solid Genome Sequencer, in which the genetic sequence of a DNA fragment is sequenced; after extension of the DNA strand complementary to the strand to be sequenced, usually The identity of the extended nucleotide is detected via a fluorescent or radioactive label attached to the complementary nucleotide. In all of these methods, genotype or sequencing data are typically determined based on fluorescence or other signals or lack thereof. These systems are often combined with low-level software packages that perform specific allelic calls (secondary genetic data) from analog output from fluorescence or other detection devices (primary genetic data). For example, in the case of a given allele on a SNP array, the software will make a call, eg, the presence or absence of a certain SNP if the measured fluorescence intensity is above or below a certain threshold. Similarly, the output of the sequencer is a chromatogram indicating the level of fluorescence detected for each dye, and the software makes the following call: a certain base pair is A or T or C or G. High-throughput sequencers typically perform a series of such measurements, calling out reads that represent the most likely structure of the sequenced DNA sequence. The direct analog output of chromatograms is defined here as primary genetic data, and base pair/SNP calls by software are considered here as secondary genetic data. In one embodiment, raw data refers to raw intensity data, which is the unprocessed output of a genotyping platform, where a genotyping platform can refer to a SNP array or a sequencing platform. Secondary genetic data refers to processed genetic data in which allelic calls have been made, or sequence data have been assigned base pairs, and/or sequence reads have been mapped to a genome.

Many higher-level applications make use of these allelic calls, SNP calls, and sequence reads, the secondary genetic data generated by genotyping software. For example, DNANEXUS, ELAND or MAQ will take sequencing reads and map them to the genome. For example, in the case of non-invasive prenatal diagnosis, complex information such as PARENTALSUPPORT ^™ can utilize a large number of SNP calls to determine the genotype of an individual. Furthermore, in the case of preimplantation genetic diagnosis, it is possible to take a set of sequence reads that are mapped to the genome, and by taking a normalized count of the reads that are mapped to each chromosome or chromosome segment, it is possible to determine The ploidy state of an individual. In the case of non-invasive prenatal diagnosis, it is possible to take a set of sequence reads already measured against DNA present in maternal plasma and map them to the genome. A normalized count of reads mapped to each chromosome or chromosome segment can then be taken and the data used to determine the ploidy state of the individual. For example, it may be possible to conclude that those chromosomes with a disproportionately large number of reads in a gestating fetus of a mother whose blood was drawn are trisomy.

In practice, however, the initial output of the measuring instrument is an analog signal. When a certain base pair is interpreted by the software related to the sequencing software, for example, the software can interpret the base pair as T, in fact, the interpretation is the most likely interpretation considered by the software. In some cases, however, the call may have a low confidence level, for example an analog signal may indicate that a particular base pair is only 90% likely to be a T, and 10% likely to be an A. In another example, genotype calling software associated with a SNP array reader can call a certain allele as G. In practice, however, the base analog signal may indicate that the allele is only 70% likely to be G, and the allele is 30% likely to be T. In these cases, when higher level applications use genotype calls and sequence calls obtained by lower level software, they lose some information. That is, primary genetic data as measured directly by a genotyping platform may be messier than secondary genetic data determined by a connected software package, but it is more informative. During the sequence mapping of secondary genetic data to the genome, multiple reads were discarded because some base reads were not sufficiently defined and/or mapped poorly. When using primary genetic data sequence reads, all or more of those reads that have been rejected when first converted to secondary genetic data sequence reads can be used by probabilistically processing the reads.

In one embodiment of the invention, the higher level software does not rely on allelic calls, SNP calls or sequence reads determined by the lower level software. In fact, the calculations of the higher level software are based on the analog signals measured directly from the genotyping platform. In one embodiment of the invention, an information-based approach such as PARENTAL SUPPORT ^™ is modified such that its ability to reconstruct the genetic data of an embryo/fetus/child is adapted to directly use raw genetic data as measured by a genotyping platform . In one embodiment of the invention, an information-based approach such as PARENTALSUPPORT ^™ enables allelic calls and/or chromosome copy number calls to be made using primary genetic data and without secondary genetic data. In one embodiment of the invention, all gene calls, SNP calls, sequence reads, sequence mappings are probabilistically by using raw intensity data as directly measured by genotyping platforms, rather than converting raw genetic data into Secondary gene interpretation to deal with. In one embodiment, the DNA measurements of the prepared samples used to calculate the allele count probabilities and determine the relative probabilities of each hypothesis comprise raw genetic data.

In some embodiments, the method can improve the accuracy of the genetic data of the target individual with the genetic data of at least one related individual, the method comprising obtaining the raw genetic data specific to the genome of the target individual and the genetic data of the related individual genetic data specific to the genome of the target individual; create a set of one or more hypotheses about which segments of which chromosomes of the related individual might correspond to those segments in the genome of the target individual; given the original genetic data of the target individual and the associated The genetic data of the individual determines the probability of each hypothesis; and using the probabilities associated with each hypothesis, the most likely state of the actual genetic material of the target individual is determined. In some embodiments, the method may determine the copy number of the chromosome segment in the genome of the target individual, the method comprising creating a set of copy number hypotheses about how many copies of the chromosome segment are present in the genome of the target individual; Raw genetic data for individuals and genetic information from one or more related individuals are incorporated into datasets; characteristics of platform responses associated with datasets are estimated, where platform responses can vary from one experiment to another; given datasets and platform responses features, computing a conditional probability for each copy number hypothesis; and determining the copy number of the chromosome segment based on the most likely copy number hypothesis. In one embodiment, the method of the present invention can determine the ploidy state of at least one chromosome in a target individual, said method comprising obtaining raw genetic data from the target individual and from one or more related individuals; for each chromosome of the target individual , creating a set of at least one ploidy state hypothesis; determining the statistical probability of each ploidy state hypothesis in said set using one or more expertise, each expertise used given the resulting genetic data; for each ploidy state assumptions, combining statistical probabilities as determined by one or more specialized techniques; and determining the ploidy state of each chromosome in the target individual based on the combined statistical probabilities assumed for each ploidy state. In one embodiment, the method of the present invention can determine the allelic status in a set of alleles of a target individual and one or both parents of the target individual, and optionally one or more related individuals, the method comprising obtaining target individual and raw genetic data from one or both parents and from any related individuals; creating a set of at least one allelic hypothesis for the target individual and one or both parents and optionally one or more related individuals, wherein said Hypotheses describe possible allelic states in the set of alleles; given the resulting genetic data, determining the statistical probability of each allelic hypothesis in the set of hypotheses; Individually, the allelic status of each allele in the allelic set is determined based on the statistical probability assumed for each allele.

In some embodiments, the genetic data of the pooled sample may comprise sequence data, where the sequence data may not uniquely map to the human genome. In some embodiments, the genetic data of the pooled sample may comprise sequence data, where the sequence data maps to multiple locations in the genome, where each possible mapping is associated with a probability that the given mapping is correct. In some embodiments, it is assumed that sequence reads are not associated with a particular location in the genome. In some embodiments, sequence reads are associated with multiple locations in the genome and associated probabilities of belonging to said locations.

Counting Methods for Determining Chromosomal Copy Number

In one aspect, the invention features a method of testing for non-normal distribution of fetal chromosomes by comparing the number of sequence markers aligned to different chromosomes (see, e.g., U.S. Patent No. 8,296,076, filed April 20, 2012, The aforementioned patents are hereby incorporated herein by reference in their entirety). As known in the art, the term "sequence marker" refers to a relatively short (eg, 15-100) nucleic acid sequence that can be used to identify a larger sequence, eg, maps to a chromosomal or genomic region or gene. In some embodiments, the method involves (i) simultaneously hybridizing a sample comprising a mixture of maternal and fetal DNA to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000 or 100,000 different primer pools of target loci are contacted to generate a reaction mixture; wherein the target loci are from a plurality of different chromosomes; and wherein the plurality of different chromosomes contain at least one suspected nonpositive gene in the sample (ii) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product; (iii) sequencing the amplification product to obtaining a plurality of sequence tags aligned with the target locus; wherein the sequence tags are of sufficient length to be assigned to a specific target locus; (iv) assigning in silico the plurality of sequence tags to their corresponding target loci; (v) Determining in silico the number of sequence markers aligned to the target locus of the first chromosome and the number of sequence markers aligned to the target locus of the second chromosome; and (vi) comparing in silico the results from step (v) Quantities to determine the presence or absence of a non-normal distribution of the first chromosome.

In one aspect, the invention provides methods for detecting the presence or absence of fetal aneuploidy by comparing the relative frequencies of target amplicons between chromosomes (see, e.g., PCT Publication No. WO2012/ 103031, the disclosure of which is hereby incorporated by reference in its entirety). In some embodiments, the method involves (i) simultaneously hybridizing a sample comprising a mixture of maternal and fetal DNA to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, Primer pools of 75,000 or 100,000 different non-polymorphic target loci are contacted to generate a reaction mixture; wherein the target loci are from a plurality of different chromosomes; (ii) subjecting the reaction mixture to primer extension reaction conditions to generate Amplification products of amplicons; (iii) quantification in silico of relative frequencies of target amplicons from first and second associated chromosomes; (iv) comparison in silico of target amplicons from first and second associated chromosomes the relative frequency of the amplicon; and (v) identifying the presence or absence of aneuploidy based on the compared relative frequencies of the first and second associated chromosomes. In some embodiments, the first chromosome is a chromosome suspected of being euploid. In some embodiments, the second chromosome is a chromosome suspected of aneuploidy.

Combined Prenatal Diagnosis Methods

There are various methods that can be used for prenatal diagnosis or prenatal screening for aneuploidy or other genetic defects. Elsewhere in this document and in U.S. Utility Application No. 11/603,406, filed November 28, 2006; U.S. Utility Application No. 12/076,348, filed March 17, 2008; and PCT Application No. PCT/S09/52730 One such method is described in , which uses the genetic data of related individuals to improve the accuracy by which the genetic data of the target individual (such as a fetus) is known or estimated. Other methods used in prenatal diagnosis involve measuring the levels of certain hormones in the maternal blood, where those hormones are associated with various genetic abnormalities. An example of this is called a triple test, a test in which the levels of several (usually two, three, four or five) different hormones in the maternal blood are measured. In cases where multiple methods are used to determine the likelihood of a given result, none of which is conclusive in itself, it is possible to combine the information given by those methods to make a more accurate statement than either method alone. Prediction. In a triple test, combining information given by the three different hormones could yield more accurate predictions of genetic abnormalities than hormone levels alone could.

Disclosed herein is a method for making more accurate predictions about the genetic status of the fetus, particularly the likelihood of a genetic abnormality in the fetus, the method comprising combining the predictions of the genetic abnormality of the fetus, wherein those predictions are made using Made in a variety of ways. A "more accurate" method may refer to a method for diagnosing an abnormality that has a lower false negative rate at a specified false positive rate. In an advantageous embodiment of the invention, one or more of said predictions are made on the basis of genetic data about the fetus, wherein genetic knowledge is determined using the PARENTAL SUPPORT ^™ method, that is to say using the Genetic Data Determine the genetic data of the fetus with greater accuracy. In some embodiments, the genetic data may include the ploidy state of the fetus. In some embodiments, the genetic data may refer to a set of allelic calls on the fetal genome. In some embodiments, some predictions may be made using triple testing. In some embodiments, some predictions can be made using measurements of other hormone levels in maternal blood. In some embodiments, predictions made by methods that consider diagnosis may be combined with predictions made by methods that consider screening. In some embodiments, the methods involve measuring maternal blood levels of alpha-fetoprotein (AFP). In some embodiments, the methods involve measuring maternal blood levels of unconjugated estriol (UE ₃ ). In some embodiments, the method involves measuring maternal blood levels of β-human chorionic gonadotropin (β-hCG). In some embodiments, the methods involve measuring maternal blood levels of invasive trophoblastic antigen (ITA). In some embodiments, the methods involve measuring maternal blood levels of statins. In some embodiments, the methods involve measuring maternal blood levels of pregnancy-associated plasma protein A (PAPP-A). In some embodiments, the methods involve measuring maternal blood levels of other hormones or maternal serum markers. In some embodiments, other methods may be used to make some predictions. In some embodiments, some predictions can be made using a well-integrated test, such as a test that combines an ultrasound and blood test at about week 12 of pregnancy with a second blood test at about week 16. In some embodiments, the method involves measuring fetal neck translucency (NT). In some embodiments, the methods involve making predictions using measured levels of the hormones described above. In some embodiments, the method involves a combination of the methods described above.

There are several ways to combine predictions, for example one could convert hormone measurements into multiples of the median (MoM) and then into likelihood ratios (LR). Similarly, other measurements can be converted to LR using a mixed model of the NT distribution. LR for NT and biochemical markers can be multiplied by age and pregnancy-related risk to derive risk for various conditions, such as trisomy 21. The Detection Rate (DR) and False Positive Rate (FPR) can be calculated by taking the proportion of risks that exceed a specified risk threshold.

In one embodiment, a method of interpreting ploidy status involves comparing the relative probabilities of each of the ploidy hypotheses determined using a joint distribution model and allele count probabilities to the ploidy hypotheses calculated using statistical techniques. Combining relative probabilities, the statistical techniques derived from other methods of determining the risk score of a fetus being trisomy include (but are not limited to): read count analysis, comparing heterozygosity rates, statistics available only when using parental genetic information, Probabilities of normalized genotype signals for certain parental backgrounds, statistics calculated using estimated fetal fractions of first or prepared samples, and combinations thereof.

Another approach may involve the case of four measured hormone levels, where the probability distribution over those hormones is known: p(x ₁ , x ₂ , x ₃ , x ₄ |e) for the euploid case; And for the case of aneuploidy, p(x ₁ , x ₂ , x ₃ , x ₄ |a). The probability distribution of the DNA measurements can then be measured, g(y|e) and g(y|a) for the euploid and aneuploid cases, respectively. Assuming they are independent with respect to the euploid/aneuploid hypothesis, they can be combined as p(x ₁ , x ₂ , x ₃ , x ₄ |a)g(y|a) and p(x ₁ , x ₂ , x ₃ , x ₄ |e)g(y|e), and are then each multiplied by the priors p(a) and p(e) given the maternal age. The highest one can then be chosen.

In one embodiment, it is possible to think of the central limit theorem to assume that the distribution over g(y|a or e) is Gaussian, and to measure the mean and standard deviation by looking at multiple samples. In another embodiment, it can be assumed that they are not independent with respect to the outcome and enough samples collected to estimate the joint distribution p(x ₁ , x ₂ , x ₃ , x ₄ |a or e).

In one embodiment, the ploidy state of the target individual is determined to be the ploidy state associated with the most probable hypothesis. In some cases, a hypothesis will have a normalized combined probability greater than 90%. Each hypothesis is associated with one or a group of ploidy states and can be chosen to be combined with a normalized probability greater than 90% or some other threshold such as 50%, 80%, 95%, 98%, 99% or 99.9% The hypothesized associated ploidy state serves as the threshold required to be interpreted as a hypothesis for the determined ploidy state.

DNA from a child from a previous pregnancy in the mother's blood

One difficulty with non-invasive prenatal diagnosis is the differentiation of fetal cells from a current pregnancy from those from a previous pregnancy. Some believe that genetic material from previous pregnancies disappears after a while, but conclusive evidence has yet to be shown. In one embodiment of the invention, it is possible to use the PARENTAL SUPPORT ^™ (PS) method and knowledge of the paternal genome to determine the presence of fetal DNA of paternal origin (that is, fetal DNA inherited from the father) present in the maternal blood. DNA). This approach can take advantage of phased parental genetic information. It is possible to phase the parental genotypes from the unphased genotype information using the genetic data of the grandparents (eg, measured from the grandfather's sperm) or genetic data from other born children or aborted samples. Unphased genetic information can also be phased by means of HapMap-based phasing or haplotype analysis of paternal cells. Successful haplotype analysis has been demonstrated by arresting cells at the mitotic stage when chromosomes are in tight bundles and using microfluidics to place separate chromosomes in separate wells. In another embodiment, it is possible to use phased parental haplotype data to detect the presence of more than one congener from the father, meaning the presence of genetic material from more than one child in the blood. By focusing on the chromosomes in the fetus that are predicted to be euploid, the possibility of the fetus having a trisomy can be ruled out. In addition, it is possible to determine whether the fetal DNA is not from the current father, in which case other methods (such as triple testing) can be used to predict genetic abnormalities.

There may be other sources of fetal genetic material available via methods other than blood draws. In the case of fetal genetic material available in maternal blood, there are two main categories: (1) whole fetal cells, such as nucleated fetal red blood cells or erythroblasts, and (2) free-floating fetal DNA. In the case of whole fetal cells, there is some evidence that fetal cells can persist in maternal blood for extended periods of time such that it is possible to isolate cells from pregnant women containing DNA from a child or fetus from a previous pregnancy. There is also evidence that free-floating fetal DNA is cleared from the system in a matter of weeks. One challenge is how to determine the identity of the individual whose genetic material the cells contain, ensuring that the genetic material being measured is not from a fetus from a previous pregnancy. In one embodiment of the invention, knowledge of the maternal genetic material can be used to ensure that the genetic material in question is not the maternal genetic material. There are various ways to accomplish this, including information-based approaches such as PARENTAL SUPPORT ^™ , as described in this document or in any of the patents mentioned in this document.

In one embodiment of the invention, blood obtained from a pregnant mother may be separated into a fraction comprising free-floating fetal DNA and a fraction comprising nucleated red blood cells. Free-floating DNA can optionally be enriched, and genotype information from the DNA can be measured. Knowledge of the maternal genotype can be used to determine various aspects of the fetal genotype based on genotype information measured from free-floating DNA. These aspects can refer to ploidy state and/or set of allelic identities. Individual nucleated red blood cells can then be genotyped using the methods described elsewhere in this document and in other referenced patents, particularly those mentioned in the first part of this document. Knowledge of the maternal genome will allow determination of whether any given single blood cell is genetically maternal. And the aspect of fetal genotype determined as described above will allow determination of whether a single blood cell is genetically derived from a currently gestating fetus. Essentially, this aspect of the invention allows the use of genetic knowledge of the mother and possibly genetic information from other related individuals (such as the father) as well as genetic information measured from free-floating DNA found in the maternal blood to determine the Whether the isolated nucleated cells found in are (a) genetically maternal, (b) genetically derived from a currently gestating fetus, or (c) genetically derived from a previously pregnant fetus.

Prenatal sex chromosome aneuploidy determination

In methods known in the art, the fact that there is fetal free-floating DNA (ff DNA) in the mother's plasma has been used when attempting to determine the sex of a gestating fetus from the mother's blood. If the Y-specific locus can be detected in maternal plasma, then this means that the gestating fetus is male. However, when using methods known in the art, the absence of detection of the Y-specific locus in plasma does not always ensure that the fetus in pregnancy is female, as in some cases the amount of fffDNA is too low to ensure The Y-specific loci could be detected in the case of male fetuses.

A novel approach is presented here that does not require the measurement of Y-specific nucleic acids, ie, DNA from exclusively paternally derived loci. A previously published Parental Support method uses crossover frequency data, parental genotype data, and information technology to determine the ploidy status of a gestating fetus. The sex of the fetus is just the ploidy state of the fetus in the sex chromosomes. XX's child is female and XY is male. The methods described herein also enable determination of the ploidy state of the fetus. It should be noted that sex determination is practically synonymous with ploidy determination of the sex chromosomes; in the case of sex determination, the child is usually assumed to be euploid, so fewer assumptions are possible.

The methods disclosed herein involve looking at the loci shared by the X and Y chromosomes to create a pre-measured baseline of fetal DNA representative of the fetus. Then, only those regions specific to the X chromosome can be queried to determine whether the fetus is female or male. In the case of males, we would expect to see less fetal DNA from loci specific to the X chromosome than from loci specific to both X and Y. In female fetuses, by contrast, we would expect the amount of DNA in each of these groups to be the same. The DNA in question can be measured by any technique that can quantify the amount of DNA present on a sample, such as qPCR, SNP arrays, genotyping arrays or sequencing. With regard to DNA derived exclusively from one individual, we would expect to see the following:

In the case where fetal DNA is mixed with maternal DNA and where the fraction of fetal DNA in the mixture is F, and where the fraction of maternal DNA in the mixture is M, so F+M = 100%, we would expect to see the following:

In cases where F and M are known, predicted ratios can be calculated and observed data can be compared to predicted data. In cases where M and F are not known, thresholds can be chosen based on historical data. In both cases, the amount of DNA measured at loci specific for X and Y can be used as a baseline, and sex testing of the fetus can be based on DNA observed at loci specific for the X chromosome only amount. If the amount is lower than baseline by an amount approximately equal to 1/2F or an amount that brings it down below a predetermined threshold, then the fetus is determined to be male; and if the amount is about equal to baseline or if not lower so that it falls below a predetermined threshold If the amount is below the threshold, then the fetus is determined to be female.

In another example, one can look only at those loci shared by the X and Y chromosomes, commonly referred to as the Z chromosome. The set of loci on the Z chromosome is always always A on the X chromosome, and always always B on the Y chromosome. If the SNP from the Z chromosome was found to have the B genotype, the fetus was interpreted as male; if the SNP from the Z chromosome was found to have only the A genotype, the fetus was interpreted as female. In another embodiment, loci found only on the X chromosome can be looked at. A context such as AA|B is especially informative because the presence of B indicates that the fetus has an X chromosome from the father. Backgrounds such as AB|B are also informative, as we would expect to see that B is usually only half present in the case of female fetuses compared to male fetuses. In another example, one can look at SNPs on the Z chromosome, where both the A and B alleles are present on the X and Y chromosomes, and where it is known which SNPs are from the paternal Y chromosome, and which are from The father's X chromosome.

In one embodiment, it is possible to amplify single nucleotide positions known to vary between homologous non-recombination (HNR) regions shared by the Y and X chromosomes. Within this HNR region, there is a large degree of sequence identity between the X and Y chromosomes. Single nucleotide positions within this region of consensus, while invariant among X and Y chromosomes in a population, differ between X and Y chromosomes. Each PCR test can amplify sequences from loci present on both the X and Y chromosomes. Within each amplified sequence will be a single base that can be detected using sequencing or some other method.

In one embodiment, the sex of the fetus can be determined from free-floating DNA of the fetus found in maternal plasma, and the method comprises some or all of the following steps: 1) Design PCR (conventional or mini-PCR, optionally 2) Obtaining maternal plasma; 3) Using HNR X/Y PCR detection, PCR amplifies the target from maternal plasma ; 4) Sequencing the amplicon; 5) Checking the sequence data for the presence of one or more internal Y alleles in the amplified sequence. The presence of one or more will indicate a male fetus. Absence of all Y alleles in all amplicons indicates a female fetus.

In one embodiment, targeted sequencing can be used to measure DNA in maternal plasma and/or parental genotype. In one embodiment, all sequences that are unequivocally derived from DNA of paternal origin can be ignored. For example, in the background AA|AB, the number of A sequences can be counted and all B sequences ignored. To determine the heterozygosity rate of the above algorithm, the number of observed A sequences can be compared to the expected number of total sequences for a given probe. There are a number of ways in which the predicted number of sequences per probe can be calculated on a per-sample basis. In one embodiment, it is possible to use historical data to determine what fraction of all sequence reads belongs to each specific probe and then use this empirical score along with the total number of sequence reads to estimate the number of sequences at each probe. Another approach could be to target some known homozygous alleles and then use historical data to correlate the number of reads at each probe with the number of reads at known homozygous alleles. For each sample, the number of reads at the homozygous allele can then be measured and this measurement, along with an empirically derived relationship, then used to estimate the number of sequence reads at each probe.

In some embodiments, it is possible to determine the sex of the fetus by combining predictions obtained by multiple methods. In some embodiments, multiple methods are taken from the methods described in the present invention. In some embodiments, at least one of the methods is taken from the methods described herein.

In some embodiments, the methods described herein can be used to determine the ploidy state of a gestating fetus. In one embodiment, the ploidy calling method uses loci specific to the X chromosome or shared by the X and Y chromosomes, but does not utilize any Y-specific loci. In one embodiment, the ploidy calling method uses one or more of: a locus specific to the X chromosome, a locus common to the X and Y chromosomes, and a locus specific to the Y chromosome. In one example, where the sex chromosome ratios are similar, such as 45,X (Turner syndrome), 46,XX (normal female) and 47,XXX (trisomy X), the allelic distribution can be compared Discrimination is achieved from predicted allele distributions under various assumptions. In another embodiment, this may be accomplished by comparing the relative number of sequence reads of the sex chromosomes relative to one or more reference chromosomes that are assumed to be euploid. It should also be noted that these methods can be extended to include aneuploid cases.

Single Gene Disease Screening

In one embodiment, the method for determining the ploidy state of a fetus can be extended to enable simultaneous testing for monogenic disorders. Single gene disease diagnosis utilizes the same targeted approach as used for aneuploidy testing and requires additional specific targets. In one embodiment, monogenic NPD diagnosis is by linkage analysis. In many cases, direct testing of cfDNA samples is unreliable because the presence of maternal DNA makes it nearly impossible to determine whether the fetus has inherited the mother's mutation. Detection of unique paternally derived alleles is less challenging, but is only sufficiently informative if the disease is dominant and carried by the father, limiting the utility of the method. In one embodiment, the method involves PCR or related amplification methods.

In some embodiments, the methods involve using information from first-degree relatives to phase abnormal alleles in the parents around very closely linked SNPs. Parental Support can then be run on the targeted sequencing data obtained from these SNPs to determine which homologues (normal or abnormal) the fetus has inherited from both parents. As long as the SNPs are sufficiently linked, the inheritance of the genotype of the fetus can be determined very reliably. In some embodiments, the method comprises (a) adding to our composite pool for aneuploidy testing a set of SNP loci to densely flank a specified set of common diseases; Genetic data for each relative, reliably phase the alleles from these added SNPs with normal and abnormal alleles; and (c) in the region surrounding the disease locus in the inherited maternal and paternal Reconstruct fetal haplotypes or phase SNP allelic sets on this congener to determine fetal genotypes. In some embodiments, additional probes that are tightly linked to disease-linked loci are added to the set of polymorphic loci used for aneuploidy testing.

Reconstructing fetal diplotypes is challenging because the sample is a mixture of maternal and fetal DNA. In some embodiments, the method incorporates relative information for phasing SNPs and disease alleles, and then considers the physical distance of the SNPs and recombination data from position-specific recombination possibilities as well as genetic inheritance from maternal plasma. The observed data are measured to obtain the most likely genotype of the fetus.

In one embodiment, multiple additional probes per disease-linked locus are included in the panel of targeted polymorphic loci; the number of additional probes per disease-linked locus can be between 4 and 10, between Between 11 and 20, between 21 and 40, between 41 and 60, between 61 and 80 or combinations thereof.

Phasing diploid data from the parents can be challenging, and there are a number of ways this can be accomplished. Some are discussed in this disclosure, others are described in more detail in other publications (see e.g. PCT Publication No. WO2009105531, filed February 9, 2009, and PCT Publication No. WO2010017214, filed August 4, 2009, which describe The disclosures of each are hereby incorporated herein by reference in their entirety). In one embodiment, the parents can be phased by inference, by measuring haploid tissue from the parents, eg, by measuring one or more sperm or eggs. In one embodiment, parenthood can be phased by inference, using measured genotype data for first-degree relatives (eg, parents-of-parents or siblings). In one example, the parents can be phased by dilution, wherein the DNA is diluted in one or more wells to the extent that no more than about one copy of each haplotype is expected in each well, and then one or more DNA in each well. In one embodiment, parental genotypes can be phased using a computer program that uses population-based haplotype frequencies to infer the most likely phasing type. In one embodiment, a parent can be phased if phased haplotype data for the other parent and unphased genetic data for one or more genetic progeny of the parent are known. In some embodiments, a genetic offspring of a parent may be one or more embryos, fetuses, and/or born children. Some of these methods and others for phasing one or both parents are disclosed in more detail, for example, in U.S. Publication No. 2011/0033862, filed August 19, 2010; Publication No. 2011/0178719; U.S. Publication No. 2007/0184467, filed November 22, 2006; U.S. Publication No. 2008/0243398, filed March 17, 2008, each of which is hereby incorporated by reference in its entirety incorporated into this article.

fetal genome remodeling

In one aspect, the invention features a method for determining the haplotype of a fetus. In various embodiments, this method allows determining which polymorphic loci (e.g., SNPs) are inherited by the fetus and reconstructing which homologues (including recombination events) are present in the fetus (and thereby inserting sequences at polymorphic genes between seats). If necessary, essentially the entire genome of the fetus can be reconstructed. If some uncertainty remains in the genome of the fetus (such as the spacing of crossovers), this can be minimized by analyzing additional polymorphic loci, if necessary. In various embodiments, polymorphic loci are selected to encompass one or more of the chromosomes at a density that reduces any uncertainty to a desired level. This method is well suited for the detection of associated polymorphisms or other mutations in the fetus because it enables their detection to be based on linkage (eg, the presence of linked polymorphic loci in the fetal genome) rather than by direct detection of associated polymorphisms in the fetal genome. Morphology or other mutations. For example, if the parents are carriers of a mutation associated with cystic fibrosis (CF), a nucleic acid sample comprising maternal DNA from the mother of the fetus and fetal DNA from the fetus can be analyzed to determine whether the fetal DNA includes Mutant haplotype. In particular, polymorphic loci can be analyzed to determine whether fetal DNA includes a haplotype containing a CF mutation without necessarily detecting the CF mutation itself in the fetal DNA.

In some embodiments, the methods involve determining a parental haplotype (eg, the haplotype of the mother or father of the fetus). In some embodiments, this determination is made without the use of data from maternal or paternal relatives. In some embodiments, dilution methods as described herein and elsewhere (see, e.g., U.S. Publication No. 2011/0033862, filed August 19, 2010, which is hereby incorporated by reference in its entirety) are used sequentially , SNP genotyping or sequencing to determine parental haplotypes. Because the DNA is diluted, it is unlikely that more than one haplotype will be in the same section (or tube). Thus, effectively a single DNA molecule can be present in a tube, which allows the determination of haplotypes on a single DNA molecule. In some embodiments, the method includes dividing the DNA sample into fractions such that at least one of the fractions includes a chromosome or a chromosome segment from a pair of chromosomes, and dividing the DNA sample into at least one of the fractions The DNA sample is genotyped (eg, to determine the presence of two or more polymorphic loci) to determine the parental haplotype. In some embodiments, genotyping involves sequencing (eg, shotgun sequencing). In some embodiments, genotyping involves using a SNP array to detect polymorphic loci, e.g., at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different polymorphisms loci. In some embodiments, genotyping involves the use of multiplex PCR. In some embodiments, the method involves simultaneously hybridizing a portion of the sample to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different polymorphic loci (e.g. SNPs) are contacted to generate a reaction mixture; and the reaction mixture is subjected to primer extension reaction conditions to generate amplification products, which are measured with a high-throughput sequencer to generate sequencing data.

In some embodiments, the mother's haplotype is determined using data from relatives of the mother by any of the methods described herein. In some embodiments, the haplotype of the father is determined using data from the father's relatives by any of the methods described herein. In some embodiments, the haplotypes of the father and mother are determined. In some embodiments, at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000 DNA samples from the mother (or father) and relatives of the mother (or father) are determined using the SNP array Or the existence of 100,000 different polymorphic loci. In some embodiments, the method involves simultaneously hybridizing DNA samples from the mother (or father) and/or relatives of the mother (or father) to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000 , 40,000, 50,000, 75,000, or 100,000 primer pools of different polymorphic loci (e.g., SNPs) are contacted to generate a reaction mixture; and the reaction mixture is subjected to primer extension reaction conditions to generate an amplification product, measured with a high-throughput sequencer The products are amplified to generate sequencing data. Parental haplotypes can be determined based on SNP arrays or sequencing data. In some embodiments, parental data may be phased by methods described or referred to elsewhere in this document.

This parental haplotype data can be used to determine whether the fetus has inherited the parental haplotype. In some embodiments, a nucleic acid sample comprising maternal DNA from the mother of the fetus and fetal DNA from the fetus is analyzed using a SNP array to detect at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000 , 75,000 or 100,000 different polymorphic loci. In some embodiments, a nucleic acid sample comprising maternal DNA from the mother of the fetus and fetal DNA from the fetus is obtained by simultaneously hybridizing the samples to at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000 , 40,000, 50,000, 75,000, or 100,000 primer libraries for different polymorphic loci (eg, SNPs) are contacted to generate reaction mixtures for analysis. In some embodiments, the reaction mixture is subjected to primer extension reaction conditions to generate amplification products. In some embodiments, the amplification products are measured with a high-throughput sequencer to generate sequencing data. In various embodiments, the SNP array or sequencing data is used to create an index for any crossover by using data on the probability of chromosomal crossovers at different locations in the chromosome (e.g. by using recombination data, such as can be found in the HapMap database). Recombination Risk Score for Spacing), the correlation between polymorphic alleles on the chromosome is modeled to determine the parental haplotype. In some embodiments, allele counts at polymorphic loci are calculated in silico based on the sequencing data. In some embodiments, a plurality of ploidy hypotheses are created in silico, each for the different possible ploidy states of the chromosome; for each ploidy hypothesis, the predicted alleles for the polymorphic loci on the chromosome are Gene counting to construct a model (e.g., joint distribution model); using the joint distribution model and allele counts, to determine in silico the relative probability of each of the ploidy hypotheses; and by selecting the ploidy state corresponding to the hypothesis with the greatest probability , to interpret the ploidy status of the fetus. In some embodiments, the steps of constructing a joint distribution model for allele counts and determining the relative probability of each hypothesis are performed using a method that does not require the use of a reference chromosome.

In some embodiments, the fetal haplotype is determined for one or more chromosomes taken from the group consisting of chromosomes 13, 18, 21, X, and Y. In some embodiments, fetal haplotypes are determined for all fetal chromosomes. In various embodiments, the methods measure substantially the entire genome of the fetus. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the fetal genome is haplotyped. In some embodiments, the haplotype determination results for the fetus include information about which of at least 1,000, 2,000, 5,000, 7,500, 10,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 different polymorphic loci are present, etc. gene information.

Composition of DNA

Measuring the allelic distribution over a set of alleles can be advantageous when performing informative analysis on sequencing data measured on a mixture of fetal and maternal blood to determine genomic information about the fetus, such as the ploidy state of the fetus of. Unfortunately, in many cases, such as when attempting to determine the ploidy state of a fetus from the mixture of DNA found in the plasma of a maternal blood sample, the amount of DNA available is insufficient for direct measurement with good fidelity Allelic distribution in a mixture. In these cases, amplification of the DNA mixture will provide a sufficient number of DNA molecules so that the desired allelic distribution can be measured with good fidelity. However, current amplification methods commonly used in DNA amplification for sequencing are often very biased, meaning that they do not amplify both alleles of a polymorphic locus by the same amount. Biased amplification can result in an allelic distribution that is very different from that in the initial mixture. Highly accurate measurements of the relative amounts of alleles present at polymorphic loci are not required for most purposes. In contrast, in one embodiment of the invention, an amplification or enrichment method that specifically enriches for polymorphic alleles and preserves allele ratios is advantageous.

Described herein are various methods that can be used to preferentially enrich a DNA sample at multiple loci in a manner that minimizes allelic bias. Some examples are the use of circularizing probes to target multiple loci, where the 3' and 5' ends of the precircularized probes are designed to hybridize to polymorphic sites away from the targeted allele bases at one or several positions. Another example is the use of PCR probes, where the 3' end of the PCR probe is designed to hybridize to a base one or a few positions away from the polymorphic site of the targeted allele. Another example is the use of a split-and-merge approach to create DNA mixtures in which preferentially enriched loci are enriched with low allelic bias, without the drawbacks of direct recombination. Another example is the use of hybrid capture methods in which the capture probes are designed such that the region of the DNA in the capture probe that is designed to hybridize to the polymorphic site flanking the target is separated by one or a few bases from the polymorphic site. Dots separated.

In cases where the allelic distribution measured at a set of polymorphic loci is used to determine the ploidy state of an individual, it is necessary to maintain the relative amounts of the alleles as in a DNA sample prepared for genetic measurements. This preparation may involve WGA amplification, targeted amplification, selective enrichment techniques, hybridization capture techniques, circularization of probes or the amount of DNA to be amplified and/or selective enhancement of DNA corresponding to certain alleles Other methods of existence of molecules.

In some embodiments of the invention, there is a set of DNA probes designed for the locus of interest where the locus has the greatest minor allele frequency. In some embodiments of the invention, there is a set of probes designed to target where the genome has the greatest likelihood of a fetus with highly informative SNPs at those loci. In some embodiments of the invention, there is a set of probes designed for a locus of interest, wherein the probes are optimized for a given subset of the population. In some embodiments of the invention, there is a set of probes designed for a locus of interest, wherein the probes are optimized for a given mix of population subsets. In some embodiments of the invention, there is a set of probes designed for a locus of interest, wherein the probes are optimized for a given pair of parents derived from genes with different minor allele frequency profiles. different population subgroups. In some embodiments of the invention, there is a circularized strand of DNA comprising at least one base pair annealed to a piece of DNA of fetal origin. In some embodiments of the invention, there is a circularized strand of DNA comprising at least one base pair annealed to a piece of DNA having placental origin. In some embodiments of the invention, there is a circularized strand of circularized DNA with at least some of the nucleotides annealing to DNA of fetal origin. In some embodiments of the invention, there is a circularized strand of circularized DNA with at least some of the nucleotides annealing to DNA of placental origin. In some embodiments of the invention, there is a set of probes, wherein some of the probes target single tandem repeats and some of the probes target single nucleotide polymorphisms. In some embodiments, loci are selected for non-invasive prenatal diagnosis. In some embodiments, the probe is used for non-invasive prenatal diagnosis. In some embodiments, loci are targeted using methods that may include circularizing probes, MIPs, probe capture by hybridization, probes on SNP arrays, or combinations thereof. In some embodiments, the probes are used as circularization probes, MIPs, capture by hybridization probes, probes on SNP arrays, or combinations thereof. In some embodiments, the loci are sequenced for non-invasive prenatal diagnosis.

In cases where the relative informativeness of the sequence is greater when combined with the relevant parental context, it follows that maximizing the number of sequence reads containing SNPs for which the parental context is known can maximize the number of sequencing read sets for mixed samples. The amount of information is at its maximum. In one embodiment, the number of sequence reads containing SNPs for which the parental context is known can be enhanced by preferentially amplifying specific sequences using qPCR. In one embodiment, the number of sequence reads containing SNPs for which the parental context is known can be enhanced by preferentially amplifying specific sequences using circularizing probes (eg, MIPs). In one embodiment, the number of sequence reads containing SNPs for which the parental context is known can be enhanced by preferentially amplifying specific sequences using capture by hybridization methods (eg, Hue Select). Different approaches can be used to enhance the number of sequence reads containing SNPs for which the parental context is known. In one example, targeting can be achieved by extension ligation, extension-free ligation, by hybridization capture, or PCR.

In a sample of fragmented genomic DNA, some DNA sequences uniquely map to individual chromosomes; other DNA sequences may be found on different chromosomes. It should be noted that DNA found in plasma (whether maternal or fetal in origin) is generally fragmented, usually to less than 500 bp in length. In a typical genomic sample, approximately 3.3% of mappable sequences would map to chromosome 13; 2.2% of mappable sequences would map to chromosome 18; 1.35% of mappable sequences would map to chromosome 21; 4.5% of mappable sequences would map to chromosome 18; Mapped sequences will map to the X chromosome in females; 2.25% of mappable sequences will map to the X chromosome (in males); and 0.73% of mappable sequences will map to the Y chromosome (in males). These are the chromosomes most likely to be aneuploid in the fetus. Also, in short sequences, approximately 1 in 20 sequences will contain a SNP, using the SNP contained on dbSNP. Given that there may be multiple SNPs that have not been discovered, the ratio is likely to be higher.

In one embodiment of the invention, targeted methods can be used to enhance the fraction of DNA in a DNA sample that maps to a given chromosome such that the fraction significantly exceeds the typical percentages listed above for genomic samples. In one embodiment of the invention, a targeted approach can be used to enhance the DNA fraction in a DNA sample such that the percentage of sequences containing a SNP is significantly greater than what would normally be found in a genomic sample. In one embodiment of the invention, targeting methods can be used to target target DNA from a chromosome or from a set of SNPs in a mixture of maternal and fetal DNA for prenatal diagnosis.

It should be noted that a method for determining fetal aneuploidy has been reported (US Patent 7,888,017) by counting the number of reads that map to a suspect chromosome and comparing it to the number of reads that map to a reference chromosome A comparison is made and an excess of reads on a suspected chromosome is used that corresponds to the hypothesis of triploidy at that chromosome in the fetus. Those methods for prenatal diagnosis will not utilize any class of targeting, nor do they describe the use of targeting for prenatal diagnosis.

By utilizing targeted approaches in sequencing mixed samples, it is possible to achieve a level of accuracy with fewer sequence reads. Accuracy can refer to sensitivity, it can refer to specificity, or it can refer to some combination thereof. The desired level of accuracy can be between 90% and 95%; it can be between 95% and 98%; it can be between 98% and 99%; it can be between 99% between 99.5% and 99.5%; it could be between 99.5% and 99.9%; it could be between 99.9% and 99.99%; it could be between 99.99% and 99.999%; it could be between Between 99.999% and 100%. An accuracy level of more than 95% can be called high accuracy.

In the prior art there are various published methods showing how the ploidy state of the fetus can be determined from a mixed sample of maternal and fetal DNA, for example: G.J.W. Liao et al. Clin Chem. 2011; 57(1) pp. Pages 92-101. These methods focus on thousands of locations along each chromosome. The number of positions along a chromosome that can be targeted yet for a given number of sequence reads to still yield highly accurate ploidy determinations for a fetus from a mixed sample of DNA is unexpectedly low. In one embodiment of the invention, accurate ploidy determination can be achieved by using targeted sequencing, using any targeted method, such as qPCR, ligand-mediated PCR, other PCR methods, capture by hybridization, or circularizing probes. , wherein the number of loci that need to be targeted along the chromosome can be between 5,000 and 2,000 loci; it can be between 2,000 and 1,000 loci; it can be between Between 1,000 and 500 loci; it could be between 500 and 300 loci; it could be between 300 and 200 loci; it could be between 200 and 150 between loci; it could be between 150 and 100 loci; it could be between 100 and 50 loci; it could be between 50 and 20 loci; It can be between 20 and 10 loci. Optimally, it may be between 100 and 500 loci. High levels of accuracy can be obtained by targeting a small number of loci and performing an unexpectedly small number of sequence reads. The number of readings can be between 100 million and 50 million readings; the number of readings can be between 50 million and 20 million readings; the number of readings can be between 20 million and 0.1 million readings; the number of readings can be between Between 10 million and 5 million readings; the number of readings can be between 5 million and 2 million readings; the number of readings can be between 2 million and 1 million; the number of readings can be between 1 million and 500,000 the number of readings may be between 500,000 and 200,000; the number of readings may be between 200,000 and 100,000; the number of readings may be between 100,000 and 50,000; the number of readings may be between 50,000 and 20,000; The number of reads may be between 20,000 and 10,000; the number of reads may be less than 10,000. Fewer read numbers are required for larger amounts of input DNA.

In some embodiments, there is a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin, wherein the percentage of sequences uniquely mapped to chromosome 13 is greater than 4%, greater than 5%, greater than 6 %, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the invention there is a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin wherein the percentage of sequences uniquely mapped to chromosome 18 is greater than 3%, greater than 4% , greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the invention there is a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin, wherein the percentage of sequences uniquely mapped to chromosome 21 is greater than 2%, greater than 3% , greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the invention, there is a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin, wherein the percentage of sequences uniquely mapped to the X chromosome is greater than 6%, greater than 7%, Greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30%. In some embodiments of the invention, there is a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin, wherein the percentage of sequences uniquely mapped to the Y chromosome is greater than 1%, greater than 2%, Greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15%, greater than 20%, greater than 25%, or greater than 30 %.

In some embodiments, a composition comprising a mixture of DNA of fetal origin and DNA of maternal origin wherein the percentage of sequences that uniquely map to a chromosome and contain at least one single nucleotide polymorphism is described Is greater than 0.2%, greater than 0.3%, greater than 0.4%, greater than 0.5%, greater than 0.6%, greater than 0.7%, greater than 0.8%, greater than 0.9%, greater than 1%, greater than 1.2%, greater than 1.4%, greater than 1.6%, greater than 1.8%, greater than 2%, greater than 2.5%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 12%, greater than 15% or greater than 20%; and wherein said chromosome is taken from the group 13, 18, 21, X or Y. In some embodiments of the invention, there is a composition comprising a mixture of fetal-derived DNA and maternal-derived DNA that uniquely maps to a chromosome and contains at least one sequence from a set of SNPs The percentage of sequences with single nucleotide polymorphisms is greater than 0.15%, greater than 0.2%, greater than 0.3%, greater than 0.4%, greater than 0.5%, greater than 0.6%, greater than 0.7%, greater than 0.8%, greater than 0.9%, Greater than 1%, greater than 1.2%, greater than 1.4%, greater than 1.6%, greater than 1.8%, greater than 2%, greater than 2.5%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8 %, greater than 9%, greater than 10%, greater than 12%, greater than 15% or greater than 20%, wherein the chromosome is taken from No. 13, No. 18, No. 21, X and Y chromosome groups, and wherein in the single nucleotide The number of SNPs in a polymorphism set is between 1 and 10, between 10 and 20, between 20 and 50, between 50 and 100, between Between 100 and 200, between 200 and 500, between 500 and 1,000, between 1,000 and 2,000, between 2,000 and 5,000, between 5,000 and 10,000, between 10,000 and Between 20,000, between 20,000 and 50,000, and between 50,000 and 100,000.

In theory, each cycle of amplification doubles the amount of DNA present; in practice, however, the degree of amplification is slightly less than two. In theory, amplification (including targeted amplification) will produce unbiased amplification of DNA mixtures; in practice, however, different alleles will often be amplified to a different extent than other alleles. When DNA is amplified, the degree of allelic bias generally increases with the number of amplification steps. In some embodiments, the methods described herein involve amplifying DNA with low levels of allelic bias. Because allelic bias increases with each cycle, allelic bias per cycle can be determined by computing the nth root of the overall bias, where n is the base-2 logarithm of the enrichment. In some embodiments, there is a composition comprising a second DNA mixture, wherein the second DNA mixture has been preferentially enriched at a plurality of polymorphic loci from the first DNA mixture, wherein the degree of enrichment is at least 10 , at least 100, at least 1,000, at least 10,000, at least 100,000, or at least 1,000,000, and wherein the ratio of alleles at each locus in the second DNA mixture to the allele at that locus in the first DNA mixture The ratios of genes differ on average by less than 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05% , 0.02% or 0.01%. In some embodiments, there is a composition comprising a second mixture of DNA, wherein the second mixture of DNA has been preferentially enriched from a first mixture of DNA at a plurality of polymorphic loci, wherein the plurality of polymorphic loci The allelic bias per cycle averaged less than 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.02%. In some embodiments, the plurality of polymorphic loci comprises at least 10 loci, at least 20 loci, at least 50 loci, at least 100 loci, at least 200 loci, at least 500 loci, at least 1,000 loci, at least 2,000 loci, at least 5,000 loci, at least 10,000 loci, at least 20,000 loci, or at least 50,000 loci.

some examples

In some embodiments, disclosed herein is a method for generating a report revealing a ploidy state determined in a gestating fetus, the method comprising: obtaining DNA containing DNA from the mother of the fetus and DNA from the fetus. A first sample of DNA; obtaining genotype data from one or both of the fetal parents; preparing a first sample by isolating DNA to obtain a prepared sample; measuring DNA at multiple polymorphic loci in a prepared sample; From the DNA measurements obtained, allele counts or allele count probabilities at multiple polymorphic loci are calculated on a computer; for different possible ploidy states of the chromosome, multiple ploidy states on the chromosome are created on the computer Ploidy assumptions for predicted allele count probabilities for multiple polymorphic loci; Construct a joint distribution model for the allele count probabilities of the ploidy hypotheses; using the joint distribution model and the allele count probabilities calculated for the prepared samples, the relative probabilities of each of the ploidy hypotheses are determined in silico; assumed ploidy status, deciphering the ploidy status of the fetus; and generating a report revealing the determined ploidy status.

In some embodiments, the method is used to determine the ploidy state of a corresponding plurality of gestating fetuses in a plurality of mothers, the method further comprising: determining the percentage of fetal-derived DNA in each of the prepared samples; and wherein the step of measuring the DNA in the prepared samples is performed by sequencing a plurality of DNA molecules in each of the prepared samples, wherein the sequenced DNA molecules are from those preparations with a smaller fraction of fetal DNA than from those preparations with Those with greater fetal DNA fractions prepared more samples.

In some embodiments, the method is used to determine the ploidy state of a corresponding plurality of gestating fetuses in a plurality of mothers, and wherein, for each of the fetuses, by sequencing a first fraction of the prepared DNA sample, Obtaining a first set of measurements to measure DNA in the prepared sample, the method further comprising: determining a first relative probability for each of the ploidy hypotheses for each of the fetuses given the first set of DNA measurements; A second fraction of prepared samples of those fetuses in which the first relative probability determination for each of the ploidy hypotheses indicates a significant but inconclusive probability corresponding to the ploidy hypothesis for aneuploid fetuses is resequenced to obtain A second set of measurements; using the second set of measurements and optionally also using the first set of measurements, determine a second relative probability of a ploidy hypothesis for the fetus; and by selecting the The hypothetical ploidy state determined by probabilistic determination) is interpreted as the ploidy state of the fetuses whose second sample was resequenced.

In some embodiments, a target composition is disclosed comprising: a preferentially enriched DNA sample, wherein the preferentially enriched DNA sample has been selected from a first DNA sample at a plurality of polymorphic loci preferential enrichment, wherein the first DNA sample consists of a mixture of maternal DNA and fetal DNA derived from maternal plasma, wherein the degree of enrichment is at least 2-fold, and wherein the average of the first sample and the preferentially enriched sample The allelic bias is selected from the group consisting of less than 2%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.02%, and less than 0.01%. In some embodiments, a method of creating such preferentially enriched DNA samples is disclosed.

In some embodiments, a method for determining the presence or absence of fetal aneuploidy in a sample of maternal tissue comprising fetal and maternal genomic DNA is disclosed, wherein the method comprises: (a) obtaining from The maternal tissue sample obtains a mixture of fetal and maternal genomic DNA; (b) selectively enriches the mixture of fetal and maternal DNA at multiple polymorphic alleles; (c) makes fetal and maternal DNA from step a The selective enrichment fragments of the mixture of maternal genomic DNA are distributed to obtain a reaction sample comprising a single genomic DNA molecule or an amplification product of a single genomic DNA molecule; (d) the selective enrichment in the reaction sample of step c) performing massively parallel DNA sequencing on the collected genomic DNA fragments to determine the sequence of said selectively enriched fragments; (e) identifying the chromosome to which the sequence obtained in step d) belongs; (f) analyzing the data of step d) to determine i ) the number of genomic DNA fragments from step d) that belong to at least one first target chromosome that is assumed to be diploid in the mother and fetus, and ii) the number of genomic DNA fragments from step d) that belong to a second target chromosome quantity, wherein said second chromosome is suspected to be aneuploid in the fetus; (g) if the second target chromosome is euploid, then using the quantity determined in step f) part i), calculate the the expected distribution of the number of second target chromosomes in the genomic DNA fragment; (h) if the second target chromosome is aneuploid, then use the first number in step f) part i) and the mixture of step b) an estimated fraction of fetal DNA found, calculating an expected distribution of the number of second target chromosomes in the genomic DNA fragments from step d); and (i) using maximum likelihood or maximum a posteriori methods to determine ) is more likely to be part of the distribution calculated in step g) or the distribution calculated in step h); thereby indicating the presence or absence of fetal aneuploidy.

Exemplary Cancer Diagnostic Methods

It should be noted that it has been shown that DNA derived from cancer living in a subject can be found in the blood of the subject. In the same way that genetic diagnosis can be made from measurements of admixture of DNA found in the maternal blood, genetic diagnosis can equally well be made from measurements of admixture of DNA found in the subject's blood. Genetic diagnosis can include aneuploidy status or genetic mutations. Any claim of the invention that reads on the basis of determining the ploidy state or genetic state of the fetus from measurements obtained on the mother's blood can equally well read on the basis of determining the ploidy state or genetic state of the cancer from measurements on the subject's blood. .

In some embodiments, the methods of the invention allow for the determination of the ploidy state of a cancer, the method comprising obtaining a mixed sample containing genetic material from a subject and genetic material from a cancer; measuring the DNA in the mixed sample; the fraction of cancer-derived DNA; and determining the ploidy state of the cancer using the measurements obtained on the pooled sample and the calculated fraction. In some embodiments, the method can further comprise administering a cancer therapy based on the determination of the ploidy state of the cancer. In some embodiments, the method can further comprise administering a cancer treatment based on the determination of the ploidy state of the cancer, wherein the cancer treatment is taken from the group comprising drugs, biological therapies, and antibody-based therapies, and combinations thereof.

Exemplary Implementations

Any of the embodiments disclosed herein may be implemented in digital electronic circuitry, integrated circuits, specially designed ASICs (Application Specific Integrated Circuits), computer hardware, firmware, software, or combinations thereof. The apparatus of the disclosed embodiments of the present invention may be implemented as a computer program product tangibly embodied in a machine-readable storage device executed by a programmable processor; and the method steps of the disclosed embodiments of the present invention may be implemented by referring to The operations of inputting data and generating output are performed by a programmable processor executing program instructions for carrying out the functions of the disclosed embodiments of the invention. The disclosed embodiments of the invention may be advantageously implemented in one or more computer programs executable and/or interpretable on a programmable system comprising at least one programmable processor, which may be a dedicated or in general, coupled to receive data and instructions from and transmit data and instructions to the storage system, at least one input device, and at least one output device. Each computer program can be implemented in a high-level procedural or object-oriented programming language, or in assembly or machine language if necessary; and in any case, the language can be a compiled or interpreted language. A computer program can be implemented in any form, including as a stand-alone program or as a module, component, subroutine or other unit suitable for use in a computing environment. A computer program can be intended to be executed or interpreted on one computer or on multiple computers at one site or distributed across multiple sites and interconnected by a communication network.

As used herein, computer-readable storage media refers to physical or tangible storage (as opposed to signals) and includes, but is not limited to, volatile and nonvolatile media implemented in any method or technology for tangible storage of information. , removable and non-removable media, such as computer readable instructions, data structures, program modules or other data. Computer-readable storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state storage technology, CD-ROM, DVD or other optical storage, cassette tape, magnetic tape, magnetic disk storage or other magnetic storage A device or any other physical or material medium that can be used to tangibly store desired information or data or instructions and which can be accessed by a computer or processor.

Any of the methods described herein may include the output of data in a tangible format, such as on a computer screen or on printed paper. In the explanation of any of the embodiments elsewhere in this document, it should be understood that the method can be combined with the output of actionable data in a format that can be acted upon by a physician. Additionally, the method may be combined with the actual execution of a clinical decision to generate clinical treatment or the execution of a clinical decision to take no action. Some of the embodiments described in this document with regard to determining genetic data about a target individual can be combined with the decision to select one or more embryos for transfer in the case of IVF, optionally with transferring the embryos to the expectant mother process composition of the uterus. Some of the embodiments described in this document with respect to determining genetic data about a target individual can be combined with notifying a medical professional of a potential chromosomal abnormality or lack thereof, optionally with the possibility of an abortion or no abortion in the case of prenatal diagnosis Decide on a combination. Some of the embodiments described herein may be combined with the output of actionable data, and the execution of a clinical decision to result in a clinical treatment or to take no action.

Exemplary diagnostic kit

In one embodiment, the invention comprises a diagnostic cartridge capable of partially or fully performing any of the methods described in the invention. In one embodiment, the diagnostic cassette may be located in a physician's office, a hospital laboratory, or any suitable location suitably close to the patient's point of care. The cassette may be capable of running the entire method in a fully automated fashion, or the cassette may require one or more steps to be performed manually by a technician. In one embodiment, the cassette may be capable of analyzing at least genotype data measured on maternal plasma. In one embodiment, the cartridge may be connected to means for transmitting the genotype data measured on the diagnostic cartridge to an external computing facility which may then analyze the genotype data and possibly also generate a report. A diagnostic cartridge may include a robotic unit capable of transferring an aqueous or liquid sample from one container to another. It can contain a variety of solid and liquid reagents. It can contain high-throughput sequencers. It can contain computers.

Experimental part

Disclosed embodiments of the present invention are described in the following examples, which are set forth to aid in the understanding of the present invention and should not be construed in any way as limiting the scope of the present invention as defined in the preceding claims . The following examples are presented in order to provide a complete disclosure and description of how the embodiments may be used by one of ordinary skill in the art, and are not intended to limit the scope of the invention, nor are they intended to represent that the following experiments were all or the only ones performed. Efforts have been made to ensure accuracy with respect to quantities used (eg, amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Parts are by volume and temperatures are in degrees Celsius unless otherwise specified. It is to be understood that method variants as described which can be obtained without changing fundamental aspects of the experiments are for illustration.

Experiment 1

The aim was to show that the Bayesian Maximum Likelihood Estimation (MLE) algorithm, which uses parental genotypes to calculate fetal fraction, improves the accuracy of non-invasive prenatal trisomy diagnosis compared to published methods.

Mock sequencing data of maternal cfDNA were created by sampling the reads obtained for trisomy 21 and the corresponding maternal cell line. The ratio of correct disomy and trisomy calls was determined from 500 simulations of various fetal fractions with respect to published methods (Yau et al. BMJ 2011;342:c7401) and our MLE-based algorithm. We validated the simulations by obtaining 5 million shotgun readings collected under an IRB-approved protocol from four pregnant mothers and corresponding fathers. Parental genotypes were obtained on a 290K SNP array. (See Figure 14)

In simulations, the MLE-based method achieved 99.0% accuracy for fetal fractions as low as 9% and reported confidence levels that corresponded well to the overall accuracy. We validated these results using four real samples where we obtained all correct calls with a calculated confidence of over 99%. In contrast, our implementation of the algorithm disclosed by Yau et al. required a fetal fraction of 18% to achieve 99.0% accuracy, and achieved only 87.8% accuracy at 9% fetal DNA.

Determination of fetal fraction from parental genotypes and MLE-based methods achieved greater accuracy than published algorithms for predicted fetal fractions in the first and second trimesters. Furthermore, the confidence measures produced by the methods disclosed herein are critical in determining the reliability of the results, especially at low fetal fractions where ploidy detection is more difficult. The published method uses a less accurate threshold method for calling ploidy based on a large disomy training data set, which is a method of pre-defining the false positive rate. In addition, published methods risk reporting false-negative results when fetal cfDNA is insufficient to make a call in the absence of a confidence measure. In some embodiments, a confidence estimate for the called ploidy state is calculated.

Experiment 2

The aim is to improve the non-invasive detection of fetal trisomies 18, 21 and X by using targeted sequencing methods together with parental genotype and Hapmap data with a Bayesian maximum likelihood estimation (MLE) algorithm, especially in the in samples composed of low fetal fractions.

Maternal samples and corresponding paternal samples from four euploid and two trisomy-positive pregnancies were obtained from patients with known fetal karyotypes according to an IRB-approved protocol. Maternal cfDNA was extracted from plasma and after preferential enrichment for the target-specific SNPs, approximately 10 million sequence reads were obtained. The parental samples were similarly sequenced to obtain genotypes.

The algorithm correctly called disomies 18 and 21 with respect to the normal chromosomes of all euploid samples and aneuploid samples. The interpretation of trisomy 18 and trisomy 21 was correct, as was the X chromosome copy number in male and female fetuses. The confidence levels generated by the algorithm exceeded 98% in all cases.

The method accurately reported the ploidy of all chromosomes tested for six samples, including samples consisting of less than 12% fetal DNA, which accounted for approximately 30% of first trimester 1 and 2 trimester samples. A key difference between the present MLE algorithm and the disclosed method is that it utilizes parental genotype and Hapmap data to improve accuracy and generate confidence measures. At low fetal fractions, all methods become less accurate; it is important to correctly identify samples where fetal cfDNA is insufficient for reliable interpretation. Others have used Y-chromosome-specific probes to estimate fetal fraction in male fetuses, but parallel parental genotyping enables estimation of fetal fraction in both sexes. Another inherent limitation of published methods using non-targeted shotgun sequencing is that the accuracy of ploidy calls for chromosomes varies due to differences in factors such as GC abundance. The targeted sequencing method of the present invention is largely independent of such chromosome scale variations and achieves a more consistent performance across chromosomes.

Experiment 3

The aim was to determine whether novel informatics could be used to analyze free-floating fetal DNA in maternal plasma for SNP loci to detect trisomy in triploid fetuses with high confidence.

Following the abnormal ultrasound, 20 mL of blood was drawn from the pregnant patient. After centrifugation, maternal DNA was extracted from buffy coat (DNEASY, QIAGEN); cell-free DNA was extracted from plasma (QIAAMP QIAGEN). Targeted sequencing was applied to SNP loci on chromosomes 2, 21 and X in both DNA samples. Maximum likelihood Bayesian estimation selects the most likely hypothesis from all possible groups of ploidy states. The method determines fetal DNA fraction, ploidy state and unambiguous confidence in ploidy determination. No assumptions were made about the ploidy of the reference chromosome. The diagnosis uses a test statistic independent of sequence read counts, which is the latest state of the art.

The method of the invention accurately diagnoses the trisomy of No. 2 and No. 21 chromosomes. The estimated child score was 11.9% [CI11.7-12.1]. The fetus was found to have one maternal and two paternal copies of chromosomes 2 and 21 with an effective confidence level of 1 (probability of error <10 ⁻³⁰ ). This was achieved with 92,600 and 258,100 reads on chromosomes 2 and 21, respectively.

This is the first demonstration of non-invasive prenatal diagnosis of trisomy from maternal blood in which the fetus is triploid, as confirmed by metaphase karyotype. Existing methods of non-invasive diagnosis would not detect aneuploidy in this sample. Current methods rely on excess sequence reads on trisomic chromosomes relative to a disomic reference chromosome; however, triploid fetuses do not have a disomic reference. Furthermore, existing methods do not achieve similarly high confidence ploidy determinations at this fraction of fetal DNA and number of sequence reads. It is straightforward to extend the method to all 24 chromosomes.

Experiment 4

The following protocol was used for the 800-fold amplification of DNA isolated from maternal plasma of euploid pregnant women and genomic DNA from the triploid 21 cell line using standard PCR (meaning no nesting was used). Library preparation and amplification involved blunting of single tube ends followed by A-tailing. Adapter ligation was run using the ligation kit found in the Agilent Huell Select kit, and PCR was run for 7 cycles. Then, 15 STA cycles (95°C, 30s; 72°C, 1min; 60°C, 4min; 65°C, 1min; 72°C) were performed using 800 different primer pairs targeting SNPs on chromosome 2, 21, and X , 30s). Reactions were run with a primer concentration of 12.5 nM. DNA was then sequenced with an Illumina IIGAX sequencer. The sequencer output 1.9 million reads, 92% of which mapped to the genome; of those reads that mapped to the genome, more than 99% mapped to one of the regions targeted by the targeting primers. The amounts of plasma DNA and genomic DNA are essentially the same. Figure 15 shows the ratio of two alleles of about 780 SNPs detected by a sequencer in genomic DNA obtained from a cell line known to be trisomy at chromosome 21. It should be noted that allele ratios are plotted here for ease of visualization, as allele distributions are not straightforward to read visually. Circles indicate SNPs on disomic chromosomes, while stars indicate SNPs on trisomic chromosomes. Figure 16 is another representation of the same data as in Panel X, where the Y-axis is the relative amount of A and B measured for each SNP, and where the X-axis is the number of SNPs where the SNPs are separated by chromosomes. In Figure 16, SNP1 to 312 are found on chromosome 2, SNP 313 to 605 are found on trisomy 21, and SNP 606 to 800 are on chromosome X. The data for chromosomes 2 and X show disomic chromosomes because the relative sequence counts are in three clusters: AA at the top of the plot, BB at the bottom of the plot, and AB in the middle of the plot. The data for trisomy 21 show four clusters: AAA at the top of the plot, AAB around the line 0.65 (2/3), ABB around the line 0.35 (1/3), and BBB at the bottom of the plot.

Figures 17A-D show data for the same 800-plex protocol, but measured on DNA amplified from four plasma samples from pregnant women. For these four samples, we expect to see seven clusters of points: (1) along the top of the plot are those loci where both the mother and fetus are AA; (2) just below the top of the plot are those loci where the mother Those loci that are AA and the fetus is AB; (3) just above the line 0.5 are those loci where the mother is AB and the fetus is AA; (4) along the line 0.5 are those where both mother and fetus are AB (5) slightly below the line 0.5 are those loci where the mother is AB and the fetus is BB; (6) slightly above the bottom of the plot are those genes where the mother is BB and the fetus is AB loci; (1) along the bottom of the figure are those loci where both mother and fetus are BB. The smaller the fetal fraction, the smaller the intervals between clusters (1) and (2), between clusters (3), (4) and (5), and between clusters (6) and (7). The interval is expected to be half the fraction of fetal-derived DNA. For example, if the DNA is 20% fetal and 80% maternal, then we would expect (1) to (7) to be centered around 1.0, 0.9, 0.6, 0.5, 0.4, 0.1, and 0.0, respectively; see eg Figure 17D , POOL1_BC5_Reference Ratio. If instead the DNA is 8% fetal and 92% maternal, then we expect (1) to (7) to be centered at 1.00, 0.96, 0.54, 0.50, 0.46, 0.04, and 0.00, respectively; see for example Figure 17B, POOL1_BC2 _Reference ratio. If no fetal DNA is detected, then we would not expect to see (2), (3), (5) or (6); alternatively, we could say that the interval is zero, and thus (1) and (2) overlap each other, As with (3), (4) and (5) and (6) and (7); see eg Figure 17C, POOL1_BC7_Reference Ratio. It should be noted, Figure 17A, that the fetal fraction of POOL1_BC1_reference ratio is about 25%.

Experiment 5

Most methods of DNA amplification and measurement introduce some allelic bias, where the detected intensities or counts of the two alleles commonly found at a locus do not represent the actual amount of the allele in the DNA sample. For example, for a single individual, at a heterozygous locus, we would expect to see a 1:1 ratio of the two alleles, which is the predicted theoretical ratio for a heterozygous locus; however, due to allelic bias, we would See 55:45, or even 60:40. It should also be noted that in the case of sequencing, simple random noise can cause significant allelic bias if the read depth is low. In one embodiment, it is possible to model the behavior of each SNP such that if a consistent bias is observed for a particular allele, this bias can be corrected for. Figure 18 shows the fraction of data that can be explained by the binomial variance before and after bias correction. In Figure 18, the stars represent the allelic bias observed on the raw sequence data of the 800-plex experiment; the circles represent the allelic bias after correction. Note that if there was no allelic bias at all, then we would expect the data to fall along the line x=y. A similar set of data generated by amplifying DNA using 150-plex targeted amplification produced data that fell very close to the line 1:1 after bias correction.

Experiment 6

Universal amplification of DNA using ligated adapters with primers specific for adapter tags has the effect of enriching the fraction of shorter DNA strands where primer annealing and extension times are limited to a few minutes. Most library protocols designed to create DNA libraries suitable for sequencing contain such steps, and exemplary protocols are disclosed and well known to those skilled in the art. In some embodiments of the invention, adapters containing universal markers are ligated to plasma DNA and amplified using primers specific for the adapter markers. In some embodiments, the universal marker can be the same marker as used for sequencing, it can be a universal marker used only for PCR amplification or it can be a set of markers. Because fetal DNA is generally short in nature, while maternal DNA can be both short and long in nature, this method has the effect of enriching the proportion of fetal DNA in the mixture. Free-floating DNA is thought to be DNA from apoptotic cells, and contains both fetal and maternal DNA, and is short, mostly below 200 bp. Cellular DNA released by cell lysis (a common phenomenon following phlebotomy) is usually almost exclusively maternal and is also very long, mostly in excess of 500 bp. Thus, a blood sample that has been left undisturbed for more than a few minutes will contain a mixture of short (fetal+maternal) and longer (maternal) DNA. Performing universal amplification on maternal plasma with a relatively short extension time followed by targeted amplification will tend to increase the relative proportion of fetal DNA when compared to plasma that has been amplified using only targeted amplification. This can be seen in Figure 19, which shows the measured fetal percentage (vertical axis) when the input is plasma DNA versus plasma DNA from a library that has been prepared using the Illumina GAIIx library preparation protocol Fetal percentage measured at time. All points fell below the line, indicating that the library preparation step enriched the fraction of fetal-derived DNA. The two plasma samples in red (indicating hemolysis and thus the amount of long maternal DNA present will increase from lysis) show a particularly pronounced enrichment of the fetal fraction when library preparation is performed prior to targeted amplification . The methods disclosed herein are particularly applicable in situations where there is hemolysis or some other situation where cells containing relatively long strands of contaminated DNA have been lysed, mixed samples of short and long DNA are contaminated . Relatively short annealing and extension times are typically between 30 seconds and 2 minutes, but they can be as short as 5 or 10 seconds or less, or as long as 5 or 10 minutes.

Experiment 7

The following protocol was used for the 1,200-plex amplification of DNA isolated from maternal plasma of a euploid pregnant woman and genomic DNA from a triploid 21 cell line using a direct PCR protocol with a semi-nested approach. Library preparation and amplification involved blunting of single tube ends followed by A-tailing. Adapter ligation was performed using a modification of the ligation kit found in the Agilent Select kit, and PCR was run for 7 cycles. In the targeted primer pool, 550 tests were performed for SNPs from chromosome 21 and 325 tests were performed for SNPs from each of chromosomes 1 and X. Both protocols involved 15 STA cycles (95°C, 30s; 72°C, 1 min; 60°C, 4min; 65°C, 30s; 72°C, 30s), using 16nM primer concentrations. The semi-nested PCR protocol involved a second amplification of 15 STA cycles (95°C, 30s; 72°C, 1min; 60°C, 4min; 65°C, 30s; 72°C, 30s), using an internal forward marker concentration of 29nM and 1 μM or 0.1 μM reverse labeling concentration. DNA was then sequenced with an Illumina IIGAX sequencer. For the direct PCR protocol, 73% of the reads mapped to the genome; for the semi-nested protocol, 97.2% of the sequence reads mapped to the genome. Thus, the semi-nested protocol yielded about 30% more information, probably primarily due to the elimination of the primers most likely to generate primer-dimers.

Read depth variability tends to be higher when using the semi-nested protocol than when using the direct PCR protocol (see Figure 20), where diamonds refer to the read depth for loci run with the semi-nested protocol and squares refer to Read depth for loci run without nesting. Regarding the diamonds, the SNPs are arranged by read depth, so the diamonds all fall on the curve, while the squares seem to be loosely correlated; the arrangement of the SNPs is arbitrary, and the height of the point indicates the read depth rather than its left-to-right position .

In some embodiments, the methods described herein can achieve excellent depth-of-read (DOR) variance. For example, in one version of this experiment (FIG. 21 ) using 1,200-plex direct PCR amplification of genomic DNA, out of 1,200 assays: 1186 assays had a DOR greater than 10; the average read depth was 400; 1063 assays (88.6%) had a read depth between 200 and 800 with an ideal window where the number of reads per allele was high enough to give meaningful data, whereas the number of reads per allele was not The marginal use of readings as high as those is particularly small. Only 12 alleles had higher read depth, the highest being 1035 reads. The standard deviation of DOR was 290, the mean DOR was 453, the coefficient of variance of DOR was 64%, there were 950,000 total reads, and 63.1% of the reads mapped to the genome. In another experiment (FIG. 22) using a 1,200-fold semi-nested scheme, the DOR was higher. The standard deviation of DOR was 583, the mean DOR was 630, the coefficient of variance of DOR was 93%, there were 870,000 total reads, and 96.3% of the reads mapped to the genome. Note that in both cases, the SNPs are ranked by the mother's read depth, so the curve represents the maternal read depth. The difference between children and fathers is not significant; it's just a trend that is significant for this explanation.

Experiment 8

In one experiment, semi-nested 1,200-plex PCR was used to amplify DNA from one cell and from three cells. This experiment is relevant for prenatal aneuploidy testing using fetal cells isolated from maternal blood or for preimplantation genetic diagnosis using biopsied blastomere or trophectoderm samples. In each case there were 3 replicates of 1 and 3 cells from 2 individuals (46XY and 47XX+21). The test targets chromosomes 1, 21, and X. Three different lysis methods were used: ARCTURUS, MPERv2 and alkaline lysis. Sequencing was run on a pool of 48 samples in one sequencing lane. The algorithm returns the correct ploidy call for each of the three chromosomes and for each of the replicates.

Experiment 9

In one experiment, four maternal plasma samples were prepared and amplified using a hemi-nested 9,600-plex protocol. Samples were prepared as follows: Up to 40 mL of maternal blood was centrifuged to separate the buffy coat and plasma. Genomic DNA in maternal samples is prepared from buffy coat and paternal DNA is prepared from blood or saliva samples. Cell-free DNA in maternal plasma was isolated using the CIRCULATING NUCLEIC ACID kit and eluted in 45 μL TE buffer according to the manufacturer's instructions. Universal junction adapters were attached to the ends of each molecule of 35 μL of purified plasma DNA and the pool was amplified using adapter-specific primers for 7 cycles. The library was purified with AGENCOURTAMPURE beads and eluted in 50 μl of water.

3 μl of DNA was amplified for 15 STA cycles using 9600 target-specific tagged reverse primers at a primer concentration of 14.5 nM and one library adapter-specific forward primer at 500 nM (first polymerase activation at 95°C for 10 min; then is 15 cycles: 95°C, 30s; 72°C, 10s; 65°C, 1min; 60°C, 8min; 65°C, 3min and 72°C, 30s; and finally extension at 72°C for 2min).

The hemi-nested PCR protocol involved a second amplification of the diluted product of the first STA result using a reverse marker concentration of 1000 nM and a concentration of 16.6u nM for each of the 9600 target-specific forward primers for 15 STA cycle (first polymerase activation at 95°C for 10min; then 15 cycles: 95°C, 30s; 65°C, 1min; 60°C, 5min; 65°C, 5min and 72°C, 30s; and finally at 72°C extension 2min).

Aliquots of the STA products were then amplified by standard PCR with 1 μM marker-specific forward and barcoded reverse primers for 10 cycles to generate barcoded sequencing libraries. Aliquots of each library were mixed with different barcoded libraries and purified using spin columns.

In this way, 9,600 primers were used in a single well reaction; the primers were designed to target SNPs found on chromosomes 1, 2, 13, 18, 21, X and Y. The amplicons were then sequenced using an Illumina GAIIX sequencer. Through the sequencer, approximately 3.9 million reads were generated per sample, of which 3.7 million (94%) mapped to the genome, and of those reads, 2.9 million reads (74%) at an average read depth of 344 and a median read depth of 255 Depth mapping to targeted SNPs. The fetal fractions of the four samples were found to be 9.9%, 18.9%, 16.3% and 21.2%.

Related maternal and paternal genomic DNA samples were amplified and sequenced using a semi-nested 9600-plex protocol. The semi-nested protocol differs in that it applies 9,600 outer forward primers and 7.3nM labeled reverse primers in the first STA. Thermal cycling conditions and composition for the second STA and barcode PCR were the same as for the half-side nested protocol.

The sequencing data were analyzed using the informatics method disclosed herein and the ploidy status of six chromosomes of fetuses whose DNA was present in 4 maternal plasma samples was called. Ploidy calls for all 28 chromosomes in the panel were correctly called with greater than 99.2% confidence, except for one chromosome which was called correctly, but with 83% confidence.

Figure 23 shows the read depth of the 9,600 multiple hemi-nested approach and the 1,200 multiple hemi-nested approach described in Experiment 7, but the number of SNPs with read depths greater than 100, greater than 200, and greater than 400 were significantly Higher than 1,200 in heavy program. The number of reads at the 90th percentile can be divided by the number of reads at the 10th percentile to obtain a dimensionless measure indicating the uniformity of the read depth; the lower the number, the more uniform (narrow) the read depth. For the method run in Experiment 9, the average 90th percentile/10th percentile ratio was 11.5; for the method run in Experiment 7, the average ratio was 5.6. For a given protocol multiplex, the narrower the read depth, the better the sequencing efficiency because fewer sequence reads are necessary to ensure that a certain percentage of reads exceeds the read number threshold.

Experiment 10

In one experiment, four maternal plasma samples were prepared and amplified using a semi-nested 9,600-plex protocol. The details of Experiment 10 are very similar to Experiment 9, with the exception of a nested scheme and including the identities of the four samples. Ploidy calls for all 28 chromosomes in the panel were correctly called with greater than 99.7% confidence. 7.6 million (97%) reads mapped to the genome and 6.3 million (80%) reads mapped to targeted SNPs. The average reading depth was 751 and the median reading depth was 396.

Experiment 11

In one experiment, three maternal plasma samples were split into five equal fractions, and each fraction was amplified using 2,400 compound primers (four fractions) or 1,200 compound primers (one fraction) and using semi-nested Amplified by the formula protocol, a total of 10,800 primers. After amplification, the fractions are pooled together for sequencing. The details of Experiment 11 are very similar to Experiment 9, with the exception of the nesting scheme and the split-and-merge approach. The ploidy calls for all 21 chromosomes in the panel were correctly called with more than 99.7% confidence, except for one missing call, where the confidence was 83%. 3.4 million reads mapped to targeted SNPs with an average read depth of 404 and a median read depth of 258.

Experiment 12

In one experiment, four maternal plasma samples were split into four equal fractions, and each fraction was amplified with 2,400 composite primers and amplified using a semi-nested protocol, for a total of 9,600 primers. After amplification, the fractions are pooled together for sequencing. The details of Experiment 12 are very similar to Experiment 9, with the exception of the nesting scheme and the split-and-merge approach. The ploidy calls for all 28 chromosomes in the panel were correctly called with more than 97% confidence, except for one missing call, where the confidence was 78%. 4.5 million reads mapped to targeted SNPs with an average read depth of 535 and a median read depth of 412.

Experiment 13

In one experiment, four maternal plasma samples were prepared and amplified using a 9,600-plex triple hemi nested protocol for a total of 9,600 primers. The details of Experiment 12 were very similar to Experiment 9, with the exception of a nested protocol involving three rounds of amplification; the three rounds involved 15, 10 and 15 STA cycles, respectively. The ploidy calls for 27 of the 28 chromosomes in the panel were correctly called with greater than 99.9% confidence, except for one that was correctly called at 94.6% and one missing call with 80.8% confidence. 3.5 million reads mapped to targeted SNPs with an average read depth of 414 and a median read depth of 249.

Experiment 14

In one experiment, 45 sets of cells were amplified using a 1,200-plex semi-nested protocol, sequenced, and ploidy determined for three chromosomes. It should be noted that this experiment is intended to simulate the conditions of preimplantation genetic diagnosis on single cell biopsies from day 3 embryos or trophectoderm biopsies from day 5 embryos. 15 individual single cells and 30 groups of three cells were placed in 45 individual reaction tubes for a total of 45 reactions, where each reaction contained cells from only one cell line, but different reactions contained cells from different cell lines cell. Cells were prepared into 5 μl of wash buffer and lysed by adding 5 μl of Arcturus PICOPURE lysis buffer (Applied Biosystems) and incubated at 56°C for 20 minutes, at 95°C for 10 minute.

DNA from single/triple cells was amplified for 25 STA cycles using 1200 target-specific forward and labeled reverse primers at a primer concentration of 50 nM (first polymerase activation at 95°C for 10 min; then 25 cycles: 95°C, 30s; 72°C, 10s; 65°C, 1min; 60°C, 8min; 65°C, 3min and 72°C, 30s; and finally extension at 72°C for 2min).

The semi-nested PCR protocol involved three parallel runs of the diluted product of the first STA result using a reverse marker-specific primer concentration of 1000 nM and a concentration of 60 nM for each of the 400 target-specific nested forward primers. Second amplification, continued for 20 STA cycles (first polymerase activation at 95°C for 10min; then 15 cycles: 95°C, 30s; 65°C, 1min; 60°C, 5min; 65°C, 5min and 72°C, 30s ; and a final extension at 72° C. for 2 min). A total of 1200 targets amplified in the first STA were thus amplified in three parallel 400-plex reactions.

Aliquots of the STA products were then amplified by standard PCR with 1 μM marker-specific forward and barcoded reverse primers for 15 cycles to generate barcoded sequencing libraries. Aliquots of each library were mixed with different barcoded libraries and purified using spin columns.

In this way, 200 primers were used in single-cell reactions; the primers were designed to target SNPs found on chromosomes 1, 21 and X. The amplicons were then sequenced using an Illumina GAIIX sequencer. Through the sequencer, approximately 3.9 million reads were generated per sample, of which 500,000 to 800,000 million reads mapped to the genome (74% to 94% of all reads per sample).

Related maternal and paternal genomic DNA samples from cell lines were analyzed and sequenced using the same semi-nested 1200-plex pool with a similar protocol, with fewer cycles and a 1200-plex second STA.

The sequencing data was analyzed using the informative method disclosed herein and the ploidy state of the three chromosomes of the sample was called.

Figure 24 shows the normalized read depth ratios (vertical axis) for three chromosomes (1=chromosome 1; 2=chromosome 21; 3=chromosome X) for six samples. The ratio was set equal to the number of reads mapped to the chromosome, normalized, and divided by the number of reads mapped to the chromosome, averaged over three wells, each containing three 46XY cells. The expected ratio of the three sets of data points corresponding to the 46XY reaction is 1:1. The three sets of data points corresponding to 47XX+21 cells predict a ratio of 1:1 for chromosome 1, 1.5:1 for chromosome 21, and 2:1 for chromosome X.

Figure 25 shows the allele ratios plotted for the three reactions for the three chromosomes (1, 21, X). The lower left response shows the response on three 46XY cells. The left region is the allele ratio of chromosome 1, the middle region is the allele ratio of chromosome 21, and the right region is the allele ratio of X chromosome. For 46XY cells, for chromosome 1, we expect to see ratios of 1, 0.5, and 0, corresponding to the SNP genotypes AA, AB, and BB. For 46XY cells, for chromosome 21 we expect to see ratios of 1, 0.5, and 0, corresponding to the SNP genotypes AA, AB, and BB. For 46XY cells, for the X chromosome, we expect to see ratios of 1 and 0, corresponding to SNP genotypes A and B. The lower right reaction shows the reaction on three 47XX+21 cells. Each allele ratio is separated by chromosome as in the lower left panel. For 47XX+21 cells, for chromosome 1, we expect to see ratios of 1, 0.5, and 0, corresponding to the SNP genotypes AA, AB, and BB. For 47XX+21 cells, for chromosome 21, we expect to see ratios of 1, 0.67, 0.33, and 0, corresponding to the SNP genotypes AAA, AAB, ABB, and BBB. For 47XX+21 cells, for the X chromosome, we expect to see ratios of 1, 0.5, and 0, corresponding to the SNP genotypes AA, AB, and BB. The upper right graph was obtained for a reaction containing 1 ng of genomic DNA from the 47XX+21 cell line. Figure 26 shows the same graph as in Figure 25, but with the reaction performed on only one cell. The left panel is the reaction containing 47XX+21 cells and the right panel is the reaction containing 46XX cells.

From the plots shown in Figures 25 and 26, it is intuitively apparent that there are two clusters of points about chromosomes where we expect to see ratios of 1 and 0, and three clusters of points about chromosomes where we expect to see The ratios for are 1, 0.5, and 0; and four clusters of points about chromosomes, where we expect to see ratios of 1, 0.67, 0.33, and 0. The parental support algorithm was able to make correct calls for all three chromosomes for all 45 reactions.

Experiment 15

In one experiment, maternal plasma samples were prepared and amplified using a hemi-nested 19,488-plex protocol. Samples were prepared as follows: Up to 20 mL of maternal blood was centrifuged to separate the buffy coat and plasma. Genomic DNA in maternal samples is prepared from buffy coat and paternal DNA is prepared from blood or saliva samples. Cell-free DNA in maternal plasma was isolated using the Qiagen Circulating Nucleic Acid Kit and eluted in 50 μL TE buffer according to the manufacturer's instructions. Universal ligation adapters were attached to the ends of each molecule of 40 μL of purified plasma DNA and pools were amplified using adapter-specific primers for 9 cycles. The library was purified with Anzin Coat Ampulex beads and eluted in 50 μl of DNA suspension buffer.

6 μl of DNA was amplified for 15 round 1 STA cycles (first polymerase activation at 95°C) using 19,488 target-specific tagged reverse primers at a primer concentration of 7.5 nM and one library adapter-specific forward primer at 500 nM 10 min; followed by 15 cycles: 96 °C, 30 s; 65 °C, 1 min; 58 °C, 6 min; 60 °C, 8 min; 65 °C, 4 min and 72 °C, 30 s; and finally extension at 72 °C for 2 min).

The hemi-nested PCR protocol involved a second amplification of the diluted product of the 1st round of STA results for 15 cycles using a reverse marker concentration of 1000 nM and a concentration of 20 nM for each of the 19,488 target-specific forward primers (2nd round of STA) (first polymerase activation at 95°C for 10min; then 15 cycles: 95°C, 30s; 65°C, 1min; 60°C, 5min; 65°C, 5min and 72°C, 30s; and finally Extension at 72°C for 2 min).

Aliquots of round 2 STA products were then amplified by standard PCR with 1 μM marker-specific forward and barcoded reverse primers for 12 cycles to generate barcoded sequencing libraries. Aliquots of each library were mixed with different barcoded libraries and purified using spin columns.

In this way, 19,488 primers were used in a single well reaction; the primers were designed to target SNPs found on chromosomes 1, 2, 13, 18, 21, X and Y. The amplicons were then sequenced using an Illumina GAIIX sequencer. Through the sequencer, approximately 10 million reads were generated per plasma sample, of which 9.4-9.6 million reads mapped to the genome (94%-96%), and of those reads, 99.95% with a mean read depth of 460 and a median of 350 Read depth mapping to targeted SNPs. For comparison, a perfect uniform distribution would be: 10M reads/19,488 targets = 513 reads/target. Regarding primer-dimers, 30,000 reads were derived from sequenced primer-dimers (0.3% of reads generated by the sequencer). Regarding genomic samples, 99.4%-99.7% of reads mapped to the genome, and of those reads, 99.99% mapped to targeted SNPs, and 0.1% of reads generated by the sequencer were primer-dimers.

For a plasma sample with 10 million sequencing reads, typically at least 19,350 (99.3%) of the 19,488 targeted SNPs were amplified and sequenced. For a DNA sample with 2M sequencing reads, typically at least 19,000 targeted SNPs (97.5%) were amplified and sequenced. The lower number may be due to sampling noise, since the number of reads was lower and the sequencer missed some amplification products. If necessary, the number of sequencing reads can be increased to increase the number of targeted SNPs that are amplified and sequenced.

Related maternal and paternal genomic DNA samples were amplified in round 1 STA using 7.5 nM semi-nested 19,488 external forward primers and labeled reverse primers. Thermal cycling conditions and composition for round 2 STA and barcode PCR were the same as the half-side nested protocol.

The mean fetal fraction of 407 samples was found to be 14.8%. The sequencing data were analyzed using the informative method disclosed herein and the DNA was called for four chromosomes (13, 18, 21, Y) of the fetus in 378 of the 407 maternal plasma samples and in 407 maternal plasma samples. Ploidy status of the X chromosome in 375 of the present plasma samples. Ploidy calls were correctly called for all 1,887 chromosomes in the panel with greater than 90% confidence. 1882 of 1887 calls were greater than 95%; and 1,862 of 1,887 calls were called with greater than 99% confidence.

Control experiments similar to those in the plasma PCR protocol were performed using water instead of DNA extracted from plasma. Based on six such trials experimented, 5%-6% of sequencing reads were primer-dimers. Other sequencing reads were due to background noise. This experiment demonstrates that even in the absence of a nucleic acid sample containing a target locus to which a primer hybridizes (rather than hybridizing to other primers and forming amplification primer-dimers), fewer primer-dimers are formed.

Experiment 16

The following experiments demonstrate exemplary methods for designing and selecting primer libraries that can be used in any of the multiplex PCR methods of the invention. The goal is to select from an initial pool of candidate primers primers that can be used to simultaneously amplify a large number of target loci (or subsets of target loci) in a single reaction. With respect to an initial set of candidate target loci, it is not necessary to design or select primers for each target locus. Preferably, primers are designed and selected for most of the most desirable loci of interest.

step 1

Based on publicly available information on desired parameters of the target locus, such as the frequency of SNPs within the target population or the heterozygosity rate of SNPs (www.ncbi.nlm.nih.gov/projects/SNP/; Sherry ST (Sherry ST), Ward MH (Ward MH), Korodov M (Kholodov M) et al. dbSNP: the NCBI database of genetic variation (dbSNP: the NCBI database of genetic variation). Nucleic Acids Res. 2001 Jan 1;29(1):308-11, each of which is incorporated herein by reference in its entirety), to select a set of candidate target loci (eg, SNPs). For each candidate locus, one or more PCR primer pairs were designed using the Primer3 program (www.primer3.sourceforge.net; libprimer3 version 2.2.3, which is hereby incorporated by reference in its entirety). If there is no viable PCR primer design for a particular locus of interest, then on further consideration, that locus of interest is eliminated.

If necessary, a "target locus score" (higher scores indicating higher desirability) can be calculated for most or all target loci, e.g. based on a weighted average of various desired parameters for the target loci, calculating target locus fraction. The parameters can be assigned different weights based on their importance to the particular application in which the primer will be used. Exemplary parameters include heterozygosity at the locus of interest, prevalence of disease associated with a sequence (e.g., polymorphism) at the locus of interest, prevalence of disease associated with a sequence (e.g., polymorphism) at the locus of interest, Penetrance, specificity of the candidate primers used to amplify the target locus, size of the candidate primers used to expand the target locus, and the size of the target amplicon.

step 2

Calculate the thermodynamic interaction scores between each primer and all primers for all other target loci from step 1 (see e.g. Allawi, H.T. and Santa Lucia, J., Jr.) (1998), "Thermodynamics of Internal C-T Mismatches in DNA (Thermodynamics of Internal C-T Mismatches in DNA)", Nucleic Acids Research 26, 2694-2701; Pere N. (Peyret, N.), Seina Villatner P.A. (Seneviratne, P.A.), Alavi H.T., and Santa Lucia J. (1999), "Nearest-Neighbor Thermodynamics and NMR of DNA sequences with internal A-A, C-C, G-G, and T-T mismatches. NMR ofDNA Sequences with Internal A-A, C-C, G-G, and T-T Mismatches)", Biochemistry (Biochemistry) 38, 3468-3477; Alavi H.T. and Santa Lucia J. (1998), "The internal A-C in DNA Nearest-Neighbor Thermodynamics of Internal A-C Mismatches in DNA: Sequence Dependence and pH Effects", Biochemistry 37, 9435-9444; Alavi H.T. and Santa Lucia Jr. J. (1998), "Nearest Neighbor Thermodynamic Parameters for Internal G-A Mismatches in DNA", Biochemistry 37, 2170-2179; and Alavi H.T. and Santa Lucia Jr. J. (1997), "Thermodynamics and NMR of Internal G-T mismatches in DNA" and "Thermodynamics and NMR of Internal G-TMismatches in DNA", Biochemistry 36, 10581-10594; MultiPLX 2.1 (Kaplinsky L( Kaplinski L), Andreson R (Andreson R), Puurand T (Puurand T), Remm M (Remm M). MultiPLX: automatic grouping and evaluation of PCR primers (MultiPLX: automatic grouping and evalu ation of PCR primers. Bioinformatics. 2005 Apr 15;21(8):1701-2, each of which is hereby incorporated herein by reference in its entirety). This step yields a 2D matrix of cross-correlation scores. The interaction scores predict the likelihood of a primer-dimer involving two interacting primers. Calculate the score as follows:

Interaction score = max(-ΔG_2, 0.8*(-ΔG_i))

in

ΔG_2 = Gibbs energy (the energy required to break the dimer) of the dimer that can be extended by PCR on both ends (i.e. the 3' end of each primer anneals to the other primer); and

ΔG_1 = Gibbs energy of a dimer that can be extended on at least one terminus by PCR.

Step 3:

For each locus of interest, if more than one primer pair design exists, choose a design using the following method:

1 For each primer pair design for a locus, find the worst-case (highest) interaction score for all primers for both primers in that design and for all designs for all other target loci.

2 Pick the design with the best (lowest) worst-case interaction score.

step 4

A graph is constructed such that each node represents a locus and its associated primer pair design (eg, maximal clique problem). Create an edge between each pair of nodes. Each edge is assigned a weight equal to the worst-case (highest) interaction score between primers associated with the two nodes connected by the edge.

step 5

When necessary, for each pair of designs for two different target loci, where one primer from one design and one primer from the other design will anneal to overlap the region of interest, between the nodes of the two designs Add a side in between. Set the weights of these edges equal to the highest weight assigned in step 4. Thus, step 5 prevents the library from having primers that would anneal to overlap the region of interest, and thus prevent interference with each other during the multiplex PCR reaction.

step 6

The initial mutual use score threshold is calculated as follows:

Weight threshold = max(edge weight)-0.05*(max(edge weight)-min(edge weight))

in

max(edgeweight) is the maximum edge weight in the graph; and

min(edgeweight) is the minimum edge weight in the graph.

The initial bounds of the threshold are set as follows:

max_weightthreshold = max(edge_weight)

min_weight threshold = min(edge weight)

step 7

Construct a new graph consisting of the same set of nodes as the graph of step 5, including only edges whose weight exceeds a weight threshold. Therefore, the step ignores interactions whose scores are equal to or lower than the weight threshold.

Step 8

Remove nodes (and all edges connected to removed nodes) from the graph of step 7 until no edges remain. Nodes are removed by iteratively applying the following procedure:

1 Find the highest degree (highest number of edges) node. If more than one exists, pick one arbitrarily.

2 Define a node set consisting of the node chosen above and all nodes connected to it, but excluding any nodes of lesser degree than the node chosen above.

3 Select the node with the lowest target locus score (lower scores indicate less desirability) from the set in step 1. Remove that node from the graph.

step 9

If the number of nodes remaining in the graph satisfies the number of target loci required for the composite PCR pool (within acceptable tolerances), then the method continues at step 10 .

If too many or too few nodes remain in the graph, then a binary search is performed to determine what threshold would leave the desired number of nodes in the graph. If there are too many nodes in the graph, the weight threshold bounds are adjusted as follows:

max_weightthreshold = weightthreshold

Otherwise (if there are too few nodes in the graph), then the weight threshold bounds are adjusted as follows:

min_weight threshold = weight threshold

Then, the weight threshold is adjusted as follows:

Weight threshold = (max_weight threshold + min_weight threshold)/2

Repeat steps 7-9.

Step 10

Select the primer pair design associated with the remaining nodes in the graph for the primer pool. This library of primers can be used in any of the methods of the invention.

If desired, this method of designing and selecting primers can be performed on primer pools in which only one primer (rather than a primer pair) is used to amplify the locus of interest. In this case, the nodes represent one primer (rather than a pair of primers) per locus of interest.

Experiment 17

Figure 27 is a graph comparing two primer libraries designed using the methods of the present invention. This graph shows the number of loci targeted by each primer pool with a specific minor allele frequency. During selection of the "new pool" library, more primers are retained. This library achieves more target loci, especially those with relatively large minor allele frequencies (they are used in some methods of the present invention, such as for the detection of fetal chromosomal abnormalities to provide more information allele) amplification.

These primer pools were used in the following multiplex PCR method. Blood (20-40 mL) was collected from each subject into two to four CELL-FREE ^™ DNA tubes (Streck). Plasma (minimum 7 mL) was isolated from each sample via a double centrifugation protocol (2,000 g, 20 min; followed by 3,220 g, 30 min), with the supernatant transferred after the first spin. cfDNA was isolated from 7-20 mL plasma using the Qiagen Ziamp Circulating Nucleic Acid Kit and eluted in 45 μL LTE buffer. Pure maternal genomic DNA was isolated from the buffy coat obtained after the first centrifugation, and pure paternal genomic DNA was similarly prepared from blood, saliva or cheek samples.

Maternal cfDNA, maternal genomic DNA, and paternal genomic DNA samples were preamplified for 15 cycles using 11,000 target-specific assays, and aliquots were transferred to a second PCR of 15 cycles using nested primers Reacting. Finally, samples were prepared for sequencing by adding barcoded markers in a third round of 12-cycle PCR. Thus, 11,000 targets were amplified in a single reaction; the targets included SNPs found on chromosomes 13, 18, 21, X and Y. The amplicons were then sequenced using an Illumina GAIIx or HISEQ sequencer. Parental genotypes were sequenced at a lower read depth than fetal genotypes (approximately 20% of cfDNA read depth).

Experiment 18

When necessary, the size and quantity of PCR products can be analyzed using standard methods, for example using the Agilent Technologies 2100 Bioanalyzer (FIG. 28A-M). For example, the direct PCR method without nesting described herein was used in 2,400-plex (Figures 28B-28G) and 19,488-plex experiments (Figures 28H to 28M). The amount of primers for Figures 28B-28D and 28H to 28J was 10 nM. The amount of primers for Figures 28E-28G and 28K to 28M was 1 nM. The amount of input DNA was 24 ng for Figures 28B, 28E, 28H, and 28K; 80 ng for Figures 28C, 28F, 28I, and 28L; and 250 ng for Figures 28D, 28G, 28J, and 28M. More input DNA yielded a greater proportion of the desired 180 bp product. The peak at 140 bp is the primer dimer product.

Experiment 19

Evidence-of-principle studies have demonstrated that T13, T18, T21, 45,X, and 47,XXY are equally accurate in all chromosomes.

patient

Pregnant couples were enrolled in specific antenatal care centers according to local law and according to protocols approved by the Institutional Review Board. Inclusion criteria were age at least 18 years, gestational age at least nine weeks, singleton pregnancy and informed consent. A blood sample was drawn from the pregnant mother, and a blood or cheek sample was collected from the father. Selection from 2 T13 (Patau syndrome) pregnant, 2 T18 (Edwards syndrome) pregnant, 2 T21 (Down syndrome) pregnant, 2 45, X pregnant, 2 47 , XXY pregnant women and samples of 90 normal pregnant women, then a population of about 500 women was tested for the chromosomal abnormalities detected by the method. When postnatal child tissue was available, a normal fetal karyotype was confirmed by molecular karyotyping of the samples. Euploid samples were drawn from low-risk women prior to invasive testing. Aneuploidy samples were drawn at least 7 days after invasive testing and aneuploidy was confirmed via cytogenetic karyotyping or fluorescence in situ hybridization at an independent laboratory.

Sample preparation and multiplex PCR

For the data in Figures 30A-E, 30G, 30H, and 31A-31G, sample preparation and 19,488-plex PCR were performed as described in Experiment 15. For the data in Figure 30F, sample preparation and 11,000-plex PCR were performed as described in Experiment 17.

Methods and Data Analysis

The algorithm considers parental genotype and crossover frequency data (such as data from the HapMap database) to calculate predicted alleles for a very large number of possible fetal ploidy states for 19,488 polymorphic loci and at various fetal cfDNA fractions distributed. (FIGS. 29A-29C). Unlike allele ratio-based methods, it also takes linkage disequilibrium into account and uses a non-Gaussian data model to describe the expected distribution of allelic measurements at SNPs given observed plateau characteristics and amplification bias. It then compares the various predicted allelic distributions to the actual allelic distribution as measured in cfDNA samples (Fig. 29C), and calculates each hypothesis (monosomy, disomy, or trisomy, where Based on various potential intersections, there are multiple hypotheses) likelihoods. The algorithm sums the likelihood for each individual monosomy, disomy, or trisomy hypothesis (FIG. 29D) and calls the ploidy state with the greatest overall likelihood as copy number and fetal fraction (FIG. 29E). While lab researchers don't disregard sample karyotypes, the algorithm reads ploidy status without human intervention and disregards the truth.

data interpretation

Graphical representation of the resulting data

To determine the ploidy state of the associated chromosomes, the algorithm considers the distribution of sequence counts for each of the two possible alleles of 3,000 to 4,000 SNPs per chromosome. It is important to note that the algorithm makes ploidy calls using methods that are not suitable for visualization. Therefore, for illustration, the data are presented here in a simplified form in the form of ratios of the two most likely alleles, labeled A and B, so that relevant trends can be more easily visualized. This simplified illustration does not take into account some of the algorithmic features. As an example, two important aspects of the algorithm that are not possible to illustrate with a visualization showing allelic ratios are: 1) the ability to exploit linkage disequilibrium, i.e. the likelihood that a measurement at one SNP will have a negative impact on neighboring SNPs. Effects of identity; and 2) Non-Gaussian data models were used to describe the expected distribution of allelic measurements at SNPs given platform characteristics and amplification bias. It should also be noted that the algorithm only considers the two most common alleles at each SNP, ignoring other possible alleles.

The graphical representations in Figures 30A-30H include samples in which two, one or three fetal chromosomes are present. In general, these graphical representations indicate euploidy (Figures 30A-30C), monosomy (Figure 30D) and trisomy (Figures 30E-30H), respectively. In all figures, each dot represents a single SNP, where the targeted SNPs for one chromosome are plotted sequentially from left to right along the horizontal axis. The vertical axis indicates the number of reads for the A allele as a fraction of the total number of reads for the A and B alleles of the SNP. It should be noted that the measurements were performed on total cfDNA isolated from maternal blood, and that cfDNA included both maternal and fetal cfDNA; thus, each point represents the combined fetal and maternal DNA contribution to the SNP. Thus, increasing the proportion of maternal cfDNA from 0% to 100% will gradually shift some points up or down the graph depending on the genotype of the mother and fetus. This is described in more detail below with the corresponding figures.

If you want to facilitate visualization, you can color-code the points according to the maternal genotype, since the maternal genotype contributes more to the position of each point and most trisomies are maternally inherited; this helps for visualizing ploidy status. Specifically, SNPs whose maternal genotype is AA can be represented in red, SNPs whose maternal genotype is AB can be represented in green, and SNPs whose maternal genotype is BB can be represented in blue.

In all cases, SNPs that were found to be homozygous for the A allele (AA) in mothers and fetuses were strongly associated with the upper limit of the plot, since the fraction of A allele reads was high, since the B allele should not be present. Conversely, SNPs found to be homozygous for the B allele in mothers and fetuses are strongly associated with the lower limit of the plot, since the fraction of A allele reads is low since only the B allele should be present. Points that do not correlate closely with the upper and lower limits of the plot represent SNPs where the mother, fetus, or both are heterozygous; these points are useful for identifying fetal ploidy, but can also be informative for determining paternal versus maternal inheritance. The points are separated based on maternal and fetal genotype and fetal fraction, and thus, the precise location of each individual point along the y-axis depends on stoichiometry and fetal fraction. For example, a locus where the mother is AA and the fetus is AB is expected to have different fractions of A allele reads depending on fetal fraction, and thus be positioned differently along the y-axis.

presence of two chromosomes

Figures 30A-30C depict the presence of two chromosomes when the sample is completely maternal (no fetal cfDNA present, Figure 30A), contains a moderate fetal cfDNA fraction (Figure 30B), or contains a high fetal cfDNA fraction (Figure 30C). Existence data.

Figure 30A shows data obtained from cfDNA isolated from blood of non-pregnant women. When no fetal cfDNA is present and the sample contains only maternal cfDNA, the plot represents euploid maternal genotypes only; the signature pattern consists of point "clusters": the red clusters that are closely related to the top of the plot (where the maternal genotype is AA SNPs), a blue cluster closely related to the bottom of the plot (where the maternal genotype is a SNP for BB), and a single concentrated green cluster (where the maternal genotype is a SNP for AB) (not shown in the color plot).

When fetal cfDNA is present, the positions of the spots are shifted so that the clusters separate into discrete "bands". It should be noted that for samples with a fetal fraction of 0%, groupings of points are called "clusters" (as in Figure 30A); and for all samples with fetal fraction > 0%, groupings of points are called "bands" (as in Figure 30B -30J). These discrete bands will be easily visible if the fetal fraction is high enough. Specifically, Figures 30B and 30C demonstrate characteristic patterns associated with two fetal chromosomes present at intermediate and high fetal fractions, respectively. This pattern includes three central green bands corresponding to heterozygous SNPs in the mother and two "marginal" bands corresponding to homozygous SNPs in the mother at the top (red) and bottom (blue) of the plot, respectively ( Color chart not shown).

Figure 30B shows data obtained from cfDNA isolated from plasma samples of women carrying euploid fetuses and a 12% fetal cfDNA fraction. Here, clusters of points closely associated with the top and bottom of the graph are each separated into two discrete bands: a red and a blue outer edge band that remains closely associated with the upper or lower bound of the graph; and a red and a blue The inner edge bands (not shown in the color map) that have been separated from the bounds of the map. These inner marginal bands centered at 0.92 and 0.08 represent the SNPs (in red) where the maternal genotype is AA and the fetal genotype is AB, and the SNPs where the maternal genotype is BB and the fetal genotype is AB, respectively (shown in blue). The central cluster of green dots expands, but separation into distinct bands is not easily seen at this fetal fraction.

At high fetal cfDNA fractions, the typical pattern (a set of three green bands with two red and two blue bordered bands) indicating the presence of two chromosomes is easily seen (not shown in the color map). Figure 30C presents data obtained from plasma samples from a woman carrying a euploid fetus with a fetal cfDNA fraction of 26%. Here, the marginal bands have been separated such that the inner bands are shifted towards the center of the plot, due to changes in the level of the B allele due to increasing fetal cfDNA fraction. Remarkably, at higher fetal fractions it is now easy to see the separation of the central green cluster into three distinct bands. In this case, the central bands of the triplets are clustered around 0.37, 0.50 and 0.63, corresponding to where the maternal genotype is AB and the fetal genotypes are AA (top), AB (middle) and BB (bottom). ) of those SNPs.

These hallmark patterns, three green bands and four bordered bands (two red and two blue), indicate the presence of two chromosomes, such as the X chromosome in autosomal euploidy or in female (XX) fetuses .

there is a chromosome

Fetal heterozygosity is not possible when the fetus has inherited only a single chromosome and thus only a single allele. Therefore, the only possible fetal SNP identities are A or B. Thus, the characteristic pattern of a maternally inherited monosomy has two central green bands representing SNPs in which the mother is heterozygous; and has only a single marginal red and blue band representing SNPs in which the mother is homozygous, and they are respectively A strong correlation was maintained with the upper and lower bounds (1 and 0) of the plot (Fig. 30D) (not shown in the color plot). It should be noted that there are no inner edge bands. This pattern indicates the presence of one chromosome, such as the X chromosome in a maternally inherited autosomal monosomy or in a male (XY) fetus.

three chromosomes

Trisomic chromosomes have three characteristic patterns. The first pattern represents a maternally inherited meiotic trisomy, in which the fetus inherits meiotic errors of two homologous, discordant chromosomes from the mother (Fig. 30E); this pattern includes two central green bands as well as marginal Two each of the red and blue bands. (not shown in color diagram) The second pattern represents a paternally inherited meiotic trisomy in which the fetus inherits two homologous, discordant chromosomes from the father (Fig. 30F); this pattern includes four central green bands and three marginal red bands and three marginal blue bands (not shown in color map). A third pattern represents a maternally (Fig. 30G) or paternally (Fig. 30H) inherited mitotic trisomy, in which the fetus inherits mitotic errors of two identical chromosomes from either the mother or the father; this pattern includes four central green bands And two each of the fringe red and blue bands. Maternally and paternally inherited mitotic trisomies can be distinguished by the position of the lateral red and blue bands such that the red and blue inner marginal bands (those not associated with the boundaries of the diagram) are closer to paternally inherited mitotic trisomies The center of the body (not shown in the color map). This is due to the paternal contribution of identical chromosomes. It should be noted that our previous results showed that at the blastomere stage, 66.7% of maternally inherited trisomies were meiotic and only 10.2% of paternally inherited trisomies were.

Regarding the Y chromosome, the PS method considers a different set of assumptions: the presence of zero, one or two chromosomes. Because there is no maternal contribution to the sequence reads at each locus and because heterozygous loci are not possible (the case where two Y chromosomes must involve two identical chromosomes), the bands remain consistent with the top of the plot (A et al. allele) or bottom (B allele) are closely related (not shown in data plots) and depend on quantitative allele count data, greatly simplifying analysis. It should be noted that because the method interrogates SNPs, it uses homologous non-recombinant SNPs from the Y chromosome, thus obtaining data on both X and Y for one probe pair.

Identify aneuploidy

Given sufficient fetal fractions, identification of autosomal aneuploidies using this graph-based visualization is straightforward and requires only identifying the abnormal number of chromosomes present in the graph, as described above. Combining copy number knowledge of the X and Y chromosomes to identify the presence of sex chromosome aneuploidy. Specifically, a plot representing a fetus with genotype 47,XXX will have a typical "three chromosomes" pattern, and a plot representing a fetus with genotype 47,XXY will have a typical "two chromosomes" pattern for the X chromosome pattern, but will also have allelic reads indicating the presence of a Y chromosome. The method is similarly capable of calling 47,XYY, where a "one chromosome" pattern indicates the presence of a single X chromosome and an allelic read indicates the presence of two Y chromosomes. A fetus with genotype 45, X will have a typical "one chromosome" pattern for the X chromosome, and data indicating zero Y chromosomes.

The effect of fetal fraction

As discussed above, the number of sequence reads from the fetus contributes to the precise location of each point in the graph along the y-axis. Because fetal fraction affects the proportion of readings derived from the fetus and the mother, it also affects the positioning of each point. At high fractions of fetal cfDNA (generally over about 20%), as in Figures 30C-30E and Figures 30G and 30H, it is readily apparent that although the point clusters are primarily based on maternal The presence of fetal DNA of alleles of this genotype shifts the cluster into multiple distinct bands. However, as the fetal fraction decreases (as in Figures 30B and 30F), the points regress toward the poles and center of the plot, resulting in tighter clusters. Specifically, the marginal red band group where the maternal genotype is AA regresses towards the top of the plot; the marginal blue band group where the maternal genotype is BB regresses towards the bottom; and the central green band group where the mother is heterozygous is in The center of the map is compressed into a single cluster (compare Figures 30B and 30C) (not shown in color map). While aneuploidy is not readily visible using this visualization technique for low fetal fraction cases, the algorithm is able to identify ploidy states with very low fetal fractions, eg 3% fetal fraction. It can do this because statistical techniques compare observed data with very accurate data models that predict allelic distribution for a given set of sample parameters including, for example, copy number, parental genotype, and fetal fraction. Data model accuracy is critical in the low fetal fraction case since the difference between allele distributions for different ploidy states is proportional to fetal fraction. Additionally, the algorithm is capable of determining when a data set contains insufficient data to make a confident determination of fetal ploidy.

result

Sequencing reads that map to targeted SNPs are considered informative and used by the algorithm. More than 95% of the target loci were observed in the sequencing results. Diagrams to visualize key ploidy calls are depicted in Figures 31A-31G. Figure 31A represents a euploid sample. Here, chromosomes 13, 18 and 21 have a typical "two chromosome" pattern (as described herein). This includes a set of three central green bands and two red and two blue fringe bands. The presence of this along with the two central green bands of the X chromosome and the Y chromosome band along the perimeter of the plot indicates a euploid XY genotype (not shown in color plot).

The graphs in Figures 31B, 31C and 31D indicate the most prevalent autosomal trisomies T13, T18 and T21, respectively. Specifically, Figure 3 IB depicts the T13 sample. Here, chromosomes 18 and 21 exhibit a typical "two chromosome" pattern, the X chromosome exhibits a typical "one chromosome" pattern, and there are reads from the Y chromosome. Taken together, this indicated disomy at chromosomes 18 and 21 and identified a fetal XY genotype. Specifically, however, chromosome 13 depicts the typical "three-chromosome" pattern. Similarly, Figure 31C depicts a T18 sample, and Figure 31D depicts a T21 sample.

The method is also capable of detecting sex chromosome aneuploidies, including 45,X (Figure 31E), 47,XXY (Figure 31F) and 47,XYY (Figure 31G). It should be noted that the method is to call copy numbers on chromosomes 13, 18, 21, X, and Y; the overall chromosome number is reported assuming the remaining chromosomes are disomy. The X chromosome region of the graph depicting the 45,X sample revealed the presence of a single chromosome. However, the absence of reads from the Y chromosome and the "two-chromosome" pattern of chromosomes 13, 18, and 21 indicated the 45,X genotype. In contrast, the 47,XXY sample produced a map that revealed the presence of two X chromosomes. The data also revealed allelic reads from the Y chromosome. Together with the presence of two copies of chromosomes 13, 18 and 21, this is indicative of a 47,XXY genotype. The presence of a "one chromosome" pattern for the X chromosome and reads indicating the presence of two Y chromosomes is indicative of a 47,XYY genotype.

discuss

This method non-invasively detects T13, T18, T21, 45,X, 47,XXY and 47,XYY from maternal blood. This method interrogates cfDNA from maternal plasma by targeted multiplex PCR amplification of 19,488 SNPs and high-throughput sequencing. This, along with complex information analysis of methods that take into account parental genotype information and many sample parameters, including fetal fraction and DNA quality, more robustly detects fetal signals and is specific for the seven most common types of aneuploidy at birth (T13, T18 Highly accurate ploidy calls were made for all five chromosomes involved in , T21, 45, X, 47, XXX, 47, XXY and 47, XYY). This approach offers multiple clinical advantages over previous methods, including, and most notably, greater clinical coverage and sample-specific computational accuracy (similar to personalized risk scores).

Increased Clinical Coverage

Given its ability to accurately detect autosomal trisomies and sex chromosome aneuploidies, this method provides aneuploidy coverage equivalent to approximately twice that of clinically available NIPT methods. The method presented here is the only non-invasive test for calling the ploidy of sex chromosomes with high accuracy. Previous DNA pooling experiments and individual plasma samples analyzed in our experimental assay suggest that this approach will detect a larger population of sex chromosome abnormalities, including 47,XXX. The method presented here also detects aneuploidy at chromosomes 13, 18 and 21 with high sensitivity and specificity, and proper primer design is expected to detect copy number at the remaining chromosomes as well.

Sample specific calculation accuracy

Notably, this method calculates the sample-specific accuracy of the ploidy call on each chromosome in each sample. The accuracy calculated by this method is expected to significantly reduce the rate of incorrect calls by identifying and flagging individual samples with poor quality DNA or low fetal fractions that have the potential to yield poorly accurate test results. In contrast, methods based on massively parallel shotgun sequencing (MPSS) generate positive or negative calls using single-hypothesis rejection tests, and their accuracy estimates are based on characteristics of published study populations rather than individual samples, assuming they have the same The groups have the same accuracy. However, individual accuracies for samples with parameters in the tails of the population distribution can vary significantly. This is exacerbated at low fetal fractions, as in early gestational age or for samples with low DNA quality. These samples are generally not identified and flagged for subsequent testing, which can result in lost interpretations. However, the methods of the present invention take into account multiple parameters, including fetal fraction and multiple DNA quality metrics, to make each chromosome copy number call, calculating the sample-specific accuracy of the call. This makes the method identify individual samples with low accuracy and flag them for subsequent testing. Doing so is expected to virtually eliminate missing calls, especially in the first trimester when fetal fractions are typically low. Assuming no calls is far preferable to missing calls because no calls only require redrawing and reanalysis.

Convert calculated accuracy to traditional risk scores

This approach can provide adjusted risks for aneuploidy in high-risk pregnancies that take into account prior risks (Benn P, Cuckle H, Pergament E ). Non-invasive prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly (Non-invasive prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly). Ultrasound Obstet Gyneco1 2012 ;39:127-130, which is hereby incorporated by reference herein in its entirety). While the method of the present invention provides calculated accuracies tailored to each patient, for clinical use these accuracies can be converted to conventional risk scores, which also represent the risk of aneuploid pregnancy but expressed in fractional form. Traditional risk scores take into account various parameters, including maternal age-related risk and serum levels of biochemical markers, thereby providing a risk score above which the mother is considered high risk and recommended for subsequent invasion diagnostic procedures. This approach significantly optimizes this risk score, thereby reducing the false positive and false negative rates and providing a more accurate assessment of individual maternal risk. The calculated accuracy as used here is the likelihood that the ploidy call is correct and is expressed as a percentage, but the calculated accuracy used in Experiment 19 does not include age-related risk. Because calculations of risk scores often include age-related risks, computational accuracy and traditional risk scores are not interchangeable; they must be combined to transform into a traditional risk score. The formula for combining age-related risk and calculated accuracy is:

wherein R ₁ is the risk score as calculated by the method of the invention and R ₂ is the risk score as calculated by the first trimester screening.

SNP-Based Approaches Offset Amplification Variation Issues

An inherent disadvantage of counting methods used by some other methods is that they determine fetal ploidy status by measuring the ratio of the number of reads that map to a relevant chromosome (eg, chromosome 21 ) to those that map to a reference chromosome. Chromosomes with high or low GC content, including chromosome 13, X, and Y, showed high variability in amplification. This produces a signal change that is comparable in magnitude to the fetal cfDNA signal, which confounds copy number calls by altering the ratio of allelic reads from the associated chromosome to the allelic reads from the reference chromosome. This results in low accuracy for Chromosome 13, X and Y. Notably, this problem is exacerbated at low fetal cfDNA fractions, as is often the case in early gestational age.

In contrast, SNP-based methods do not rely on consistent amplification levels between chromosomes and are thus expected to provide equally accurate results for all chromosomes. Because the present method looks in part at the relative counts of different alleles at polymorphic loci, which differ by definition by only a single nucleotide, it does not require the use of a reference chromosome, and this precludes reliance on quantification of read counts Chromosome-to-chromosome amplification variation is inherent to the method. Unlike quantitative methods that require a euploid reference chromosome, the methods of the present invention are expected to be able to detect triploidy as well as copy number neutral abnormalities such as uniparental disomies.

The importance of early detection

Notably, the combined birth prevalence of sex chromosome aneuploidies was higher than the most common autosomal aneuploidies (Figure 32). However, no routine non-invasive screening method that reliably detects sex chromosome abnormalities currently exists. Thus, prenatal testing for sex chromosome abnormalities is generally performed as a side effect of routine testing for Down syndrome or other autosomal aneuploidies; a large proportion of cases are missed entirely. Early and accurate detection is critical for many of these disorders in which early therapeutic intervention improves clinical outcome. Turner syndrome, for example, is often not diagnosed until adolescence, but its overall birth prevalence is 1 in 2,500 women. Growth hormone therapy is known to prevent short stature caused by the condition, but treatment is significantly more effective when started before the age of 4 years. In addition, estrogen replacement therapy can stimulate secondary sexual characteristics in patients with Turner syndrome, but retreatment must begin before the age of ten, before the syndrome is usually detected. Taken together, this underscores the importance of early, routine, and safe detection of sex chromosome aneuploidy. This approach provides the first method with the potential to serve as a routine screen for sex chromosomal abnormalities.

additional application

Because this method utilizes targeted amplification, it is uniquely poised to detect submicroscopic abnormalities such as microdeletions and microduplications. While non-targeted approaches such as MPSS have been shown to detect DiGeorge microdeletion syndrome, this requires sufficiently high levels of genome coverage that the approach is not feasible. This is because the effectiveness of non-targeted amplification on submicroscopic regions will be orders of magnitude smaller, since a very small fraction of sequencing reads will be informative. Additionally, the fact that currently available methods have difficulty accurately identifying the ploidy state of the sex chromosomes suggests that they will also suffer from variable amplification problems on smaller chromosome segments.

Similarly, SNP-based methods can detect UPD disorders, which are copy number neutral abnormalities that would not be possible with current non-invasive methods that rely on enumeration or those that rely on cytogenetic karyotyping and/or fluorescence in situ hybridization. Traditional invasive methods (such as amniocentesis and CVS) to detect. This is because SNP-based methods are able to uniquely distinguish individual haplotypes, whereas clinically available MPSS-based and targeted methods amplify non-polymorphic loci and thus are not able to determine, for example, whether related chromosomes are derived from the same parent. This means that these microdeletions/microduplications and UPD syndromes, including Prader-Willi syndrome, Angelman syndrome, and Beckwith-Wiedemann syndrome, generally cannot be diagnosed prenatally , and are often initially misdiagnosed postpartum. This significantly delays therapeutic intervention. Additionally, because this approach targets SNPs, this approach will also facilitate parental haplotype reconstitution, allowing detection of fetal inheritance at individual disease-linked loci (Kitzman JO, Snyder MW MW), Ventura M (Ventura M) et al. Noninvasive whole-genome sequencing of a human fetus. SciTransl Med 2012;4:137ra76, hereby incorporated herein by reference in its entirety).

The results presented here demonstrate the extended scope of this approach for identifying prenatal aneuploidies. Specifically, by amplifying and sequencing 19,488 SNPs, this method enables the determination of copy number on chromosomes 13, 18, 21, X, and Y, and uniquely predicts that detection cannot be performed by any other clinically available method. Other chromosomal abnormalities such as triploidy and UPD detected by non-invasive methods obtained. The increased clinical coverage and robust sample-specific computational accuracy suggest that this approach could provide a viable adjunct to invasive tests for the detection of fetal chromosomal aneuploidy.

All patents, published applications, and published references cited herein are hereby incorporated by reference in their entirety. Although the inventive method has been described in connection with specific embodiments thereof, it will be appreciated that further modifications can be made thereto. Furthermore, this application is intended to cover any variations, uses, or adaptations of the methods of the invention, including the invention as they come within known or customary practice in the art to which the methods of the invention pertain and as falling within the scope of the following claims Deviate. For example, any of the methods disclosed herein for use with DNA can be readily adapted for use with RNA by including a reverse transcription step for converting RNA to DNA. The illustrated examples using polymorphic loci can be readily adapted for amplification of non-polymorphic loci, if necessary.

Claims (102)

1. a method for the target gene seat in amplification of nucleic acid sample, described method comprises:

A () makes described nucleic acid samples contact to produce reaction mixture with the test primer storehouse hybridizing at least 1,000 different target gene seat simultaneously; And

B () makes described reaction mixture experience primer extension reaction condition to produce the amplified production comprising target amplicon.

2. method according to claim 1, wherein at least 5,000 different target gene seat is amplified.

3. method according to claim 1, wherein at least 10,000 different target gene seat is amplified.

4. method according to claim 1, wherein at least 20,000 different target gene seat is amplified.

5. method according to claim 1, wherein at least 30,000 different target gene seat is amplified.

6. method according to claim 1, wherein the described amplified production of at least 90% is target amplicon.

7. method according to claim 1, wherein the described amplified production of at least 95% is target amplicon.

8. method according to claim 1, wherein the described amplified production of at least 99% is target amplicon.

9. method according to claim 1, wherein the described target gene seat of at least 90% is amplified.

10. method according to claim 1, wherein the described target gene seat of at least 95% is amplified.

11. methods according to claim 1, wherein the described target gene seat of at least 99% is amplified.

12. methods according to claim 1, the described amplified production being wherein less than 20% is test primer dimer.

13. methods according to claim 1, the described amplified production being wherein less than 10% is test primer dimer.

14. methods according to claim 1, the described amplified production being wherein less than 1% is test primer dimer.

15. methods according to claim 1, the described amplified production being wherein less than 0.1% is test primer dimer.

16. methods according to claim 1, wherein said test primer is selected from candidate drugs storehouse based on one or more parameter.

17. methods according to claim 16, wherein said test primer is selected from candidate drugs storehouse based on the ability of described candidate drugs formation primer dimer at least partly.

18. methods according to claim 17, wherein said selection comprises

I () calculates major part from two kinds of candidate drugs in described storehouse or the undesirable mark that likely combines on computers, wherein each undesirable mark is based, at least in part, between described two kinds of candidate drugs and forms dimeric likelihood;

(ii) from described candidate drugs storehouse, the highest candidate drugs of undesirable mark is removed;

(iii) if the described candidate drugs removed in step (ii) is the member of primer pair, from described candidate drugs storehouse, another member of described primer pair is so removed; And

(iv) optionally repeating step (ii) and (iii).

19. methods according to claim 17, wherein said selection comprises

I () calculates major part from two kinds of candidate drugs in described storehouse or the undesirable mark that likely combines on computers, wherein each undesirable mark is based, at least in part, between described two kinds of candidate drugs and forms dimeric likelihood;

(ii) from described candidate drugs storehouse, undesirable mark is removed in the maximum quantity combination as two kinds of candidate drugs higher than the candidate drugs of the part of the first minimum threshold;

(iii) if the described candidate drugs removed in step (ii) is the member of primer pair, from described candidate drugs storehouse, another member of described primer pair is so removed; And

(iv) optionally repeating step (ii) and (iii).

20. methods according to claim 19, comprise the quantity by remaining candidate drugs in the following described storehouse of further minimizing: described first minimum threshold used in step (ii) is reduced to the second lower minimum threshold and repeating step (ii) and (iii) until in described storehouse the undesirable mark of remaining candidate drugs combination be all equal to or less than described second minimum threshold, or until in described storehouse the quantity of remaining candidate drugs reduce to desired quantity.

21. methods according to claim 19, comprise described first minimum threshold used in step (ii) is increased to the second higher minimum threshold and repeating step (ii) and (iii) until in described storehouse the undesirable mark of remaining candidate drugs combination be all equal to or less than described second minimum threshold, or until in described storehouse the quantity of remaining candidate drugs reduce to desired quantity.

22. methods according to claim 18 or 19, wherein candidate drugs selects from the group of two or more candidate drugs with the equal undesirable mark removed from described storehouse based on one or more parameter.

23. methods according to claim 1, wherein the concentration of often kind of test primer is less than 100nM.

24. methods according to claim 1, wherein the concentration of often kind of test primer is less than 10nM.

25. methods according to claim 1, wherein the concentration of often kind of test primer is less than 2nM.

26. methods according to claim 1, wherein said test primer storehouse comprises the test primer pair that at least 1,000 comprises positive test primer and negative testing primer, and wherein often pair of test primer hybridization is to target gene seat.

27. methods according to claim 1, wherein said test primer storehouse comprises the independent test primer that at least 1,000 hybridizes to different target locus separately.

28. methods according to claim 1, the GC content of wherein said test primer, between 30% and 80%, comprises end points.

29. methods according to claim 1, the scope of the GC content of wherein said test primer is less than 20%.

30. methods according to claim 1, the melting temperature(Tm) of wherein said test primer, between 40 DEG C and 80 DEG C, comprises end points.

31. methods according to claim 1, the scope of the melting temperature(Tm) of wherein said test primer is less than 5 DEG C.

32. methods according to claim 1, the length of wherein said test primer, between 17 and 35 Nucleotide, comprises end points.

33. methods according to claim 1, wherein said test primer comprises non-targeted specific marker.

34. methods according to claim 33, wherein said mark forms inner loop structure.

35. methods according to claim 35, wherein said mark is between two DNA lands.

36. methods according to claim 34, wherein said test primer comprises and has specific 5th ' district to target gene seat, do not have specificity and form the interior region of ring structure and have specific 3rd ' district to described target gene seat described target gene seat.

37. methods according to claim 36, the length in wherein said 3rd ' district is at least 7 Nucleotide.

38. according to method according to claim 37, and the length in wherein said 3rd ' district, between 7 and 20 Nucleotide, comprises end points.

39. method according to claim 33, wherein said test primer comprises target gene seat is not had to specific 5th ' district, is then have specific region to target gene seat, do not have specificity and form the interior region of ring structure and have specific 3rd ' district to described target gene seat described target gene seat.

40. methods according to claim 1, the scope of the length of wherein said target amplicon is less than 15 Nucleotide.

41. methods according to claim 1, wherein said primer extension reaction condition is polymerase chain reaction condition (PCR).

42. methods according to claim 41, wherein the length of annealing steps is greater than 10 minutes.

43. methods according to claim 1, comprise further and determine at least one target amplicon of presence or absence.

44. methods according to claim 1, comprise the sequence measuring at least one target amplicon further.

45. methods according to claim 1, wherein said target gene seat is present on identical relative chromosome.

46. methods according to claim 1, at least some in wherein said target gene seat is present on different relative chromosome nucleic acid.

47. methods according to claim 1, wherein said nucleic acid samples comprises the nucleic acid through fragmentation or digestion.

48. methods according to claim 1, wherein nucleic acid samples comprises genomic dna, cDNA or mRNA.

49. methods according to claim 1, wherein nucleic acid samples comprises from single celled DNA.

50. methods according to claim 1, wherein nucleic acid samples comprises or derives from blood, blood plasma, saliva, sperm, cell culture supernatant, mucus secretion, dental plaque, stomach intestinal tissue, ight soil, urine or forensic samples.

51. methods according to claim 1, wherein said target gene seat is the section of people's nucleic acid.

52. methods according to claim 1, wherein said target gene seat comprises single nucleotide polymorphism.

Select the method for testing primer from candidate drugs storehouse for 53. 1 kinds, described method comprises:

A () calculates major part from two kinds of candidate drugs in described storehouse or the undesirable mark that likely combines on computers, wherein each undesirable mark is based, at least in part, between described two kinds of candidate drugs and forms dimeric likelihood;

B () removes the highest candidate drugs of undesirable mark from described candidate drugs storehouse;

If c described candidate drugs that () removes in step (b) is the member of primer pair, from described candidate drugs storehouse, so remove another member of described primer pair; And

D () be repeating step (b) and (c) optionally, thus select test primer storehouse.

Select the method for testing primer from candidate drugs storehouse for 54. 1 kinds, described method comprises:

A () calculates major part from two kinds of candidate drugs in described storehouse or the undesirable mark that likely combines on computers, wherein each undesirable mark is based, at least in part, between described two kinds of candidate drugs and forms dimeric likelihood;

B () to remove in the maximum quantity combination as two kinds of candidate drugs undesirable mark higher than the candidate drugs of the part of the first minimum threshold from described candidate drugs storehouse;

If c described candidate drugs that () removes in step (b) is the member of primer pair, from described candidate drugs storehouse, so remove another member of described primer pair; And

D () be repeating step (b) and (c) optionally, thus select test primer storehouse.

55. methods according to claim 54, comprise the quantity by remaining candidate drugs in the following described storehouse of further minimizing: described first minimum threshold used in step (b) is reduced to the second lower minimum threshold and repeating step (b) and (c) until in described storehouse the undesirable mark of remaining candidate drugs combination be all equal to or less than described second minimum threshold, or until in described storehouse the quantity of remaining candidate drugs reduce to desired quantity.

56. methods according to claim 54, comprise described first minimum threshold used in step (b) is increased to the second higher minimum threshold and repeating step (b) and (c) until in described storehouse the undesirable mark of remaining candidate drugs combination be all equal to or less than described second minimum threshold, or until in described storehouse the quantity of remaining candidate drugs reduce to desired quantity.

57. methods according to claim 53 or 54, wherein candidate drugs selects from the group of two or more candidate drugs with the equal undesirable mark removed from described candidate drugs storehouse based on one or more other parameter.

58. methods according to claim 53 or 54, wherein said undesirable mark is selected from by the parameter of the following group formed based on one or more at least partly: the heterozygosis rate of described target gene seat, to at the polymorphism of described target gene seat or the relevant incidence rate that suddenlys change, to described target gene seat polymorphism or suddenly change disease penetrance relevant, described candidate drugs is to the specificity of described target gene seat, the size of described candidate drugs, the melting temperature(Tm) of described target amplicon, the GC content of described target amplicon, the amplification efficiency of described target amplicon and the size of described target amplicon.

59. methods according to claim 58, wherein said undesirable mark is selected from by the parameter of the following group formed based on one or more at least partly: the heterozygosis rate of described target gene seat, described candidate drugs are to the size of the GC content of the melting temperature(Tm) of the size of the specificity of described target gene seat, described candidate drugs, described target amplicon, described target amplicon, the amplification efficiency of described target amplicon and described target amplicon; And wherein said test primer is used to increase and comprises from least 1,000 target gene seat in the maternal DNA of the pregnant mothers of fetus and the sample of foetal DNA to determine presence or absence fetal chromosomal abnormalities simultaneously.

60. methods according to claim 59, comprise the described DNA molecular joined to by universal primer binding site in described sample; Use at least 1,000 Auele Specific Primer and a universal primer to increase the DNA molecular engaged, produce first group of amplified production; And use first group of amplified production described at least 1,000 pair of primer amplified, produce second group of amplified production.

61. methods according to claim 58, wherein said undesirable mark is selected from by the parameter of the following group formed based on one or more at least partly: the heterozygosis rate of described target gene seat, described candidate drugs are to the size of the GC content of the melting temperature(Tm) of the size of the specificity of described target gene seat, described candidate drugs, described target amplicon, described target amplicon, the amplification efficiency of described target amplicon and described target amplicon; And wherein said test primer is used to increase simultaneously the DNA of the hypothesis father comprised from fetus sample at least 1,000 target gene seat and amplification simultaneously comprise from the described target gene seat in the maternal DNA of the described pregnant mothers of fetus and the sample of foetal DNA, thus determine that whether described hypothesis father is the natural father of described fetus.

62. methods according to claim 58, wherein said undesirable mark is selected from by the parameter of the following group formed based on one or more at least partly: the heterozygosis rate of described target gene seat, described candidate drugs are to the size of the specificity of described target gene seat, the size of described candidate drugs and described target amplicon; And wherein said test primer is used at least 1,000 the target gene seat using the annealing time of at least 5 minutes to increase in legal medical expert's nucleic acid samples simultaneously.

63. methods according to claim 58, comprise at least 1,000 target gene seat using described test primer simultaneously to increase in contrast nucleic acid samples with produce first group of target amplicon and described target gene seat simultaneously in amplification assay nucleic acid samples to produce second group of target amplicon; And more described first and second groups of target amplicon are not still stored in another sample to determine whether target gene seat is present in a sample, or whether target gene seat is present in described control sample and described test sample with different levels.

64. methods according to claim 63, wherein said test sample is from the doubtful individuality with the risk of relative disease or phenotype or relative disease or phenotypic increase; And one or more in wherein said target gene seat comprises the risk polymorphism that is relevant or that be correlated with described relative disease or phenotype to described relative disease or phenotypic increase.

65. methods according to claim 58, comprise use described test primer to increase to comprise simultaneously at least 1,000 target gene seat in the control sample of RNA with produce first group of target amplicon and the described target gene seat that comprises in the test sample of RNA of simultaneously increasing to produce second group of target amplicon; And more described first and second groups of target amplicon with determine described rna expression between described control sample and described test sample horizontal in presence or absence difference.

66. methods according to claim 65, wherein said RNA is mRNA.

67. methods according to claim 65, wherein said test sample is from the doubtful individuality with the risk of cancer of cancer or increase; And one or more in wherein said target gene seat comprises polymorphism that is relevant to the risk of cancer increased or that be correlated with cancer or other suddenlys change.

68. methods according to claim 65, wherein said test sample is the individuality from suffering from cancer after diagnosing; And between wherein said control sample and test sample Discrepancy Description target gene seat in described rna expression is horizontal comprise with increase polymorphism that the risk of cancer that reduces is relevant or other suddenly change.

69. methods according to claim 53 or 54, before being included in step (b) further, remove the primer pair producing the target amplicon overlapping with the target amplicon produced by another primer pair from described storehouse.

70. methods according to claim 53 or 54, in wherein said storehouse, remaining described candidate drugs can increase at least 1,000 different target gene seat simultaneously.

71. methods according to claim 53 or 54, comprise further:

E () makes the nucleic acid samples comprising target gene seat contact to produce reaction mixture with described candidate drugs remaining in described storehouse; And

F () makes described reaction mixture experience primer extension reaction condition to produce the amplified production comprising target amplicon.

72. 1 kinds of primer storehouses, its at least 1,000 different target gene seat that simultaneously increases is test primer dimer to make the described amplified production being less than 30%.

73. 1 kinds of primer storehouses, it increases at least 1,000 different target gene seat to make the described amplified production of at least 80% be target amplicon simultaneously.

74. 1 kinds of primer storehouses, it increases target gene seat to make at least 1 simultaneously, and in 000 different target gene seat, the described target gene seat of at least 80% is amplified.

75. storehouses according to any one of claim 72 to 74, wherein at least 5,000 different target gene seat is amplified.

76. storehouses according to any one of claim 72 to 74, wherein at least 10,000 different target gene seat is amplified.

77. storehouses according to any one of claim 72 to 74, wherein at least 20,000 different target gene seat is amplified.

78. storehouses according to any one of claim 72 to 74, wherein at least 30,000 different target gene seat is amplified.

79. storehouses according to any one of claim 72 to 74, wherein the described amplified production of at least 90% is target amplicon.

80. storehouses according to any one of claim 72 to 74, wherein the described amplified production of at least 95% is target amplicon.

81. storehouses according to any one of claim 72 to 74, wherein the described amplified production of at least 99% is target amplicon.

82. storehouses according to any one of claim 72 to 74, wherein the described target gene seat of at least 90% is amplified.

83. storehouses according to any one of claim 72 to 74, wherein the described target gene seat of at least 95% is amplified.

84. storehouses according to any one of claim 72 to 74, wherein the described target gene seat of at least 99% is amplified.

85. storehouses according to any one of claim 72 to 74, the described amplified production being wherein less than 20% is primer dimer.

86. storehouses according to any one of claim 72 to 74, the described amplified production being wherein less than 10% is primer dimer.

87. storehouses according to any one of claim 72 to 74, the described amplified production being wherein less than 1% is primer dimer.

88. storehouses according to any one of claim 72 to 74, the described amplified production being wherein less than 0.1% is primer dimer.

89. storehouses according to any one of claim 72 to 74, comprise at least 1,000 primer pair, wherein each primer pair comprises the forward primer and reverse primer that hybridize to target gene seat.

90. storehouses according to any one of claim 72 to 74, comprise the independent primer that at least 1,000 hybridizes to different target locus.

91. 1 kinds of test kits for the target gene seat in amplification of nucleic acid sample, comprise (i) storehouse according to any one of claim 72 to 74 and (iii) and use described storehouse to increase the specification sheets of described target gene seat.

92. 1 kinds for measuring the method for the chromosomal ploidy state in the fetus in breeding, described method comprises:

A () makes nucleic acid samples contact to produce reaction mixture with the primer storehouse hybridizing at least 1,000 different polymorphic locus simultaneously; Wherein said nucleic acid samples comprises from the maternal DNA of mother of described fetus and the foetal DNA from described fetus; And

B () makes described reaction mixture experience primer extension reaction condition to produce amplified production;

C () measures described amplified production to produce sequencing data with high-flux sequence instrument;

D () calculates on computers based on described sequencing data and counts at the allelotrope of described polymorphic locus;

E () creates multiple separately about the ploidy hypothesis of described chromosomal different possibility ploidy state on computers;

F () is supposed for often kind of ploidy, be that the expectation allelotrope counting at the described polymorphic locus place on described karyomit(e) builds simultaneous distribution model on computers;

G () uses described simultaneous distribution model and described allelotrope to count the relative probability of each measured on computers in described ploidy hypothesis; And

H () is by selecting the ploidy state corresponding to the hypothesis with maximum probability, the ploidy state of fetus described in interpretation.

93. 1 kinds of test chromosome comprise maternal and fetus DNA mixture sample in the method for skewed distribution, described method comprises:

A () makes described sample contact to produce reaction mixture with the primer storehouse hybridizing at least 1,000 different target gene seat simultaneously; Wherein said target gene seat is from multiple different karyomit(e); And wherein said multiple different karyomit(e) comprise at least one doubtful there is skewed distribution in described sample the first chromosome and the second karyomit(e) of at least one supposition normal distribution in described sample;

B () makes described reaction mixture experience primer extension reaction condition to produce amplified production;

C () checks order to obtain to described amplified production the sequence mark that multiple and described target gene seat aims at; The length of wherein said sequence mark is enough to distribute to specific targets locus;

D described multiple sequence mark is distributed to the target gene seat of its correspondence by () on computers;

E quantity that () measures the sequence mark aimed at the target gene seat of described the first chromosome on computers and the quantity of sequence mark of aiming at described second chromosomal target gene seat; And

F () compares quantity from step (e) on computers to determine the skewed distribution of the first chromosome described in presence or absence.

94. 1 kinds for detecting the method for presence or absence fetus dysploidy, described method comprises:

A sample that () makes to comprise the mixture of the DNA of maternal and fetus and the primer storehouse simultaneously hybridizing at least 1,000 different non-polymorphic target gene seat contact to produce reaction mixture; Wherein said target gene seat is from multiple different karyomit(e);

B () makes described reaction mixture experience primer extension reaction condition to produce the amplified production comprising target amplicon;

C () carries out quantitatively to the relative frequency from described the first and second relevant chromosomal target amplicon on computers;

D () compares the described relative frequency from described the first and second relevant chromosomal target amplicon on computers; And

E () differentiates presence or absence dysploidy based on described relevant first and second chromosomal compared relative frequencies.

Suppose that whether father is the method for the natural father of the fetus bred in pregnant mothers body for determining for 95. 1 kinds, described method comprises:

A () is increased from the multiple polymorphic locuses on the genetic material of described hypothesis father simultaneously, comprise at least 1,000 different polymorphic locus, thus produce first group of amplified production;

(b) increase simultaneously the blood sample deriving from described pregnant mothers DNA biased sample on corresponding multiple polymorphic locuses to produce second group of amplified production; Wherein said DNA biased sample comprises foetal DNA and maternal DNA;

C (), based on described first and second groups of amplified productions, use genotype measuring result measures the probability that described hypothesis father is the natural father of described fetus on computers; And

D () uses the described hypothesis father measured to be that the probability of the natural father of described fetus determines that whether described hypothesis father is the natural father of described fetus.

96. according to the method described in claim 95, comprises further and increases from multiple polymorphic locuses corresponding on the genetic material of described mother to produce the 3rd group of amplified production simultaneously; Wherein based on described first, second, and third group of amplified production, use genotype measuring result measures the probability that described hypothesis father is the natural father of described fetus.

97. 1 kinds of methods measuring the amount of two or more the target gene seats in nucleic acid samples, described method comprises:

A () uses pcr amplification to comprise the nucleic acid samples of the first standard gene seat, the second standard gene seat, first object locus and the second target gene seat to form amplified production; Wherein said first standard gene seat and described first object locus have the Nucleotide of equal amts but its sequence is different at one or more Nucleotide place; And wherein said second standard gene seat and described second target gene seat have the Nucleotide of equal amts but its sequence is different at one or more Nucleotide place;

B () is checked order to determine to compare the standard ratio of the first increased standard gene seat compared to the relative quantity of the second increased standard gene seat to described amplified production; The difference of amplification in PCR efficiency of the wherein said standard ratio described first standard gene seat of instruction and described second standard gene seat;

C () measures and compares the target rate of increased first object locus compared to the relative quantity of the second increased target gene seat; And

(d) based on the described standard ratio adjustment from step (b) from the described target rate of step (c) to determine the relative quantity of first object locus described in described sample and described second target gene seat.

98. according to the method described in claim 97, comprises the absolute magnitude measuring first object locus and described second target gene seat described in described sample further.

99. according to the method described in claim 97, comprises and measures presence or absence target gene seat in described sample.

100. according to the method described in claim 97, comprises at least 1,000 the different target gene seat that simultaneously increases.

101. according to the method described in claim 97, and wherein said target gene seat is present on identical relative chromosome.

102. according to the method described in claim 97, and at least some in wherein said target gene seat is present on different relative chromosome.