BR112019003704B1 - METHOD FOR PERFORMING A GENETIC ANALYSIS ON A TARGET REGION OF DNA FROM A TEST SAMPLE - Google Patents

Abstract

The present invention includes compositions and methods useful for detecting a mutational alteration, SNP, translocation, inversion, deletion, copy number change, or other genetic variation within a cellular genomic DNA or cell-free DNA (cfDNA) sample. In some embodiments, the compositions and methods of the present invention provide an extremely high level of resolution that is particularly useful in detecting copy number variations in a small fraction of the total cfDNA of a biological sample (e.g., blood).

Description

REFERENCE TO RELATED ORDERS

[0001] This application claims priority to U.S. Provisional Patent Application 62/379,593, filed August 25, 2016, and U.S. Provisional Patent Application 62/481,538, filed April 4, 2017, each of which is incorporated herein by reference in its entirety.

LISTING OF SEQUENCES RELATING TO THE DECLARATION

[0002] The Sequence Listing associated with this application is provided in text format rather than a paper copy, and is incorporated herein by reference into the specification. The name of the text file containing the sequence listing is CLFK_005_02WO_ST25. The text file is 2,238 KB, was created on August 24, 2017, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

[0003] The invention relates generally to compositions and methods for the quantitative genetic analysis of biological samples, for example, direct tissue biopsies or peripheral blood. In particular, the present invention relates to methods for detecting target-specific copy number alteration, as well as genetic characterization and analysis of biological samples.

FUNDAMENTALS

It is becoming increasingly clear that most, if not all, of the most common human cancers are diseases of the human genome. Somatic mutations are thought to accumulate over an individual's lifetime, some of which increase the likelihood that the cell they harbor will develop into a tumor. With just the wrong combination of accumulated mutational events, a precancerous growth loses the restraints that prevent uncontrolled proliferation, and the resulting cell mass becomes cancer. The constellations of mutations that are necessary and sufficient to cause cancer are often collectively referred to as "driver mutations." One of the themes emerging from recent intensive molecular analysis is that cancer, once considered a tissue-specific disease, is in fact a group of related diseases, each with a unique molecular pathology. The Human Genome Project laid the foundation for genome-wide analysis of cancers.

[0005] Changes in gene copy number are a fundamental driver of biological diversity. In the context of evolution, gene duplication and functional divergence are well-recognized drivers of species diversity. In the context of human disease, gene loss and gene amplification within somatic cells are hallmarks of diseased tissues, such as cancer. Certain therapeutic agents act specifically on cells with these genomic gain and/or loss mutations. However, identifying these copy number variations is difficult because these mutations are often present only in the DNA of diseased or cancerous cells and are not found in other cells in the body. While diseased tissue or cells are the primary source of mutated DNA, obtaining DNA through a biopsy is invasive, risky, and often impossible. The observation that dying tumor or cancer cells release small pieces of their DNA into the bloodstream, called cell-free DNA or circulating DNA, has enabled the development of genetic tests that can be performed using less invasive techniques, such as a blood sample. However, only small amounts of DNA can be obtained from isolating cell-free DNA from a sample, and only a portion of the total DNA will carry the disease-associated mutation. For example, in the context of cancer genomics, diagnostically significant tumor mutations are often found only at minor allele frequencies significantly below 50%. This is in contrast to conventional SNP genotyping, where allele frequencies are typically ~100%, 50%, or 0%.

[0006] Thus, there is a need for genomic techniques capable of detecting changes in the number of genetic copies at specific target sites.

BRIEF SUMMARY

[0007] Methods for detecting rare mutations in cfDNA have been previously described in PCT International Publication WO 2016/028316. However, these techniques still lack the sensitivity required to detect the rarest copy number losses at very small allele frequencies. Provided herein are compositions and methods for detecting target-specific copy number alteration that are applicable to various sample types, including direct tissue biopsies, peripheral blood, and in particular, cfDNA. The compositions and methods described herein are sufficiently sensitive to detect copy number alterations that are present in only a small fraction of the total DNA.

[0008] The present invention includes, inter alia, compositions and methods that are useful for detecting a mutational alteration, SNP, translocation, inversion, deletion, copy number change, or other genetic variation within a sample of cellular genomic DNA (e.g., from a tissue biopsy sample) or cfDNA (e.g., from a blood sample). In particular, the compositions and methods of the present invention provide an extremely high level of resolution that is particularly useful in detecting copy number variations in a small fraction of the total cfDNA of a biological sample (e.g., blood).

[0009] Particular embodiments are drawn to a method for performing a genetic analysis on a target DNA region of a test sample that includes: (a) generating a genomic DNA library comprising a plurality of DNA library fragments, wherein each of the DNA library fragments comprises a genomic DNA fragment from the test sample and an adapter; (b) contacting the genomic DNA library with a plurality of capture probes that specifically bind to a target DNA region, forming complexes between the capture probes and DNA library fragments comprising the target DNA region; and (c) performing a quantitative genetic analysis of the genomic DNA fragments comprising the target DNA region; wherein the adapter is a DNA polynucleotide comprising: an amplification region, a sample labeling region, and an anchor region; wherein the amplification region comprises a polynucleotide sequence capable of serving as a primer recognition site for PCR amplification; wherein the sample tag comprises a polynucleotide sequence that encodes an identity of the unique library DNA fragment and encodes an identity of the test sample; wherein the anchor region comprises a polynucleotide sequence that encodes the identity of the test sample and wherein the anchor region is capable of binding to the genomic DNA fragment; and wherein the genetic analysis is performed to detect a genetic alteration indicative of a disease state.

[0010] In some embodiments, the genetic alteration indicative of a disease state is selected from a single nucleotide variant (SNV), an insertion less than 40 nucleotides in length, a deletion of a region of DNA less than 40 nucleotides in length, and/or a copy number change. In particular embodiments, the genetic alteration indicative of a disease state is a copy number change. In some embodiments, the test sample is a tissue biopsy. In various embodiments, the tissue biopsy is taken from a tumor or tissue suspected of being a tumor. In certain embodiments, the genomic DNA is cell-free DNA (cfDNA) or cellular DNA. In particular embodiments, the genomic DNA is cfDNA that is isolated from the test sample; and wherein the test sample is a biological sample selected from the group consisting of: amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebrospinal fluid, ocular fluid, urine, saliva, feces, mucus and sweat.

[0011] In certain embodiments, the genomic DNA fragments are obtained in the steps comprising; (i) isolating cellular DNA from the test sample; and (ii) fragmenting the cellular DNA to obtain the genomic DNA fragments. In particular embodiments, step (ii) is performed by contacting cellular DNA with at least one digestion enzyme. In some embodiments, step (ii) is performed by applying mechanical stress to the cellular DNA. In certain embodiments, the mechanical stress is applied by sonicating the cellular DNA.

[0012] In particular embodiments, the sample tag further comprises a unique molecule identifier (UMI) that facilitates identification of the unique genomic DNA fragment.

[0013] In some embodiments, the amplification region is between 10 and 50 nucleotides in length. In particular embodiments, the amplification region is between 20 and 30 nucleotides in length. In certain embodiments, the amplification region is 25 nucleotides in length.

[0014] In some embodiments, the sample tag is between 5 and 50 nucleotides in length. In particular embodiments, the sample tag is between 5 and 15 nucleotides in length. In certain embodiments, the sample tag is 8 nucleotides in length. In some embodiments, the UMI multiplier is adjacent to or contained within the sample tag region.

[0015] In certain embodiments, the UMI multiplier is between 1 and 5 nucleotides in length. In particular embodiments, the UMI multiplier is 3 nucleotides in length and comprises one of 64 possible nucleotide sequences.

[0016] In some embodiments, the anchor region is between 1 and 50 nucleotides in length. In particular embodiments, the anchor region is between 5 and 25 nucleotides in length. In certain embodiments, the anchor region is 10 nucleotides in length.

[0017] Particular embodiments of the present invention are designed as methods wherein the step of (a) generating a genomic DNA library comprising a plurality of DNA library fragments comprises ligating the genomic DNA fragments with a plurality of adapters. In certain embodiments, the genomic DNA fragments are end-repaired prior to ligating the genomic DNA fragments with a plurality of adapters. In particular embodiments, the amplification regions of each adapter of the plurality of adapters comprise an identical nucleotide sequence.

[0018] In certain embodiments, the sample tag region of each adapter of the plurality of adapters comprises one of between 2 and 1,000 nucleotide sequences. In particular embodiments, the sample tag region of each adapter of the plurality of adapters comprises one of between 50 and 500 nucleotide sequences. In various embodiments, the sample tag region of each adapter of the plurality of adapters comprises one of between 100 and 400 nucleotide sequences. In some embodiments, the sample tag region of each adapter of the plurality of adapters comprises one of between 200 and 300 nucleotide sequences. In certain embodiments, the sample tag region of each adapter of the plurality of adapters is 8 nucleotides in length. In some embodiments, each sequence of the nucleotide sequences is distinct from any other sequence of the 240 nucleotide sequences by a Hamming distance of at least two.

[0019] In particular embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region. In some embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to the sample tag region. In certain embodiments, the UMI multiplier of each adapter of the plurality of adapters is between 1 and 5 nucleotides in length. In some embodiments, the UMI multiplier of each adapter of the plurality of adapters is three nucleotides in length.

[0020] In particular embodiments, the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences, and each sample region of a given sequence is paired to only one of the four anchor regions of a given sequence.

[0021] In some embodiments, the amplification regions of each adapter of the plurality of adapters comprises an identical nucleotide sequence; the sample tag region of each adapter of the plurality of adapters is 8 nucleotides in length; the nucleotide sequence of each sample tag is distinct from any other nucleotide sequence of the sample tags of the plurality of adapters by a Hamming distance of at least two; each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region; the UMI multiplier of each adapter of the plurality of adapters is three nucleotides in length; and the UMI multiplier of each of the possible nucleotide sequences is paired with each sample tag region of the plurality of adapters; the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences; and each sample region of a given sequence is paired with only one of the four anchor regions of a given sequence.

[0022] Particular embodiments of the present invention are designed for a method wherein the step of ligating the genomic DNA fragments with a plurality of adapters comprises: (i) ligating an oligonucleotide comprising at least a portion of an anchor region of each genomic DNA fragment, wherein the oligonucleotide comprising at least a portion of an anchor region is a DNA duplex comprising a 5' phosphorylated linker strand duplexed with a partner strand, wherein the partner strand is blocked from ligation by chemical modification at its 3' end and wherein the linker strand is ligated to the genomic DNA fragment; (ii) contacting the genomic DNA fragments ligated with the oligonucleotides comprising at least a portion of the anchor region with DNA oligonucleotides encoding full-length adapter sequences for each adapter nucleotide sequence of the plurality of adapters; and (iii) contacting the genomic DNA fragments and the DNA oligonucleotides encoding the full-length adapter sequence with T4 polynucleotide kinase, Taq DNA ligase, and full-length Bst polymerase under conditions suitable for DNA ligation; thereby ligating the plurality of adapters to the genomic DNA fragments. In some embodiments, the genomic DNA fragments are cfDNA. In certain embodiments, the target region of DNA is analyzed for a copy number alteration.

[0023] In particular embodiments, step (c) performing a quantitative genetic analysis of the genomic DNA fragments comprising the target DNA region comprises purifying the complexes formed between the capture probes and the DNA library fragments comprising the target DNA region. In certain embodiments, step (c) comprises purifying the complexes formed between the capture probes and the DNA library fragments comprising the target DNA region, performing primer extension and/or amplification of the DNA library fragments comprising the region of interest from the genomic DNA library. In some embodiments, step (c) comprises purifying the complexes formed between the capture probes and the DNA library fragments comprising the target DNA region, performing primer extension, and amplification of the DNA library fragments comprising the region of interest from the genomic DNA library. In certain embodiments, step (c) comprises sequencing the DNA from the DNA library fragments comprising the target DNA region to generate a plurality of sequencing reads.

[0024] In some embodiments, the present invention is designed for a method in which the genomic analysis comprises determining a copy number alteration of a DNA region of interest, and in which step (c), performing a quantitative genetic analysis of the genomic DNA fragments comprising the target DNA region, comprises determining a copy number of the region of interest present in the genomic DNA library derived from the test sample, and comparing it with a copy number of the region of interest present in the genomic DNA library derived from a reference sample, in which the reference sample comprises a known copy number of the target DNA region.

[0025] In some embodiments, determining the copy number of the region of interest comprises sequencing DNA from fragments of DNA libraries comprising the target region of DNA to generate a plurality of sequencing reads, wherein each sequencing read comprises a unique molecular identification element (UMIE). In some embodiments, the UMIE includes sequencing information of the adapter and at least a portion of the genomic DNA sequence. In some embodiments, sequencing reads comprising identical UMIEs are identified as a unique genomic sequence (UGS).

[0026] In some embodiments, the methods of determining the copy number further comprise determining a raw genomic depth (RGD) for each of the capture probes contacted with the genomic DNA library. In some embodiments, determining the RGD comprises determining the average number of UGSs associated with each capture probe sequence within a group of sample replicates. In some embodiments, capture probes associated with a highly variable number of UGSs are identified as noisy probes and are removed from further calculations. In some embodiments, determining the RGD further comprises calculating an RGD for a sample, comprising calculating a numerical average of all RGDs for all capture probes in the sample. In some embodiments, RGD values for noisy probes are not included in the calculation of an RGD for a sample.

[0027] In some embodiments, the RGDs for the capture probes are normalized across all samples in an experimental group by converting the RGD for each capture probe into a probe-specific normalized read count, comprising (i) multiplying each capture probe RGD in a sample by a normalization constant, wherein the normalization constant comprises any real number; and (ii) dividing the product of (i) by the RGD calculated for the corresponding sample; or (iii) dividing the product of (i) by an average RGD calculated from a subset of probes. In some embodiments, the subset of probes is a set of control probes.

[0028] In some embodiments, the probe-specific normalized read counts are converted to a copy number value comprising (i) multiplying the probe-specific, normalized read counts of probes targeting autosomal and/or X-linked regions by 2 in samples derived from females; (ii) multiplying the probe-specific, normalized read counts of probes targeting Y-linked and/or X-linked regions by 1 in samples derived from males; (iii) averaging the products of (i) and/or (ii) across all samples in an experiment; and (iv) dividing the product of (i) and/or (ii) by the average of (iii). In some embodiments, the approximate copy number values for all probes targeting a specific gene are averaged.

[0029] In some embodiments, the present invention is designed for a method for highly sensitive detection of copy number gain and copy number loss comprising (i) determining an RGD for a capture probe; (ii) normalizing the RGD for the capture probe across all samples in an experimental group, converting the RGD for the capture probe to a probe-specific normalized read count; (iii) calculating an approximate copy number value for each probe-specific normalized read count; and (iv) averaging the approximate copy number values for all probes targeting a specific gene.

[0030] In some embodiments, the present invention is designed for a method for measuring chromosome stability comprising (i) designing and validating a set of one or more chromosome stability probes, wherein the chromosome stability probes are uniformly distributed throughout human chromosomes; (ii) performing targeted sequencing on patient samples using one or more chromosome stability probes; (iii) determining an approximate copy number value for each chromosome probe; (iv) determining a genomic phenotype of a patient sample, wherein fluctuations in the copy number values of one or more chromosome probes in the patient sample indicate genomic instability.

[0031] In some embodiments, the present invention is directed to a method of treating a cancer in a subject in need thereof, wherein the subject has been identified as having a destabilized genome according to the method of claim 62, wherein the method of treating the cancer comprises administering a pharmaceutically effective amount of a PARP inhibitor.

[0032] In some embodiments, the present invention is designed for a method wherein the genomic analysis comprises determining a copy number alteration of a DNA region of interest, and wherein step (c) performs a quantitative genetic analysis of the genomic DNA fragments comprising the target DNA region, comprises determining a copy number of the region of interest present in the genomic DNA library derived from the test sample, and comparing it with a copy number of the region of interest present in the genomic DNA library derived from a reference sample, wherein the reference sample comprises a known copy number of the target DNA region. In some embodiments, the region of interest is a gene or a portion of the gene. In particular embodiments, the gene is associated with a disease. In certain embodiments, the disease is a cancer. In various embodiments, the gene is BRCA2, ATM, BRCA1, BRIP1, CHEK2, FANCA, HDAC2, and/or PALB2.

[0033] Particular embodiments are designed for a genomic DNA library comprising a plurality of DNA library fragments, wherein each of the DNA library fragments comprises an adapter and a genomic DNA fragment; wherein the adapter is a DNA polynucleotide comprising: an amplification region, a sample tag region, and an anchor region; wherein the amplification region comprises a polynucleotide sequence capable of serving as a primer recognition site for PCR amplification; wherein the sample tag comprises a polynucleotide sequence encoding an identity of the unique library DNA fragment and encoding an identity of the test sample; and wherein the anchor region comprises a polynucleotide sequence encoding the identity of the test sample, and wherein the anchor region is capable of binding to the genomic DNA fragment. In some embodiments, the sample tag further comprises a unique molecule identifier (UMI), wherein the UMI facilitates identification of the unique genomic DNA fragment. In particular embodiments, the amplification region is between 10 and 50 nucleotides in length. In particular embodiments, the amplification region is 25 nucleotides in length. In particular embodiments, the sample tag is between 5 and 50 nucleotides in length. In certain embodiments, the sample tag is 8 nucleotides in length. In some embodiments, the UMI multiplier is adjacent to or contained within the sample tag region. In particular embodiments, the UMI multiplier is between 1 and 5 nucleotides in length. In certain embodiments, the anchor region is between 1 and 50 nucleotides in length. In some embodiments, the anchor region is 10 nucleotides in length. In particular embodiments, the amplification regions of each adapter of the plurality of adapters comprise an identical nucleotide sequence. In some embodiments, each nucleotide sequence of the sample tags is distinct from any other sequence of the sample nucleotide sequences by a Hamming distance of at least two. In certain embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region. In particular embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to the sample tag region. In some embodiments, the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences, and wherein each sample region of a given sequence is matched to only one of the four anchor regions of a given sequence. In some embodiments, the genomic DNA fragment is cfDNA.

[0034] In certain embodiments, the amplification regions of each adapter of the plurality of adapters comprises an identical nucleotide sequence; the sample tag region of each adapter of the plurality of adapters is 8 nucleotides in length; the sample tag region of each adapter of the plurality of adapters comprises a nucleotide sequence that is distinct from any other nucleotide sequence of the sample tags of the plurality of adapters by a Hamming distance of at least two; each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region, the UMI multiplier of each adapter of the plurality of adapters is three nucleotides in length, and the UMI multiplier of each of the possible nucleotide sequences is paired with each of the sample tag regions of the plurality of adapters, the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences, and each sample region of a given sequence is paired with only one of the four anchor regions of a given sequence. In some embodiments, the genomic DNA fragment is cfDNA.

[0035] Certain embodiments are designed for a plurality of genomic DNA libraries, comprising more than one genomic library described herein. In some embodiments, the nucleic acid sequences of the sample tag regions of a genomic DNA library belonging to the plurality of genomic DNA libraries are different from the nucleic acid sequences of the sample tag regions of other genomic DNA libraries belonging to the plurality of genomic DNA libraries. In particular embodiments, the nucleic acid sequences of the amplification regions of a genomic DNA library belonging to the plurality of genomic DNA libraries are identical to the nucleic acid sequences of the amplification regions of other genomic DNA libraries belonging to the plurality of genomic DNA libraries.

[0036] Certain embodiments are designed for a method for the genetic analysis of a DNA target region of cell-free DNA (cfDNA) comprising: (a) generating a DNA library as described herein; (b) contacting the cfDNA library with a plurality of capture probes that specifically bind to a DNA target region, thereby forming complexes between the capture probes and the DNA library fragments comprising the DNA target region; and (c) performing a quantitative genetic analysis of the cfDNA fragments comprising the DNA target region; thereby performing genetic analysis of the DNA target region.

[0037] Certain embodiments are directed to a method of predicting, diagnosing, or monitoring a genetic disease in a subject, comprising: (a) obtaining a test sample from the subject; (b) isolating genomic DNA from the test sample; (c) generating a DNA library comprising a plurality of DNA library fragments, wherein each of the DNA library fragments comprises a genomic DNA fragment from the test sample and an adapter; (d) contacting the cfDNA library with a plurality of capture probes that specifically bind to a target DNA region, thereby forming complexes between the capture probes and the DNA library fragments comprising the target DNA region; and (e) performing a quantitative genetic analysis of one or more target genetic loci associated with the genetic disease in the cfDNA clone library, wherein the identification or detection of one or more genetic lesions at the one or more target genetic loci is prognostic for, diagnostic of, or monitors the progression of the genetic disease. In particular embodiments, quantitative genetic analysis comprises DNA sequencing to generate a plurality of sequencing reads.

[0038] Particular embodiments are designed for a set of adapters encoding an identification of a unique genomic DNA fragment and an identity of a test sample, for use in generating a genomic DNA library, wherein each adapter in said set of adapters is a DNA polynucleotide comprising: an amplification region, a sample tag region, and an anchor region; wherein the amplification region comprises a polynucleotide sequence capable of serving as a primer recognition site for PCR amplification; wherein the sample tag comprises a polynucleotide sequence encoding the identity of the original library DNA fragment and encoding the identity of the test sample; and wherein the anchor region comprises a polynucleotide sequence encoding the identity of the test sample, and wherein the anchor region is capable of binding to the genomic DNA fragment. In some embodiments, the sample tag further comprises a unique molecule identifier (UMI), wherein the UMI facilitates identification of the unique genomic DNA fragment. In various embodiments, the amplification region is between 10 and 50 nucleotides in length. In certain embodiments, the amplification region is 25 nucleotides in length. In particular embodiments, the sample tag is between 5 and 50 nucleotides in length. In some embodiments, the sample tag is 8 nucleotides in length. In particular embodiments, the UMI multiplier is adjacent to or contained within the sample tag region. In some embodiments, the UMI multiplier is between 1 and 5 nucleotides in length. In particular embodiments, the anchor region is between 1 and 50 nucleotides in length. In some embodiments, the anchor region is 10 nucleotides in length. In certain embodiments, the amplification regions of each adapter of the plurality of adapters comprise an identical nucleotide sequence.

[0039] In some embodiments, each nucleotide sequence of the sample tags is distinct from any other nucleotide sequence of the sample tags of the set of adapters by a Hamming distance of at least two. In various embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region. In particular embodiments, each of the plurality of adapters comprises a UMI multiplier that is adjacent to the sample tag region.

[0040] In some embodiments, the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences, and wherein each sample region of a given sequence is paired to only one of the four anchor regions of a given sequence. The set of adapters of claim 75, wherein the amplification regions of each adapter of the plurality of adapters comprise an identical nucleotide sequence; wherein the sample tag region of each adapter is 8 nucleotides in length, wherein each nucleotide sequence of the sample tags is distinct from any other nucleotide sequence of the sample tags of the set of adapters by a Hamming distance of at least two, wherein each of the plurality of adapters comprises a UMI multiplier that is adjacent to or contained within the sample tag region, wherein the UMI multiplier of each adapter of the plurality of adapters is three nucleotides in length, wherein the UMI multiplier comprises one of 64 possible nucleotide sequences, and wherein the UMI multiplier of each of the 64 possible nucleotide sequences is paired with each of the sample tag regions of the plurality of adapters, wherein the anchor tag region of each adapter of the plurality of adapters comprises one of four nucleotide sequences; and in which each sample region of a given sequence is paired with only one of the four anchor regions of a given sequence.

BRIEF DESCRIPTION OF THE VARIOUS VIEWS OF THE DRAWINGS

[0041] FIG. 1 shows the structure of the copy number loss (CNL) assay. Each gene (rows) exhibits a characteristic unique read value that is represented here by a shadow. Each sample (columns) is interrogated on the same panel of genes.

[0042] FIG. 2 shows a diagram illustrating the CNL test signal drivers.

[0043] FIG. 3 shows a diagram illustrating the steps of an illustrative CNL assay performed on cell-free DNA (cfDNA).

[0044] FIG. 4A-4E show diagrams of an illustrative first-generation adapter (FIGs. 4A and 4B) and an adapter of the present invention (FIGS. 4C-4E). FIG. 4A shows the design of the first-generation adapter. FIG. 4B shows that in the first-generation adapters, there was a collection of 249 possible sequence tags, each 5 nucleotides (nt) in length that is linked to a single anchor sequence. FIG. 4C shows a diagram of a second-generation adapter. FIG. 4D shows an illustrative set of adapters that are applied to a single sample consisting of four sets of 8mer tag sequences with each set having 60 members. Each set of 60 tags is specific to one of the four anchor sequences. FIG. 4E shows an illustrative DNA sequence of an adapter.

[0045] FIGS. 5A - FIGS. 5B show a diagram illustrating that shifting the position of the UMI multiplier on the sample label can increase the number of unique sample labels.

[0046] FIGS. 6A and B show a diagram illustrating the process of constructing genomic libraries for a CNL assay. FIG. 6A shows the step where the 10 nt anchor sequence is ligated to the 3' ends of the genomic fragments. FIG. 6B shows the step where the full-length genomic adapters are ligated to the initial anchor sequence.

[0047] FIG. 7 shows DNA entries into CNL libraries. Agarose gel images are shown with marker sizes (bp) indicated on the left.

[0048] FIGS. 8A - FIGS. 8C show conventional box and plot plots of gene copies measured in eight samples, as determined by CNL analysis.

[0049] FIGS. 9A-FIG. 9B show Logio P-value plots that quantify significant deviation from normal in CNL measurements for fragmented genomic samples. The SNP percentages at the top show the minor allele frequencies of rare heterozygous SNPs that are present in the ΔATM and ΔBRCA2 samples.

[0050] FIGS. 10A-FIG. 10B show Log10 P-value plots that quantify significant deviation from normal in CNL measurements for cfDNA samples contaminated with fragmented genomic DNA. The SNP percentages at the top show the minor allele frequencies of rare heterozygous SNPs that are present in the ΔATM and ΔBRCA2 samples.

[0051] FIGS. 11A-11D illustrate the targeted hybrid capture platform. FIG. 11A shows the conversion of cfDNA into a genomic library by the addition of adapter sequences that provide universal single-primer PCR amplification sequences, sample multiplexing tags, and unique molecular identifiers for each genomic clone. FIG. 11B shows denatured amplified genome hybridized with target-specific capture probes and primer extension. FIG. 11C shows an asymmetric paired-end sequencing scheme. FIG. 11D shows mapping statistics for 377,711,020 Illumina NextSeq reads from a typical targeted capture sequencing run. 98.5% of the reads map to the intended targets. After deduplication, 20.40% of the reads (77,053,048) are derived from unique genomic clones.

[0052] FIGS. 12A - FIGS. 12H show linker oligonucleotide sequences from Clusters 1 - 3.

[0053] FIGS. 13A - FIGS. 13H show adaptor oligonucleotide sequences from Clusters 4 - 6.

[0054] FIGS. 14A - FIGS. 14I show adapter oligonucleotide sequences from Clusters 7 - 9.

[0055] FIGS. 15A - FIGS. 15H show adapter oligonucleotide sequences from Clusters 10 - 12.

[0056] FIGS. 16A - FIGS. 16H show adaptor oligonucleotide sequences from Clusters 13 - 15.

[0057] FIGS. 17A - FIGS. 18H show adapter oligonucleotide sequences from Clusters 16 - 18.

[0058] FIGS. 18A - FIGS. 18H show adaptor oligonucleotide sequences from Clusters 19 - 21.

[0059] FIGS. 19A - FIGS. 19H show adaptor oligonucleotide sequences from Clusters 22 - 24.

[0060] FIGS. 20A - FIGS. 20H show adaptor oligonucleotide sequences from Clusters 25 - 27.

[0061] FIGS. 21A - FIGS. 21H show adaptor oligonucleotide sequences from Clusters 28 - 30.

[0062] FIGS. 22A - FIGS. 22H show adaptor oligonucleotide sequences from Clusters 31 - 32.

[0063] FIGS. 23A-23C show targeted sequencing of the TP53 gene. FIG. 23A illustrates the BedFile display of capture probes. FIG. 23B illustrates the depth of coverage at each base position on a scale of 0 to 8000 unique reads. FIG. 23C illustrates a display of the UCSC gene model of known splice variants of TP53. The thicker rectangular regions represent the amino acid coding regions for the protein encoded by TP53.

[0064] FIGS. 24A-24C illustrate the normalized raw unique read density for a single probe, TP53r10_1, across 16 samples. FIG. 24A illustrates the number of raw unique reads captured by the TP53r10_1 probe for 16 independent samples after removing redundant reads by “deduplication.” FIG. 24B shows the overall average of unique reads across 2596 capture probes for all 16 samples. FIG. 24C shows the normalized unique read depth across 16 samples (calculated as: [TP53r10_1 probe sample n unique reads x constant + overall average /sample n unique reads]).

[0065] FIG. 25 shows the overall consistency of normalized unique read counts for all 16 samples within any given TP53 probe, despite significant average depth variation between probes. Normalized unique read counts for all 16 samples are shown as well-spaced bar graph “pillars”; results for all 45 probes targeting TP53 are shown. Two probes that exhibit “noisy” counting behavior are highlighted with arrows. Counts from such probes often appear as outliers in subsequent copy number analysis.

[0066] FIG. 26 illustrates the sample-to-sample consistency of normalized probe-by-probe unique read counts across a large panel of 2596 probes. Scatterplots of three representative samples are shown. Each point represents a different probe. The x-axis is the average normalized unique read depth per probe across 16 samples. The y-axis is the normalized unique read depth per probe for three different individual samples. Consistent probe-by-probe unique read counts support quantitative analysis of chromosomal copy variation.

[0067] FIGS. 27A-27C illustrate the copy number analysis of cfDNA from a healthy female and male donor and from a patient with advanced prostate cancer. FIG. 27A shows the analysis of cfDNA from a healthy donor. The x-axis is a series of control probes targeting regions of all 22 autosomal chromosomes, a series of probes targeting the X-linked AR gene, and a series of probes targeting the coding regions of the TP53 gene. The Y-axis shows the calculated ploidy for each probe. This approximation is calculated for each probe by normalizing the observed unique read counts to a series of control samples whose ploidy is known ([unique read count for probe_Y from sample _Z] x 2 + [average unique read count for probe_Y for multiple control samples]). FIG. 27B illustrates that the X-linked AR gene exhibits a haploid copy number in healthy males. FIG. 27C illustrates copy number analysis of cfDNA from a patient with advanced prostate cancer and shows evidence of highly significant aneuploidy among the control probes, amplification of the AR gene, and loss of the TP53 gene.

[0068] FIG. 28 shows whole-genome aneuploidy analysis of a prostate patient cfDNA library relative to a control sample. The approximate ploidy for each of the 239 control probes is shown sorted by chromosome. The patient chromosome 2 probes show consistent copy loss, and most of the chromosome 5 probes show copy gain. Significant deviations from approximate ploidy are seen for many, but not all, of the patient control probes.

[0069] FIG. 29 shows the analytical validation of copy number loss detection. Genomic DNA from the immortalized line NA02718 (monoallelic ΔATM) and NA09596 (monoallelic ΔBRCA2) was spiked into the 16% “gold standard” genomic DNA of NA12878, resulting in the equivalent of a biallelic deletion minor allele frequency of 8%. After targeted sequencing and CNV analysis, the probe-by-probe ploidies for the two target genes were averaged. Two unperturbed control genes, BRIP1 and HDAC2, are shown for comparison.

DETAILED DESCRIPTION A. OVERVIEW

[0070] The present invention includes, inter alia, compositions and methods that are useful for detecting a mutational alteration, SNP, translocation, inversion, deletion, copy number change, or other genetic variation within a sample of cellular genomic DNA (e.g., from a tissue biopsy sample) or cfDNA (e.g., from a blood sample). The compositions and methods of the present invention are particularly useful in detecting incredibly difficult-to-detect copy number variations in cfDNA from a biological sample (e.g., blood) with exquisite resolution. In particular, some embodiments of the present invention relate to a method for detecting the copy number of a target DNA region from a test sample by generating a genomic DNA library consisting of genomic DNA fragments ligated to an adapter, capturing target DNA regions with a plurality of capture probes, isolating fragments from the DNA library comprising the target DNA region, and performing a quantitative genetic analysis of the target DNA region to thereby determine the copy number of the target DNA region. The adapters described herein allow identification of the individual DNA fragment being sequenced, as well as the identity of the sample or source of the genomic DNA.

[0071] The present invention contemplates, in part, compositions and methods for detecting target-specific copy number changes that are applicable to various sample types, including, but not limited to, direct tissue biopsies and peripheral blood. In the context of cancer genomics, and in particular, cell-free DNA (cfDNA) assays for the analysis of solid tumors, the amount of tumor DNA is often a very small fraction of the total DNA. Furthermore, copy number loss is difficult to detect in genomic DNA assays, and in particular, genomic DNA assays in which the copy number change may only be present in a portion of the total genomic DNA of a sample, e.g., cfDNA assays. For example, most of the cell-free DNA extracted from a cancer patient will be derived from normal sources and will have a diploid copy number (except for X-linked genes in male subjects). In a cancer patient, the tumor-derived DNA fraction generally has a minor allele frequency, such as a patient in whom 2% of circulating DNA extracted from plasma is tumor-derived. The loss of one copy of a tumor suppressor gene (e.g., BRCA1 in breast cancer) means that the minor allele frequency for the absence of detectable genomic fragments is 1%. In this scenario, a modified copy number loss assay must be able to discriminate between 100 copies (normal) and 99 copies (heterozygous gene loss). Thus, particular embodiments contemplate that the methods and compositions of the present invention allow for the detection of copy number alterations with sufficient resolution to detect copy number alterations at minor allele frequencies, even in the context of cfDNA.

[0072] To achieve this level of discrimination, the present invention provides novel sample adapter designs. The adapters of the present invention are designed to include features that are critical for successful copy number loss assay performance, including (i) uniform performance across adapters; (ii) a high number of unique molecule identifiers (UMIs); (iii) high-efficiency binding; and (iv) accommodation of sample multiplexing. For example, the adapters of the present invention provide the following:

[0073] Uniform performance across adapters: Bioinformatics analysis typically analyzes both intra-sample probe performance and inter-sample probe performance. Thus, it is contemplated that any performance fluctuation between adapter sets in samples will negatively impact the ability to detect the subtle variations required by CNL analysis. In the present invention, this uniformity of performance is achieved by having multiple anchor tags that are all represented in each sample tag set, with the fixed sample tag regions (which serve to identify the sample and genomic fragments) being randomly selected for each pool, and a UMI multiplier that increases the unique sample tag sequences to identify the genomic fragments.

[0074] High number of Unique Molecule Identifiers (UMIs): Although adapters must be functionally equivalent from a molecular biology perspective, they must possess a very large number of unique sequence tags (>10,000) that enhance the identification of unique genomic fragments. In this context, by "enhancement" we mean that each fragment of the genomic clone has a particular pair of fragmentation sites corresponding to the position in the genomic sequence where the double-stranded DNA was cleaved. This cleavage site is used to differentiate unique genomic clones since each clone likely has a different cleavage site. However, in libraries containing thousands of independent clones, uniquely derived fragments have the same cleavage sites. Genomic clones (i.e., fragments) that share the same cleavage site can be classified as unique or redundant relative to other clone sequences derived from the same sample. By attaching adapters that introduce a high diversity of sequence tags, different genomic clones that share the same cleavage site are more likely to be identified as unique. In this system, the UMI is created by combining the sample tag region with the UMI multiplier. The combination of the UMI and the cleavage site creates a unique molecular identifier element (UMIE), which facilitates the classification of sequence reads as redundant or unique reads. Particular embodiments contemplate that the UMI multiplier could comprise longer or shorter sequences to increase or decrease the overall complexity of the UMI.

[0075] High-efficiency ligation: Adapters must ligate to genomic fragments with high efficiency. In most oncology applications, the amounts of available cellular DNA or cfDNA are limited, and therefore, the conversion of these genomic fragments into genomic library clones must be highly efficient. To achieve this, in some aspects of the present invention, the adapter systems described herein convert from about 25% to about 50% or more of the input genomic fragments into genomic library clones.

[0076] Accommodating sample multiplexing: In general, there should be groupings of different adapter sets where each unique adapter in the set is ligated to a different sample. At the same time, each member of the adapter set should have essentially identical behavior (from a sequence count perspective) to all other members in a set. To achieve this, in some embodiments, the sample tag regions have a Hamming distance of 2 between any other possible sample tag combinations, reducing the chance of a read being falsely assigned to the wrong sample. In some embodiments, each adapter set is divided into sets that are paired with specific anchor regions, allowing for a further reduction in the possibility of an error in sample demultiplexing. For example, in an 8mer tag with a Hamming distance of 2, the total number of possible sequences is 16,384.

[0077] In a particular embodiment, pre-specified sets of adapter oligonucleotides are provided. These pre-specified pools are used to represent a single sample. That is, each adapter sequence in each set of adapter oligonucleotides X (16,384 in the example given above) is distinct from each adapter sequence in all other sets used to identify other samples. One of skill in the art will recognize that the number of distinct pre-specified sets that are possible for the adapter oligonucleotides will depend on the length of the sample tag and/or the UMI multiplier.

[0078] Thus, in certain embodiments, the adapters comprise a sequence, i.e., the sample tag and the adjacent and/or encompassed UMI multiplier that represents or identifies the sample and uniquely identifies the genetic fragment. This is in stark contrast to current systems used in the art that use a randomly generated tag to identify the sequence and a separate barcode or sequencer indexing to enable multiplexing.

[0079] An illustrative embodiment for detecting target-specific copy number changes within DNA obtained from a sample is shown in FIG. 3. While FIG. 3 generates a DNA library from cfDNA, this illustrative procedure can be used with DNA from other sources, e.g., fragmented cellular DNA. As shown in FIG. 3, cfDNA is collected (top panel). Next, a genomic library is generated from the cfDNA by conjugating genomic library adapters (gray circles) of the present invention to the genomic DNA. Genomic DNA fragments are captured with capture probes (black circles) that recognize the genomic region of interest. The genomic DNA of interest is sequenced, and data analysis is performed for copy loss analysis and/or characterization of the genomic DNA of interest.

[0080] The practice of particular embodiments of the invention will employ, unless specifically indicated otherwise, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); and Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998).

B. DEFINITIONS

[0081] Unless otherwise defined, all technical and scientific terms in this document have the same meaning as is commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, preferred embodiments of compositions, methods, and materials are described herein. For purposes of the present invention, the following terms are defined below.

[0082] The articles “a,” “an,” and “the” are used here to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0083] The use of the alternative (e.g., “or”) should be understood to mean one, both, or any combination of the alternatives.

[0084] The term “and/or” should be understood as meaning one or both of the alternatives.

[0085] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% over a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0086] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms “includes,” “has,” “contains,” and “comprises” are used synonymously.

[0087] By “consisting of” is meant including, and limited to, what follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0088] By “consisting essentially of” is meant to include any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending on whether or not they affect the activity or action of the listed elements.

[0089] Reference throughout this specification to “the embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “another embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the preceding phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0090] As used herein, the term "isolated" means material that is substantially or essentially free of components that normally accompany it in its native state. In particular embodiments, the term "obtained" or "derived" is used synonymously with isolated.

[0091] As used herein, the term "DNA" refers to deoxyribonucleic acid. In various embodiments, the term DNA refers to genomic DNA, recombinant DNA, synthetic DNA, or cDNA. In one embodiment, DNA refers to genomic DNA or cDNA. In particular embodiments, the DNA comprises a "target region." DNA libraries contemplated herein include genomic DNA libraries and cDNA libraries constructed from RNA, e.g., an RNA expression library. In various embodiments, the DNA libraries comprise one or more additional DNA sequences and/or tags.

[0092] The terms “target genetic locus” and “target DNA region” are used interchangeably herein and refer to a region of interest within a DNA sequence. In various embodiments, targeted genetic analyses are performed at the target genetic locus. In particular embodiments, the target DNA region is a region of a gene that is associated with a particular genetic state, genetic condition, genetic diseases; fetal testing; genetic mosaicism; paternity testing; predicting response to drug treatment; diagnosing or monitoring a medical condition; microbiome profiling; tracking pathogens; or monitoring organ transplantation. In other embodiments, the target DNA region is a DNA sequence that is associated with a particular human chromosome, such as a particular autosomal or X-linked chromosome, or a region thereof (e.g., a unique chromosomal region).

[0093] As used herein, the terms "circulating DNA", "circulating cell-free DNA", and "cell-free DNA" are often used interchangeably and refer to DNA that is extracellular DNA, DNA that has been extruded from cells, or DNA that has been released from necrotic or apoptotic cells. This term is often used in contrast to "cellular genomic DNA" or "cellular DNA", which are used interchangeably herein and refer to genomic DNA that is contained within the cell (i.e., the nuclease) and is accessible only to molecular biological techniques such as those described herein, by lysis or otherwise disrupting the integrity of the cell.

[0094] A "subject," "individual," or "patient," as used herein, includes any animal that exhibits a symptom of a condition that can be detected or identified with the compositions contemplated herein. Suitable subjects include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals (such as horses, cows, sheep, pigs), and domestic animals or pets (such as a cat or dog). In particular embodiments, the subject is a mammal. In certain embodiments, the subject is a non-human primate, and in preferred embodiments, the subject is a human.

[0095] As used herein, the term "paired" when used in relation to two different polynucleotide sequences or DNA regions comprising different polynucleotide sequences, means that the two different polynucleotide sequences or DNA regions comprising different polynucleotide sequences are present on the same polynucleotide. For example, if a particular DNA tag region is said to be paired with a particular DNA amplification region, it is understood that the sample tag region and the amplification tag are present on the same DNA polynucleotide molecule.

C. METHODS OF ANALYZING THE NUMBER OF COPIES

[0096] In various embodiments, a method is provided for analyzing the copy number of a DNA target region. In certain embodiments, the copy number analysis is performed by generating a genomic DNA library of DNA library fragments that each contain a genomic DNA fragment and an adapter, isolating the DNA library fragments containing the DNA target regions, and performing a quantitative genetic analysis of the DNA target region. By “quantitative genetic analysis” is meant an analysis performed by any molecular biological technique that is capable of quantifying changes in a DNA (e.g., a gene, genetic locus, target region of interest, etc.) including but not limited to DNA mutations, SNPs, translocations, deletions, and copy number variations (CNVs). In certain embodiments, the quantitative genetic analysis is performed by sequencing, e.g., next-generation sequencing.

[0097] Next-generation DNA sequencing (NGS) is ideal for two diagnostic applications. The first is large-scale DNA sequence determination. In the current context, this capability allows the search for rare and actionable variants that guide effective treatment decisions. The second is gene copy number counting. The output of millions of independent sequences can enable accurate measurement of gene copy number on a genome-wide scale. The emergence of non-invasive prenatal testing for fetal trisomy from maternal blood samples is a testament to this capability. RNAseq, gene expression technology using NGS, is another example, although the input is RNA (cDNA) rather than genomic DNA. Comparisons of current capture methods are described in Samorodnitsky et al. J Mol Diagn. 2015 Jan;17(1):64-75.

[0098] The present invention extends the counting capability of NGS into the realm of targeted hybrid capture methods. The methods described herein are effective for detecting copy number variation, at least in part, because they possess the following four qualities: (a) The present methods differentiate between unique clones and redundant clones. NGS sequencing of amplified genomic DNA library fragments results in a plurality of individual NGS reads, each comprising adapter-encoded sequence information linked to a specific human genomic sequence. These elements define the identity of each clone. Because the captured genomic regions are amplified by PCR, it is not uncommon for the same clone to be found multiple times in a subsequent NGS analysis. Groups of reads that are derived from a single cloning and capture process are termed "redundant reads." Two or more redundant reads are identified as redundant reads based on the sequencing information provided by the unique molecular identification elements (UMIE). UMIE refers to the combination of sequence information from adapter tags and the start of the genomic DNA sequence. Two or more reads containing identical UMIEs are identified as redundant reads. Redundant reads are grouped together, and a single representative consensus sequence is assembled from families of redundant reads. This consensus sequence is referred to as a “unique read” or “unique genomic sequence” (UGS). Each unique read represents a separate clone from the original DNA sample. The process of identifying and grouping families of redundant clones and generating a single single-read representative of that family is defined as “deduplication.” The adapters used to create genomic libraries possess a very deep repertoire of unique sample tag information (15,360 codes per adapter). When applied in conjunction with the exact mapping coordinates of each captured genomic clone (which can span >100 different positions relative to a capture probe), each unique clone generated in a genomic library and subsequently recovered by a target-specific capture probe has an extremely high probability of being distinguishable from all other unique clones spanning the same capture environment. The ability to differentiate between unique clones and redundant clones is central to the methods described here. (b) Adapters used to create genomic libraries allow for multiplexing of samples without creating adapter-to-adapter variability in copy number counts. A central basis of copy number determination is the simultaneous analysis of a set of samples that were all processed in a single sequencing run. This allows positive and negative controls to be included alongside clinical samples. A major issue with previous adapter design iterations induced subtle changes in gene copy counts between identical control samples, setting a signal-to-noise uncertainty threshold too high to be clinically useful in blood-based solid tumor genotyping assays. The present invention overcomes this issue and substantially reduces the signal-to-noise threshold, such that single-copy gene loss is detectable at an allele frequency of less than 2%. This improved signal recognition allows the methods of the present invention to have significant clinical utility in circulating tumor DNA assays. (c) The proprietary targeted hybrid capture method used here should produce highly "uniform" on-target read coverage across all targets. Methods that rely on counting single genomic fragments to estimate copy number, such as those described here, should reach near saturation in finding all possible unique fragments. Near-saturation is only achieved by oversampling, i.e., gathering more sequencing reads than the number of unique reads that will ultimately be found. To be practical, scalable, and cost-effective, the unique reads in a targeted hybrid capture library must exhibit sufficient uniformity that <10-fold oversampling of on-target reads, and preferably <4-fold oversampling of on-target reads, will capture >90% of unique on-target reads across all target locations. (d) The targeted hybrid capture method (See U.S. Patent Publication 2014-0274731) must have high on-target capture rates. To be practical, scalable, and cost-effective, in other words, to be a distinguishing feature of the present disclosure over other art in the field, the method must achieve >90%, preferably >95%, on-target reads. With on-target mapping rates greater than 95%, the requirement for 4 to 10 times oversampling of on-target readings and the overall oversampling requirement are the same.

[0099] In some embodiments, the copy number of the target DNA region present in the sample is determined by quantitative genetic analysis. In some embodiments, the copy number of the target DNA region is determined by comparing the copy number of the target DNA regions present in the sample and comparing them to the target DNA regions present in one or more samples with known copy number.

[00100] Particular embodiments contemplate that the compositions and methods described herein are particularly useful for detecting copy number alterations in a genomic DNA sample, wherein only a portion of the total genomic DNA in the sample has a copy number alteration. For example, a significant tumor mutation may be present in a sample, e.g., a cell-free DNA sample, that is present at a minor allele frequency that is significantly less than 50% (e.g., in the range of 0.1% to >20%), in contrast to conventional SNP genotyping, wherein allele frequencies are generally ~100%, 50%, or 0%. One of skill in the art will recognize that the compositions and methods of the present invention are also useful in detecting other types of mutations, including single nucleotide variants (SNVs), short insertions (e.g., less than 40 base pairs (bp)) and deletions (indels), and genomic rearrangements including oncogenic gene fusions.

[00101] In certain embodiments, the compositions and/or methods of the present invention described herein are useful for, capable of, suitable for, and/or able to detect, identify, observe, and/or reveal a copy number change of one or more target DNA regions present in less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.2%, or less than about 0.1% of the total genomic DNA of the sample. In some embodiments, the methods of the present invention are useful for, capable of, suitable for, and/or able to detect, identify, observe, and/or reveal a copy number change of one or more target DNA regions present in between about 0.01% to about 100%, about 0.01% to about 50%, and or about 0.1% to about 20% of the total genomic DNA of the sample.

[00102] Particular embodiments are represented by the conceptual framework illustrated in FIG. 1. In FIG. 1, each gene is represented by a row and each patient sample is represented as a column. In any genomic DNA sample, the number of fragments counted for each individual gene will have some variability, and for any region of DNA of interest, e.g., a gene, copy number perturbations are detected as significant deviations in the fragment count relative to the normalized counts for the target region of DNA in other samples. Such an assay requires that the gene-by-gene fragment count profile within a sample be reproducible, and also requires that the sample-by-sample count profiles be highly comparable. Both of these assay requirements require excellent discrimination in signal-to-noise counting.

[00103] Some embodiments contemplate that the assay elements that contribute to increasing the signal-to-noise ratio are the genomic input, the number of probes, and the sequencing depth, as illustrated in FIG. 2.

[00104] In particular embodiments, a method for genetic analysis of cfDNA comprises: generating and amplifying a cfDNA library, determining the number of genome equivalents in the cfDNA library; and performing a quantitative genetic analysis of one or more genomic target loci.

[00105] Particular embodiments contemplate that any of the methods and compositions described herein are effective for use to efficiently analyze, detect, diagnose, and/or monitor genetic states, genetic conditions, genetic diseases, genetic mosaicism, fetal diagnostics, paternity testing, microbiome profiling, pathogen screening, and organ transplant monitoring using genomic DNA, e.g., cellular or cfDNA, where all or where only a portion of the total genomic DNA of the sample has a trait of interest, e.g., a genetic lesion, mutation, single nucleotide variant (SNV). In some embodiments, a trait of interest is a genetic trait associated with a disease or condition. For example, a significant tumor mutation may be present in a sample, e.g., a cfDNA sample, that is present at a minor allele frequency that is significantly less than 50% (e.g., in the range of 0.1% to >20%), in contrast to conventional SNP genotyping, where allele frequencies are usually ~100%, 50%, or 0%.

[00106] In certain embodiments, the compositions and/or methods of the present invention described herein are useful for, capable of, suitable for, and/or capable of detecting, identifying, observing, and/or revealing a genetic lesion of one or more target DNA regions present in less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.2%, or less than about 0.1% of the total genomic DNA of the sample. In some embodiments, the methods of the present invention are useful for, capable of, suitable for, and/or capable of detecting, identifying, observing, and/or revealing a genetic lesion of one or more target DNA regions present in between about 0.01% to about 100%, about 0.01% to about 50%, and or about 0.1% to about 20% of the total genomic DNA of the sample.

1. GENERATING A DNA LIBRARY

[00107] In particular embodiments, genetic analysis methods contemplated herein comprise generating a DNA library comprising treating cfDNA or fragmented cellular genomic DNA with one or more end repair enzymes to generate end-repaired DNA and ligating one or more adapters to each end of the end-repaired DNA to generate the DNA library. Genomic DNA

[00108] In particular embodiments, the methods and compositions contemplated herein are designed to effectively analyze, detect, diagnose, and/or monitor copy number alteration using genomic DNA as an analyte. In certain embodiments, the copy number analysis is performed by generating a genomic DNA library from genomic DNA obtained from a test sample, e.g., a biological sample such as a tissue biopsy. In certain embodiments, the genomic DNA is circulating or cell-free DNA. In some embodiments, the genomic DNA is cellular genomic DNA.

[00109] In certain embodiments, the genomic DNA is obtained from a tissue sample or biopsy taken from a tissue, including but not limited to bone marrow, esophagus, stomach, duodenum, rectum, colon, ileum, pancreas, lung, liver, prostate, brain, nerves, meningeal tissue, kidney tissue, endometrial tissue, cervical tissue, breast, lymph node, muscle, and skin. In certain embodiments, the tissue sample is a biopsy of a tumor or a suspected tumor. In particular embodiments, the tumor is cancerous or suspected of being cancerous. In particular embodiments, the tissue sample comprises cancerous cells or cells suspected of being cancerous.

[00110] Methods for purifying genomic DNA from cells or biological tissue consisting of cells are well known in the art, and one skilled in the art will recognize ideal procedures or commercial kits depending on the tissue and the conditions under which the tissue is obtained. Some embodiments contemplate that purification of cellular DNA from a tissue will require cell disruption or cell lysis to expose the cellular DNA within, for example, by chemical and physical methods, such as mixing, grinding, or sonicating the tissue sample; removing membrane lipids by adding a detergent or surfactants that also serve in cell lysis; optionally removing proteins, for example, by adding a protease; removing RNA, for example, by adding an RNase; and purifying DNA, for example, from detergents, proteins, salts, and reagents used during the cell lysis step. DNA purification can be accomplished by precipitation, for example with ethanol or isopropanol; by phenol-chloroform extraction.

[00111] In particular embodiments, cellular DNA obtained from tissues and/or cells is fragmented before and/or during the obtaining, generation, manufacturing, formation, and/or production of a genomic DNA library as described herein. One of skill in the art will understand that there are several suitable techniques for DNA fragmentation and is able to recognize and identify suitable techniques for fragmenting cellular DNA for the purpose of generating a genomic DNA library for DNA sequencing, including but not limited to next generation sequencing. Certain embodiments contemplate that cellular DNA may be fragmented into fragments of appropriate and/or sufficient length to generate a library by methods including, but not limited to, physical fragmentation, enzymatic fragmentation, and chemical shearing.

[00112] Physical fragmentation may include, but is not limited to, acoustic shearing, sonication, and hydrodynamic shearing. In some embodiments, cellular DNA is fragmented by physical fragmentation. In particular embodiments, cellular DNA is fragmented by acoustic shearing or sonication. Particular embodiments contemplate that acoustic shearing and sonication are common physical methods used to shear cellular DNA. The Covaris® instrument (Woburn, MA) is an acoustic device for disrupting DNA at 100-5kb bp. Covaris also manufactures tubes (gTubes) that process samples in the 6-20 kb range for paired-end libraries. The Bioruptor® (Denville, NJ) is a sonication device used to shear chromatin, DNA, and tissue damage. Small volumes of DNA can be sheared at 150-1kb in length. Digilab's Hydroshear (Marlborough, MA) uses hydrodynamic forces to shear DNA. Nebulizers (Life Tech, Grand Island, NY) can also be used to atomize liquids using compressed air, shearing DNA into 100-3kb fragments in seconds. Nebulization is inexpensive, but the process can cause a loss of approximately 30% of the original sample's cellular DNA. In certain embodiments, cellular DNA is fragmented by sonication.

[00113] Enzymatic fragmentation may include, but is not limited to, treatment with a restriction endonuclease, e.g., DNase I, or treatment with a non-specific nuclease. In some embodiments, cellular DNA is fragmented by enzymatic fragmentation. In particular embodiments, cellular DNA is fragmented by treatment with a restriction endonuclease. In some embodiments, cellular DNA is fragmented by treatment with a non-specific nuclease. In certain embodiments, cellular DNA is fragmented by treatment with a transposase. Certain embodiments contemplate that enzymatic methods for shearing cellular DNA into small pieces include DNAse I, a combination of maltose-binding protein (MBP)-T7 Endo I, and a non-specific nuclease Vibrio vulnificus (Vvn) from New England Biolabs (Ipswich, MA) Fragmentase Tagging Technology, and Nextera (Illumina, San Diego, CA). The combination of nonspecific nuclease and T7 Endo works synergistically to produce nonspecific nicks and counternicks, generating fragments that dissociate 8 nucleotides or less from the nick site. Tagging uses a transposase to simultaneously fragment and insert adapters into double-stranded DNA.

[00114] Chemical fragmentation may include treatment with heat and a divalent metal cation. In some embodiments, genomic DNA is fragmented by chemical fragmentation. Particular embodiments contemplate that chemical shearing is more commonly used for the disruption of long RNA fragments as opposed to genomic DNA. Chemical fragmentation is typically accomplished by heat digestion of the DNA with a divalent metal cation (magnesium or zinc). The length of the DNA fragments can be adjusted by increasing or decreasing the incubation time.

[00115] In particular embodiments, the methods and compositions contemplated herein are designed to effectively analyze, detect, diagnose, and/or monitor copy number alteration using cell-free DNA (cfDNA) as an analyte. The size distribution of cfDNA ranges from about 150 bp to about 180 bp fragments. cfDNA fragmentation can be the result of endonucleolytic and/or exonucleolytic activity and presents a formidable challenge to accurate, reliable, and robust cfDNA analysis. Another challenge to cfDNA analysis is its short half-life in the bloodstream, on the order of about 15 minutes. Without wishing to be bound by any particular theory, the present invention contemplates, in part, that cfDNA analysis is like a “liquid biopsy” and is a real-time snapshot of current biological processes.

[00116] Furthermore, because cfDNA is not found in cells and can be obtained from a number of suitable sources, including, but not limited to, biological fluids and stool samples, it is not subject to existing limitations that plague next-generation sequencing analysis, such as access to the analyzed tissues.

[00117] Illustrative examples of biological fluids that are suitable sources from which to isolate cfDNA in particular embodiments include, but are not limited to, amniotic fluid, blood, plasma, serum, semen, lymph fluid, cerebral spinal fluid, ocular fluid, urine, saliva, mucus, and sweat. In particular embodiments, the biological fluid is blood or blood plasma.

[00118] In certain embodiments, commercially available kits and other methods known to one of skill in the art can be used to isolate cfDNA directly from a subject's biological fluids or from a previously obtained and optionally stabilized biological sample, for example, by freezing and/or adding enzyme chelating agents including, but not limited to, EDTA, EGTA, or other chelating agents specific for divalent cations.

(a) Generating end-repaired cfDNA

[00119] In particular embodiments, generating a genomic DNA library comprises end repairing isolated cfDNA or fragmented cellular DNA. The cellular cfDNA or fragmented DNA is processed by end repair enzymes to generate end-repaired cfDNA with blunt ends, 5' overhangs, or 3' overhangs. In some embodiments, end repair enzymes can yield, for example. In some embodiments, the end-repaired cfDNA or cellular DNA contains blunt ends. In some embodiments, the end-repaired cellular DNA or cfDNA is processed to contain blunt ends. In some embodiments, the blunt ends of the end-repaired cfDNA or cellular DNA are further modified to contain a single base pair overhang. In some embodiments, the end-repaired cfDNA or cellular DNA containing blunt ends can be further processed to contain an adenine (A)/thymine (T) overhang. In some embodiments, the end-repaired cfDNA or cellular DNA containing blunt ends can be further processed to contain adenine (A)/thymine (T) overhang as the single base pair overhang. In some embodiments, the end-repaired cfDNA or cellular DNA has untemplated 3' overhangs. In some embodiments, the end-repaired cfDNA or cellular DNA is processed to contain 3' overhangs. In some embodiments, the end-repaired cfDNA or cellular DNA is processed with terminal transferase (TdT) to contain 3' overhangs. In some embodiments, a G-tail can be added by TdT. In some embodiments, the end-repaired cfDNA or cellular DNA is processed to contain overhangs using partial digestion with any known restriction enzymes (e.g., with the enzyme Sau3A and the like).

(b) Attaching adapter molecules to the end-repaired cfDNA

[00120] In particular embodiments, generating a cfDNA library comprises ligating one or more adapters to each end of the end-repaired cfDNA. The present invention contemplates, in part, an adapter module designed to accommodate large numbers of genome equivalents in cfDNA libraries. The adapter modules are configured to measure the number of genome equivalents present in cfDNA libraries and, by extension, the sensitivity of sequencing assays used to identify sequence mutations.

[00121] As used herein, the terms "adapter" and "adapter module" are used interchangeably, and refer to a polynucleotide comprising at least three elements: an amplification region, a sample tag region, and an anchor region. In particular embodiments, the adapter comprises an amplification region, a sample tag region, and an anchor region. In some embodiments, the adapter also comprises a unique molecule identifier (UMI). In particular embodiments, the adapter comprises one or more amplification regions, one or more sample tag regions, one or more UMIs, and/or one or more anchor regions. In some embodiments, the adapter comprises, in the order of 5' to 3', an amplification region, a sample tag region, a UMI, and an anchor region. In particular embodiments, the adapter comprises, in the order of 5' to 3', an amplification region, a sample tag region, a UMI, and an anchor region. In certain embodiments, the UMI is contained within the sample tag region and the adapter comprises, in the order of 5' to 3', an amplification region, an integrated sample tag/UMI region, and an anchor region.

[00122] As used herein, the term "amplification region" refers to an element of the adapter molecule that comprises a polynucleotide sequence capable of serving as a primer recognition site for PCR amplification. In particular embodiments, an adapter comprises an amplification region that comprises one or more primer recognition sequences for amplifying a single primer from a genomic DNA library. In some embodiments, the amplification region comprises one, two, three, four, five, six, seven, eight, nine, ten, or more primer recognition sequences for amplifying a single primer from a genomic DNA library.

[00123] In some embodiments, the amplification region is between 5 and 50 nucleotides, between 10 and 45 nucleotides, between 15 and 40 nucleotides, or between 20 and 30 nucleotides in length. In some embodiments, the amplification region is 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, about 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, or 40 or more nucleotides. In particular embodiments, the amplification region is 25 nucleotides in length.

[00124] As used herein, the term "sample tag" or "sample tag region" is used interchangeably and refers to an adapter element comprising a polynucleotide sequence that uniquely identifies the particular DNA fragment as well as the sample from which it was derived.

[00125] In certain embodiments, the sample tag region is between 3 and 50 nucleotides, between 3 and 25 nucleotides, or between 5 and 15 nucleotides in length. In some embodiments, the sample tag region is 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, about 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides or more in length.

[00126] In certain embodiments, the adapter comprises a UMI multiplier, wherein the UMI multiplier is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides in length.

[00127] In certain embodiments, each nucleotide position of the UMI multiplier can comprise any of adenine, guanine, cytosine, or thymine. Thus, in some embodiments, a UMI multiplier comprising n number of nucleotides can comprise any of n4 possible nucleotide sequences. In some embodiments, the UMI multiplier is one nucleotide in length and comprises one of four possible sequences. In some embodiments, the UMI multiplier is two nucleotides in length and comprises one of sixteen possible sequences. In some embodiments, the UMI multiplier is three nucleotides in length and comprises one of 64 possible sequences. In some embodiments, the UMI multiplier is four nucleotides in length and comprises one of 256 possible sequences. In some embodiments, the UMI multiplier is five nucleotides in length and comprises one of 1,024 possible sequences. In some embodiments, the UMI multiplier is six nucleotides in length and comprises one of 4,096 possible sequences. In some embodiments, the UMI multiplier is seven nucleotides in length and comprises one of 16,384 possible sequences. In some embodiments, the UMI multiplier is eight nucleotides in length and comprises one of 65,5336 possible sequences. In some embodiments, the UMI multiplier is nine nucleotides in length and comprises one of 262,144 possible sequences. In some embodiments, the UMI multiplier is ten or more nucleotides in length and comprises one of 1,048,576 possible sequences.

[00128] In particular embodiments, the adapter comprises a UMI multiplier, wherein the UMI multiplier is adjacent to or contained within the sample tag region (FIG. 5A). Illustrative examples of UMI multipliers adjacent to or contained within the sample tag are shown in FIG. 5B. In FIG. 5B, an 8-mer sample tag region is shown with an adjacent UMI multiplier (top and bottom rows) or a UMI multiplier embedded within the sample tag (middle 7 rows). In some embodiments, such an adapter comprises a sample tag that is eight nucleotides in length and a UMI multiplier that is three nucleotides in length and comprises one of 64 possible sequences, and wherein the UMI multiplier is adjacent to or contained within the sample tag region. In some embodiments, identical processes ligate the full-length adapter to the other end of the genomic fragments.

[00129] In particular embodiments, an adapter module comprises one or more anchor sequences. As used herein, an “anchor region” and “anchor sequence” are used interchangeably and refer to a nucleotide sequence that hybridizes to a partner oligonucleotide. In some embodiments, the anchor region comprises the following three properties: (1) each anchor sequence is part of a family of two or more anchor sequences that collectively represent each of the four possible DNA bases at each site within the extension; this feature, balanced base representation, is useful for calibrating appropriate base calling in sequencing reads in particular embodiments; (2) each anchor sequence is composed of only two of the four possible bases, and these are specifically chosen to be an equal number of A+C or an equal number of G+T; an anchor sequence formed from only two bases reduces the possibility that the anchor sequence participates in the formation of secondary structure that would prevent proper adapter function; and (3) because each anchor sequence is composed of equal numbers of A + C or G + T, each anchor sequence shares approximately the same melting temperature and duplex stability as any other anchor sequence in a set of four.

[00130] In some embodiments, the anchor sequences are between 1 and 50 nucleotides in length. In some embodiments, the anchor sequences are between 4 and 40 nucleotides in length. In certain embodiments, the anchor region is between 5 and 25 nucleotides in length. In embodiments, the anchor region is at least 4 nucleotides, at least 6 nucleotides, at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, or at least 16 nucleotides in length. In particular embodiments, the anchor region is 10 nucleotides in length.

[00131] In particular embodiments, a ligation step comprises ligating/ligating an adapter module to the end-repaired cfDNA or cellular DNA to generate a labeled genomic DNA library. In some embodiments, a single adapter module is employed. In some embodiments, two, three, four, or five adapter modules are utilized. In some embodiments, an adapter module of identical sequence is ligated to each end of the fragmented and end-repaired DNA.

[00132] In some embodiments, a plurality of adapter species is attached to an end-repaired cellular or cell-free genomic DNA fragment. Each of the plurality of adapters may comprise one or more amplification regions for amplifying the cellular DNA or cfDNA library, one or more example tag regions for identifying the cfDNA or cellular genomic DNA fragment and identifying the individual sample; and one or more sequences for DNA sequencing.

[00133] In some embodiments, a plurality of adapter species is ligated to an end-repaired cellular or cell-free genomic DNA fragment of a sample, and the plurality of adapters all comprise amplification regions of an identical nucleotide sequence.

[00134] In certain embodiments, genomic DNA from a sample is ligated with a plurality of adapters comprising sample tag sequences that are all different from other sample tag region sequences on adapters that are ligated to genomic DNA fragments from other samples.

[00135] In particular embodiments, a plurality of adapter species are ligated to end-repaired cellular or cell-free genomic DNA fragments of a sample, and the plurality of adapters all comprise one or more sample tag regions comprising one of between 2 and 10,000 nucleotide sequences, one of between 5 and 5,000 nucleotide sequences, one of between 25 and 1,000 nucleotide sequences, one of between 50 and 500 nucleotide sequences, one of between 100 and 400 nucleotide sequences, or one of between 200 and 300 nucleotide sequences. In some embodiments, the sample tag region of each adapter is 8 nucleotides in length, and each sample tag region of the plurality of adapters comprises one of 240 nucleotide sequences.

[00136] In certain embodiments, a plurality of adapter species are ligated to an end-repaired cellular, genomic, or cell-free cellular DNA fragment of a sample, and the sample tag regions of the plurality of adapters comprise nucleotide sequences that are different from each other by a Hamming distance of 1, 2, 3, 4, or greater than 4. In particular embodiments, the Hamming distance is 2.

[00137] In particular embodiments, the sample tag regions of the plurality of adapters that are ligated to genomic DNA fragments of a sample are 8 nucleotides in length and comprise one of 240 nucleotide sequences that are different from each other by a Hamming distance of 2.

[00138] In certain embodiments, the sample tag region serves to identify individual genomic DNA fragments and to identify the individual sample, i.e., the source of the genomic library. For example, when the sample tags of a plurality of adapters ligated to a sample have one of 240 possible sequences, each sample is identified as having one of 240 possible tags, and each sample is assigned a set of 240 tags that are distinct from any other sample by a Hamming distance of two (i.e., two base changes are required to change one tag into another). These same tags are used to enumerate clone diversity and therefore also serve as sequence tags, i.e., to identify genomic DNA fragments. To further increase the diversity of possible sequence tags, UMI multipliers can be added. For example, a UMI multiplier can be added to the adapter region comprising 3 nucleotides consisting of the 64 possible 3-base combinations. Furthermore, the plurality of adapters may comprise more than one anchor sequence. For example, a plurality of adapters may contain four different anchor sequences used simultaneously. These anchor sequences can also be used during multiple sample removal to reduce errors.

[00139] FIG. 4 shows an illustrative comparison between a first generation adapter (FIG. 4A and 4B) and an adapter of the present invention (FIG. 4C - FIG. 4E). FIGS. 4A and FIG. 4B show an example of a first generation adapter that is 40 nt in length and consisted of a distinct PCR amplification sequence, sequence tag, and sample tag. Here, the sample is identified by a fixed sequence (sequence tag) that is present in all adapters that are used to generate a DNA library from the sample. Individual genomic fragments are identified by separate and distinct sequences (sequence tag). FIGS. 4C - FIG. 4E show an illustrative example of an adapter of the present invention. The sequence tag is combined with the sample tag. There is an additional 3 nt sequence, the UMI multiplier, consisting of the 64 possible 3-base combinations. The 10 nt anchor sequence is one of four distinct sequences.

[00140] Thus, in the illustrative example (See FIG. 4C - FIG. 4E), a set of adapters that are used in connection with a single sample comprises 240 sample tag sequences that can be divided into four sets of sample tag sequences with each set comprising 60 tags (one for each nucleotide, A, C, T, and G). Thus, each set of 60 tags is specific to one of the four anchor sequences. In total, a set of 240 possible sample tag configurations is possible per sample. Specifically, in this scenario, the 240 sample tag sequences are divided into four sets of 60 sequences, with each set targeting a specific anchor region. Therefore, the sample ID involves not only the tag sequence information of eight nucleotide samples, but also the associated anchor sequence information. Furthermore, the position of sequences within the read is fixed, and therefore, sample tags and anchor sequences must have a fixed position within a sequencing read in order to pass inclusion filters for downstream consideration. Furthermore, the inclusion of the UMI multiplier increases the sequence tag diversity from 240 to 240 x 64 = 15,360 possible sequence tags.

[00141] Ligation of one or more adapters contemplated herein can be accomplished by methods known to those of skill in the art. In particular embodiments, one or more adapters contemplated herein are ligated to end-repaired cfDNA comprising blunt ends. In certain embodiments, one or more adapters contemplated herein are ligated to end-repaired cfDNA comprising complementary ends appropriate for the ligation method employed. In certain embodiments, one or more adapters contemplated herein are ligated to end-repaired cfDNA comprising a 3' overhang.

[00142] In some embodiments, ligating the genomic DNA fragments of a plurality of adapters includes the steps of ligating the end-repaired cfDNA fragments or cellular DNA to an oligonucleotide containing at least a portion of an anchor region. In some embodiments, the oligonucleotide contains the complete anchor region. In particular embodiments, the oligonucleotide is a DNA duplex comprising a 5' phosphorylated linker strand duplexed with a partner strand, wherein the partner strand is blocked from ligation by chemical modification at its 3' end and wherein the linker strand is linked to the genomic DNA fragment. In certain embodiments, the DNA fragments ligated with at least a portion of the anchor region are then annealed to DNA oligonucleotides encoding the full-length adapter sequences. In particular embodiments, one or more polynucleotide kinases, one or more DNA ligases, and/or one or more DNA polymerases are added to the genomic DNA fragments and DNA oligonucleotides encoding the full-length adapter sequence. In some embodiments, the polynucleotide kinase is T4 polynucleotide kinase. In some embodiments, the DNA ligase is Taq DNA ligase. In certain embodiments, the DNA polymerase is Taq polymerase. In particular embodiments, the DNA polymerase is full-length Bst polymerase.

[00143] FIG. 6 shows an illustrative method for ligating a plurality of adapters to the 3' end of repaired DNA fragments. In the first step, the anchor sequence is attached to the 3' ends of the genomic fragments. In this step, the anchor moiety is a DNA duplex in which the ten-nucleotide 5' phosphorylated "linker strand" is duplexed with an eight-nucleotide "partner strand" that is blocked from ligation by chemical modification at its 3' end. The anchor duplex is blunt at the phosphorylated/blocked end and can therefore ligate to blunt-ended genomic fragments. In the next step, sets of oligonucleotides encoding the complete adapter sequences are annealed to the initial anchor sequence. The combined action of T4 polynucleotide kinase, Taq DNA ligase, and full-length Bst polymerase ligates this oligonucleotide through ligation as illustrated for the top strand and extends the initial anchor sequence by DNA polymerization on the bottom strand to complete the full-length adapter sequence. Identical procedures can be used to ligate full-length adapters to the 5' end of genomic fragments.

2. DNA LIBRARY AMPLIFICATION

[00144] In particular embodiments, the genetic analysis methods contemplated herein comprise amplifying a genomic DNA library, e.g., a cellular DNA library or a cfDNA library, to generate a DNA clone library or a library of DNA clones, e.g., a cfDNA clone library or a cfDNA clone library, or a cellular DNA clone library or a library of cellular DNA clones. Each molecule of the DNA library comprises an adapter ligated to each end of an end-repaired DNA fragment, and each adapter comprises one or more amplification regions. In some embodiments, different adapters are ligated to different ends of the end-repaired cfDNA. In particular embodiments, different adapters are ligated to different ends of the end-repaired cellular DNA.

[00145] In some embodiments, the same adapter is ligated to both ends of the DNA fragment. Ligation of the same adapter to both ends of the end-repaired DNA allows for PCR amplification with a single primer sequence. In particular embodiments, a portion of the library of ligated cfDNA adapters will be amplified using standard PCR techniques with a single driving amplification primer sequence. In one embodiment, the single primer sequence is about 25 nucleotides, optionally with a designed Tm of >55°C under standard ionic strength conditions.

[00146] In particular embodiments, picograms of the initial genomic DNA library, e.g., a cellular DNA library or cfDNA library, are amplified into micrograms of DNA clones, implying a 10,000-fold amplification. The amount of amplified product can be measured using methods known in the art, e.g., quantification on a Qubit 2.0 or Nanodrop instrument.

3. DETERMINING THE NUMBER OF GENOME EQUIVALENTS

[00147] In various embodiments, a method for genetic analysis of genomic DNA comprises determining the number of genome equivalents in the DNA clone library. As used herein, the term "genome equivalent" refers to the number of genome copies in each library. A key challenge faced by the compositions and methods contemplated herein is achieving sufficient assay sensitivity to detect and analyze rare genetic mutations or differences in genetic sequence. To determine assay sensitivity on a sample-by-sample basis, the number of different and distinct sequences present in each sample is measured by measuring the number of genome equivalents present in a sequencing library. To establish sensitivity, the number of genome equivalents must be measured for each sample library.

[00148] The number of genome equivalents can be determined by qPCR assay or using bioinformatics-based counting after sequencing. In the clinical sample process flow, qPCR measurement of genomic equivalents is used as a QC step for DNA libraries, e.g., cfDNA libraries or genomic DNA libraries. It establishes an expectation for assay sensitivity prior to sequence analysis and allows a sample to be excluded from analysis if its corresponding DNA clone library does not have the required depth of genome equivalents. Finally, bioinformatics-based counting of genomic equivalents is also used to identify genome equivalents—and thus assay sensitivity and false negative estimates—for each DNA clone library.

[00149] The empirical qPCR assay and statistical counting assays should be well correlated. In cases where sequencing does not reveal the sequence depth in a DNA clone library, reprocessing of the DNA clone library and/or additional sequencing may be required.

[00150] In one embodiment, genome equivalents in a cellular DNA or cfDNA clone library are determined using a quantitative PCR (qPCR) assay. In a particular embodiment, a standard library of known concentration is used to construct a standard curve, and measurements from the qPCR assay are fitted to the resulting standard curve, and a value for genome equivalents is derived from the fit. The present inventors have discovered that a repeat-based qPCR assay comprising a primer that specifically hybridizes to a common sequence in the genome, e.g., a repeat sequence, and another primer that binds to the primer binding site on the adapter, measured an 8-fold increase in genome equivalents compared to methods using only the adapter-specific primer (present at both ends of the cfDNA clone). The number of genome equivalents measured by the repeat-based assays provides more consistent library-to-library performance and better alignment between qPCR estimates of genome equivalents and tag equivalents counted bioinformatically in sequencing runs.

[00151] Illustrative examples of repeats suitable for use in the repeat-based genome-wide equivalent assays contemplated herein include, but are not limited to: short interspersed nuclear elements (SINEs), e.g., Alu repeats; long interspersed nuclear elements (LINEs), e.g., LINE1, LINE2, LINE3; microsatellite repeat elements, e.g., short tandem repeats (STRs), simple sequence repeats (SSRs); and mammalian interspersed repeats (MIRs).

[00152] In one embodiment, the repetition is an Alu repetition.

4. QUANTITATIVE GENETIC ANALYSIS

[00153] In various embodiments, a method for genetic analysis of genomic DNA, e.g., cellular genomic or cfDNA, comprises quantitative genetic analysis of one or more target genetic loci from DNA library clones. The quantitative genetic analysis comprises one or more of, or all of, the following steps: capturing DNA clones comprising a target genetic locus; amplifying the captured target genetic locus; sequencing the captured amplified target genetic locus; and bioinformatic analysis of the resulting sequence reads. As used herein, the terms "DNA library clone" refer to a DNA library fragment in which the combination of the adapter and the genomic DNA fragment results in a unique DNA sequence (e.g., a DNA sequence that can be distinguished from that of another DNA library clone).

(a) Capture of the target genetic locus

[00154] The present invention contemplates, in part, a capture probe module designed to retain the efficiency and reliability of larger probes, but which minimizes the generation of uninformative sequence in a genomic DNA library comprising smaller DNA fragments, e.g., a cfDNA clone library. A “capture probe” or “capture probe module,” as used herein, is used interchangeably and refers to a polynucleotide comprising a capture probe sequence and a tail sequence. In particular embodiments, the capture probe module sequence or a portion thereof serves as a primer binding site for one or more sequencing primers.

[00155] In particular embodiments, a capture probe module comprises a capture probe. As used herein, a “capture probe” refers to a region capable of hybridizing to a specific DNA target region. In some embodiments, capture probes are used with genomic DNA libraries constructed from cellular DNA. In particular embodiments, capture probes are used with genomic DNA libraries constructed from cfDNA. Because the average size of cfDNA is about 150 to about 170 bp and is highly fragmented, certain embodiments are targeted compositions, and the methods contemplated herein comprise using relatively small, high-density capture probes to interrogate DNA target regions of interest. In some embodiments, the capture probes are capable of hybridizing to DNA target regions that are distributed throughout chromosome segments at a uniform density. A set of such capture probes is referred to herein as “chromosome stability probes.” Chromosome stability probes are used to interrogate copy number variations on a genome-wide scale to provide a measure of chromosome copy number in the genome (e.g., chromosome ploidy).

[00156] A particular concern with the use of high-density capture probes is that, in general, capture probes are designed using specific "sequence rules." For example, regions of redundant sequence or those exhibiting extreme base composition biases are typically excluded in capture probe design. However, the present inventors have found that a lack of flexibility in capture probe design rules does not substantially affect probe performance. In contrast, capture probes chosen strictly by positional constraint provided on-target sequence information; exhibit very little off-target and unmappable read capture; and produce uniform, useful, on-target reads, with only a few exceptions. Furthermore, the high redundancy in close probe spacing more than compensates for the occasional low-performing capture probe.

[00157] In particular embodiments, a target region is targeted by a plurality of capture probes, wherein any two or more capture probes are designed to bind to the target region within 10 nucleotides of each, within 15 nucleotides of each, within 20 nucleotides of each, within 25 nucleotides of each, within 30 nucleotides of each, within 35 nucleotides of each, within 40 nucleotides of each, within 45 nucleotides of each, or within 50 nucleotides or more of each, as well as all intervening nucleotide lengths.

[00158] In one embodiment, the capture probe is about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, or about 45 nucleotides. nucleotides.

[00159] In one embodiment, the capture probe is about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, or about 100 nucleotides. In another embodiment, the capture probe is about 100 nucleotides to about 500 nucleotides, about 200 nucleotides to about 500 nucleotides, about 300 nucleotides to about 500 nucleotides, or about 400 nucleotides to about 500 nucleotides, or any intervening range thereof.

[00160] In a particular embodiment, the capture probe is 60 nucleotides. In another embodiment, the capture probe is substantially shorter than 60 nucleotides, but hybridizes comparatively as well as, or better than, a 60 nucleotide capture probe targeting the same target DNA region. In certain embodiments, the capture probe is 40 nucleotides.

[00161] In certain embodiments, a capture probe module comprises a tail sequence. As used herein, the term "tail sequence" refers to a polynucleotide at the 5' end of the capture probe module, which in particular embodiments can serve as a primer binding site. In particular embodiments, a sequencing primer binds to the primer binding site in the tail region.

[00162] In particular embodiments, the tail sequence is about 5 to about 100 nucleotides, about 10 to about 100 nucleotides, about 5 to about 75 nucleotides, about 5 to about 50 nucleotides, about 5 to about 25 nucleotides, or about 5 to about 20 nucleotides. In certain embodiments, the third region is about 10 to about 50 nucleotides, about 15 to about 40 nucleotides, about 20 to about 30 nucleotides, or about 20 nucleotides, or any intervening number of nucleotides.

[00163] In particular embodiments, the tail sequence is about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, or about 40 nucleotides.

[00164] In various embodiments, the capture probe module comprises a specific member of a binding pair to enable isolation and/or purification of one or more captured fragments from a labeled and/or amplified genomic DNA library (e.g., a cellular or cfDNA library) that hybridize to the capture probe. In particular embodiments, the capture probe module is conjugated to biotin or another suitable hapten, e.g., dinitrophenol, digoxigenin.

[00165] In various embodiments, the capture probe module is hybridized to a labeled and optionally amplified DNA library to form a complex. In some embodiments, the multifunctional capture probe module substantially hybridizes to a specific genomic target region in the DNA library.

[00166] Hybridization or hybridization conditions may include any reaction conditions in which two nucleotide sequences form a stable complex; for example, the labeled DNA library and the capture probe module form a stable labeled DNA library-capture probe module complex. Such reaction conditions are well known in the art, and those skilled in the art will appreciate that such conditions may be modified as appropriate, for example, decreased annealing temperatures with shorter length capture probes, and within the scope of the present invention. Substantial hybridization can occur when the second region of the capture probe complex exhibits 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92% 91%, 90%, 89%, 88%, 85%, 80%, 75%, or 70% sequence identity, homology, or complementarity to a region of the labeled DNA library.

[00167] In particular embodiments, the capture probe is about 40 nucleotides and has an optimal annealing temperature of about 44°C to about 47°C.

[00168] In certain embodiments, the methods contemplated herein comprise isolating a labeled cfDNA library-capture probe module complex. In particular embodiments, methods for isolating DNA complexes are well known to those of skill in the art, and any methods considered appropriate by one of skill in the art can be employed with the methods of the present invention (Ausubel et al., Current Protocols in Molecular Biology, 2007-2012). In particular embodiments, the complexes are isolated using biotin-streptavidin isolation techniques.

[00169] In particular embodiments, removal of the single-stranded 3' ends of the isolated labeled DNA library-capture probe module complex fragments is contemplated. In certain embodiments, the methods comprise enzymatically processing 3'-5' exonuclease of the isolated labeled DNA library-multifunctional capture probe module complex to remove the single-stranded 3' ends.

[00170] In certain other embodiments, the methods comprise performing 5'-3' DNA polymerase extension of the multifunctional capture probe using the isolated labeled DNA library fragments as a template.

[00171] In certain other embodiments, the methods comprise creating a hybrid capture probe-isolated labeled DNA target molecule, e.g., a labeled cfDNA target molecule or a labeled cellular DNA target molecule, through the joint action of a 5' FLAP endonuclease, DNA polymerization, and nick closure by a DNA ligase.

[00172] A variety of enzymes may be utilized for the enzymatic 3'-5' exonuclease processing of the isolated labeled DNA library-multifunctional capture probe module complex. Illustrative examples of suitable enzymes, which exhibit 3'-5' exonuclease enzymatic activity, that may be employed in particular embodiments include, but are not limited to: T4 or Exonucleases I, III, V (See also, Shevelev IV, Hübscher U., Nat Rev Mol Cell Biol. 3(5):364-76 (2002)). In particular embodiments, the enzyme comprising the 3'-5' exonuclease activity is T4 polymerase. In particular embodiments, an enzyme may be employed that exhibits 3'-5' exonuclease enzymatic activity and is capable of primer template extension, including for example T4 or Exonucleases I, III, V. Id.

[00173] In some embodiments, the methods contemplated herein comprise performing sequencing and/or PCR on the enzymatically processed 3'-5' exonuclease complex discussed supra and elsewhere herein. In particular embodiments, a tail portion of a capture probe molecule is copied so as to generate a hybrid nucleic acid molecule. In one embodiment, the generated hybrid nucleic acid molecule comprises the target region capable of hybridizing to the capture probe module and the complement of the capture probe module tail sequence.

[00174] In a certain embodiment, the genetic analysis comprises a) hybridizing one or more capture probe modules to one or more target genetic loci of a plurality of genomic DNA library clones, to form one or more capture probe module-DNA library clone complexes; b) isolating the one or more capture probe module-DNA library clone complexes from a); c) enzymatically processing the one or more capture probe module-DNA library clone complexes from step b); d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the capture probe molecule is copied to generate amplified hybrid nucleic acid molecules, wherein the amplified hybrid nucleic acid molecules comprise a target sequence at the target genomic locus capable of hybridizing to the capture probe and the complement of the tail sequence of the capture probe module; and e) performing quantitative genetic analysis on the amplified hybrid nucleic acid molecules from d).

[00175] In a particular embodiment, methods are contemplated for determining the copy number of a specific target genetic locus comprising: a) hybridizing one or more capture probe modules to one or more target genetic loci of a plurality of DNA library clones to form one or more capture probe module-DNA library clone complexes; b) isolating the one or more capture probe module-DNA library clone complexes from a); c) enzymatically processing the one or more capture probe module-DNA library clone complexes from step b); d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the capture probe molecule is copied to generate amplified hybrid nucleic acid molecules, wherein the amplified hybrid nucleic acid molecules comprise a target sequence at the target genetic locus capable of hybridizing to the capture probe and the complement of the tail sequence of the capture probe module; e) perform PCR amplification of the hybrid amplified nucleic acid molecules in d); and f) quantify the PCR reaction in e), wherein quantification allows a determination of the copy number of the specific target region.

[00176] In one embodiment, the enzymatic processing of step c) comprises performing 3'-5' exonuclease enzymatic processing on one or more capture probe module-DNA library clone complexes of b) using an enzyme with 3'-5' exonuclease activity to remove single-stranded 3' ends; creating one or more hybrid capture probe module-cfDNA library clone molecules through the concerted action of a 5' FLAP endonuclease, DNA polymerization, and nick closure by a DNA ligase; or performing 5'-3' DNA polymerase probe extension using the DNA clone isolated in the complex as a template.

[00177] In one embodiment, the enzymatic processing of step c) comprises performing 5'-3' DNA polymerase extension of the capture probe using the DNA clone isolated in the complex as a template.

[00178] In particular embodiments, the PCR may be performed using any conventional PCR reaction conditions well known to those of skill in the art. In certain embodiments, the PCR reaction in e) employs two PCR primers. In one embodiment, the PCR reaction in e) employs a first PCR primer that hybridizes to a repeat within the target genetic locus. In a particular embodiment, the PCR reaction in e) employs a second PCR primer that hybridizes to the hybrid nucleic acid molecules at the target genetic locus/tail junction. In certain embodiments, the PCR reaction in e) employs a first PCR primer that hybridizes to the target genetic locus and a second PCR primer that hybridizes to the amplified hybrid nucleic acid molecules at the target genetic locus/tail junction. In particular embodiments, the second primer hybridizes to the target genetic locus/tail junction such that at least one or more nucleotides of the primer hybridize to the target genetic locus and at least one or more nucleotides of the primer hybridize to the tail sequence.

[00179] In certain embodiments, the amplified hybrid nucleic acid molecules obtained from step e) are sequenced and the sequences aligned horizontally, i.e., aligned with each other but not aligned to a reference sequence. In particular embodiments, steps a) to e) are repeated one or more times with one or more capture probe modules. The capture probe modules may be the same or different and designed to target both cfDNA strands of a target genetic locus. In some embodiments, when the capture probes are different, they hybridize to overlapping or adjacent target sequences within a target genetic locus in the labeled cfDNA clone library. In one embodiment, a high-density capture probe strategy is utilized in which a plurality of capture probes hybridize to a target genetic locus, and in which each of the plurality of capture probes hybridizes to the target genetic locus within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 bp or more of any other capture probe that hybridizes to the target genetic locus in a labeled DNA clone library, including all intervening distances.

[00180] In some embodiments, the method may be performed using two capture probe modules per target genetic locus, wherein one hybridizes to the "Watson" strand (non-coding strand or template strand) upstream of the target region and one hybridizes to the "Crick" strand (coding strand or non-template strand) downstream of the target region.

[00181] In particular embodiments, the methods contemplated herein may further be performed multiple times with any number of capture probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more capture probe modules per target genetic locus, any number of which hybridize to the Watson or Crick strand in any combination. In some embodiments, the obtained sequences may be aligned to each other in order to identify any of several differences.

[00182] In certain embodiments, a plurality of target genetic loci are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 10,000, 50,000, 100,000, 500,000, or more in a single reaction, using one or more capture probe modules.

(b) Sequencing

[00183] In particular embodiments, the quantitative genetic analysis comprises sequencing a plurality of hybrid nucleic acid molecules, as discussed elsewhere, supra, to generate sequencing depths sufficient to obtain a plurality of unique sequencing reads. The terms “unique reads” or “unique genomic sequences” (UGS) are used interchangeably herein and are identified by grouping individual redundant reads together into a “family.” Redundant reads are sequence reads that share an identical UMIE (e.g., share the same read code and the same DNA sequencing start position within the genomic sequence) and are derived from a single connection event and are therefore amplification-derived “siblings.” A single consensus representative of a family of redundant reads is carried forward as a unique read or UGS. Each unique read or UGS is considered a unique connection event. The sum of unique reads corresponding to a given capture probe is called the “raw genomic depth” (RGD) for that specific capture probe. Each capture probe produces a set of unique reads that are computationally distilled from the total reads by clustering into families. The unique reads for a given sample (i.e., raw genomic depth for a sample) are then calculated as the average of all observed unique reads on a probe-by-probe basis. Unique reads are important because each unique read must be derived from a single genomic DNA clone. Each unique read represents the input and analysis of a haploid equivalent of genomic DNA. The sum of the unique reads is the sum of the haploid genomes analyzed. The number of genomes analyzed, in turn, defines the sensitivity of the sequencing assay. By way of a non-limiting example, if the average unique read count is 100 genome equivalents, then this particular assay has a sensitivity of being able to detect a mutant read in 100, or 1%. Any observation less than this is not defensible.

[00184] Instances where there is an obvious change in copy number (e.g., occurrences of noisy probes) are excluded from the dataset used to calculate the sample mean. Here, a "noisy probe" refers to a probe that captures a highly variable number of unique reads among a large set of identical samples (e.g., a highly variable number of unique reads among 12-16 replicate samples). In some embodiments, the number of unique reads associated with a noisy probe is increased compared to the average number of unique reads for the sample by 50% or more. In some embodiments, the number of unique reads associated with a noisy probe is decreased compared to the average number of unique reads for the sample by 50% or more. In some embodiments, about 2% to about 4% of the probes used in a particular analysis are identified as noisy probes and are excluded from calculations to determine the average number of unique readings for a given sample.

[00185] In some embodiments, sequencing reads are identified as “on-target reads” or “off-target reads.” On-target reads have a genomic DNA sequence that maps within the vicinity of a capture probe used to create the genomic library. In some embodiments, where each genomic sequence is physically linked to a specific capture probe and where the sequence of the genomic segment and the capture probe are determined as a unified piece of information, an on-target read is defined as any genomic sequence whose start coordinates map within 400 bp, and more generally within 200 bp, of the 3' end of the corresponding capture probe. Off-target reads are defined as having genomic sequence that aligns to the reference genome at a location >500 base pairs (and more often mapping to entirely different chromosomes) relative to the capture probe.

[00186] In particular embodiments, the quantitative genetic analysis comprises multiplex sequencing of hybrid nucleic acid molecules derived from a plurality of samples.

[00187] In various embodiments, the quantitative genetic analysis comprises obtaining one or more or a plurality of clones from labeled DNA libraries, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a sequence of a target genetic locus and the second DNA sequence comprises a capture probe sequence; performing a paired-end sequencing reaction on the one or more clones and obtaining one or more sequencing reads, or performing a sequencing reaction on the one or more clones in which a single long sequencing read exceeding about 100, 200, 300, 400, 500, or more nucleotides is obtained, wherein the read is sufficient to identify both the first DNA sequence and the second DNA sequence; and ordering or grouping the sequencing reads from the one or more clones according to the probe sequences of the sequencing reads.

(c) Bioinformatics analysis

[00188] In various embodiments, the quantitative genetic analysis further comprises bioinformatics analysis of sequencing reads. Bioinformatics analysis excludes any purely mental analysis performed in the absence of a sequencing composition or method. In certain embodiments, bioinformatics analysis includes, but is not limited to: sequence alignments; genome equivalents analysis; single nucleotide variant (SNV) analysis; gene copy number variation (CNV) analysis; chromosomal copy number measurement; and detection of genetic lesions. In particular embodiments, bioinformatics analysis is useful for quantifying the number of genome equivalents analyzed in the cfDNA clone library; for detecting the genetic status of a target genetic locus; for detecting genetic lesions at a target genetic locus; and measuring copy number fluctuations within a target genetic locus.

[00189] Sequence alignments can be performed between the sequence reads and one or more human reference DNA sequences. In particular embodiments, sequencing alignments can be used to detect genetic lesions at a target genomic locus, including, but not limited to, the detection of a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a copy number change, or a gene fusion. Detection of genetic lesions that are causal or prognostic indicators can be useful in the diagnosis, prognosis, treatment, and/or monitoring of a particular genetic condition or disease.

[00190] Also contemplated herein are methods for sequence alignment analysis that can be performed without the need for an alignment of a reference sequence, referred to herein as horizontal sequence analysis. Such analysis can be performed on any sequences generated by the methods contemplated herein or any other methods. In particular embodiments, sequence analysis comprises performing sequence alignments on reads obtained by the methods contemplated herein.

[00191] In one embodiment, genome equivalents in a cfDNA clone library are determined using bioinformatics-based counting after sequencing. Each sequencing read is associated with a particular capture probe, and the collection of reads assigned to each capture probe is analyzed in groups. Within a group, sets of individual reads share the same read code and the same DNA sequence starting position within the genomic sequence. These individual reads are grouped into a "family," and a single consensus representative of that family is transmitted as a "unique read." All individual reads that constitute a family are derived from a single ligation event and are therefore "siblings" derived from amplification of one another. Each unique read is considered a unique ligation event, and the sum of unique reads is considered equivalent to the number of genome equivalents analyzed.

[00192] As the number of unique clones approaches the total number of possible sequence combinations, probability dictates that the same coding and start site combinations will be created by independent events, and that these independent events will be inappropriately clustered into individual families. The net result will be an underestimate of the genome equivalents analyzed, and rare mutant reads may be dismissed as sequencing errors because they overlap with wild-type reads that have the same identifiers.

[00193] In particular embodiments, to provide accurate analysis for cfDNA clone libraries, the number of genome equivalents analyzed is about 1/10, about 1/12, about 1/14, about 1/16, about 1/18, about 1/20, about 1/25, or less, the number of possible unique clones. It should be understood that the procedure described above is merely illustrative and not limiting.

[00194] In some embodiments, the number of genome equivalents to be analyzed may need to be increased. To expand the depth of genome equivalents, at least two solutions are contemplated. The first solution is to use more than one set of adapters per sample. By combining adapters, it is possible to multiply the total number of possible clones and therefore expand the comfortable limits of the genomic input. The second solution is to expand the read length by 1, 2, 3, 4, or 5 or more bases. The number of possible read lengths that differ by at least 2 bases from any other read length is scaled as 4(n-1), where n is the number of bases within a read length. Thus, in a non-limiting example, if a read length is 5 nucleotides and 4(5-1) = 256, then the inclusion of additional bases expands the available repertoire by a factor of four for each additional base.

[00195] In one embodiment, the quantitative genetic analysis comprises bioinformatic analysis of sequencing reads to identify rare single nucleotide variants (SNV).

[00196] Next-generation sequencing has an inherent error rate of approximately 0.02-0.02%, meaning that 1 in 200 to 1 in 500 base calls are incorrect. To detect variants and other mutations that occur at lower frequencies than this, for example, at frequencies of 1 in 1,000 sequences, it is necessary to invoke molecular annotation strategies. By way of a non-limiting example, the analysis of 5,000 unique molecules using targeted sequence capture technology would generate—at sequencing depths >50,000 reads—a collection of 5,000 unique reads, with each unique read belonging to a "family" of reads that all have the same read code. An SNV that occurs within a family is a candidate for being a rare variant. When that same variant is observed in more than one family, it becomes a very strong candidate for being a rare variant that exists within the initial sample. In contrast, variants that occur sporadically within families are likely sequencing errors, and variants that occur within one and only one family are either rare or the result of a base change that occurred ex vivo (e.g., oxidation of a DNA base error or introduced by PCR).

[00197] In one embodiment, methods of detecting SNVs include inputting 10 times as much genomic input (genomes or genome equivalents) as the desired target sensitivity of the assay. In a non-limiting example, if the desired sensitivity is 2% (2 in 100), then the experimental target is an input of 2,000 genomes.

[00198] In particular embodiments, bioinformatic analysis of desequencing data is used to detect or identify SNVs associated with a genetic state, condition, or disease, genetic mosaicism, fetal testing, paternity testing, predicting response to drug treatment, diagnosing or monitoring a medical condition, microbiome profiling, pathogen tracking, and organ transplant monitoring.

[00199] In various embodiments, a method for copy number determination analysis is provided comprising obtaining one or more or a plurality of clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a sequence of a target genetic locus and the second DNA sequence comprises a capture probe sequence. In related embodiments, a paired-end sequencing reaction on the one or more clones is performed and one or more sequencing reads are obtained. In another embodiment, a sequencing reaction on the one or more clones is performed in which a single long sequencing read of greater than about 100 nucleotides is obtained, wherein the read is sufficient to identify the first DNA sequence and the second DNA sequence. The sequencing reads from the one or more clones can be ordered or grouped according to the probe sequence of the sequencing reads.

[00200] Copy number analyses include, but are not limited to, analyses that examine the number of copies of a particular gene or mutation occurring in a given genomic DNA sample and may further include the quantitative determination of the number of copies of a given gene or sequence in a given sample. In particular embodiments, copy number analysis is used to detect or identify gene amplification associated with genetic states, conditions, or diseases, fetal testing, genetic mosaicism, paternity testing, predicting response to drug treatment, diagnosing or monitoring a medical condition, microbiome profiling, pathogen tracking, and organ transplant monitoring.

[00201] In some embodiments, copy number analysis is used to measure chromosomal instability. In such embodiments, capture probe sets comprising chromosome stability probes are used to determine copy number variations at a uniform density across all sets of chromosomes. Copy number analyses are performed for each chromosome stability probe, and the chromosome stability probes are then sorted according to their chromosomal target. This allows for the visualization of copy number losses or gains across the genome and can serve as a measure of chromosome stability.

[00202] In particular embodiments, bioinformatics analysis of desequencing data is used to detect or identify one or more genetic sequences or lesions at a target locus, including, but not limited to, the detection of a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a copy number change, or a gene fusion. Detection of genetic lesions that are causal or prognostic indicators can be useful in the diagnosis, prognosis, treatment, and/or monitoring of a particular genetic condition or disease. In one embodiment, genetic lesions are associated with genetic states, conditions, or diseases, fetal testing, genetic mosaicism, paternity testing, predicting response to drug treatment, diagnosing or monitoring a medical condition, microbiome profiling, pathogen tracking, and organ transplant monitoring.

D. CLINICAL APPLICATIONS OF QUANTITATIVE CNL ASSESSMENTS

[00203] In various embodiments, the present invention contemplates a method for detecting, identifying, predicting, diagnosing, or monitoring a condition or disease in a subject by detecting a mutational alteration, SNP, translocation, inversion, deletion, copy number change, or other genetic variation in a region of interest. E. CLINICAL APPLICATIONS OF QUANTITATIVE GENETIC ANALYSIS

[00204] In various embodiments, the present invention contemplates a method for detecting, identifying, predicting, diagnosing, or monitoring a condition or disease in a subject.

[00205] In particular embodiments, a method for detecting, identifying, predicting, diagnosing, or monitoring a genetic state, condition, or disease in a subject comprises performing a quantitative genetic analysis of one or more target genetic loci in a DNA clone library to detect or identify a sequence change at one or more target genetic loci. In some embodiments, the change is a copy number change.

[00206] In one embodiment, a method for detecting, identifying, predicting, diagnosing, or monitoring a genetic state, condition, or disease comprises isolating or obtaining cellular DNA or cfDNA from a biological sample of a subject; treating the cellular DNA or cfDNA with one or more end repair enzymes to generate end-repaired DNA; ligating one or more adapters to each end of the end-repaired DNA to generate a genomic DNA library; amplifying the DNA library to generate a DNA clone library; determining the number of genome equivalents in the DNA clone library; and performing a quantitative genetic analysis of one or more target genetic loci in a DNA clone library to detect or identify a sequence change, e.g., a SNP, a translocation, an inversion, a deletion, or a copy number change, at one or more target genetic loci.

[00207] In one embodiment, a method for detecting, identifying, predicting, diagnosing, or monitoring a genetic state, condition, or disease selected from the group consisting of: genetic diseases; genetic mosaicism; fetal testing; paternity testing; parentage testing; predicting response to drug treatment; diagnosing or monitoring a medical condition; microbiome profiling; pathogen screening; and organ transplant monitoring comprising isolating or obtaining genomic DNA from a biological sample of a subject; treating the DNA with one or more end repair enzymes to generate end-repaired DNA; ligating one or more adapters to each end of the end-repaired DNA to generate a genomic DNA library; amplifying the genomic DNA library to generate a DNA clone library; determining the number of genome equivalents in the DNA clone library; and perform a quantitative genetic analysis of one or more target genetic loci in a DNA clone library to detect or identify a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a copy number change, or a gene fusion in the sequence at one or more target genetic loci.

[00208] Illustrative examples of genetic diseases that may be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to, cancer, Alzheimer's disease (APOE1), Charcot-Marie-Tooth disease, Leber hereditary optic neuropathy (LHON), Angelman syndrome (UBE3A, ubiquitin protein ligase E3A), Prader-Willi syndrome (region on chromosome 15), β-thalassemia (HBB, β-globin), Gaucher disease (type I) (GBA, Glucocerebrosidase), Cystic fibrosis (epithelial chloride channel CFTR), sickle cell disease (HBB, β-globin), Tay-Sachs disease (HEXA, Hexosaminidase A), Phenylketonuria (PAH, Phenylalanine Hydrolysate), Familial hypercholesterolemia (LDLR, low-density lipoprotein receptor), adult polycystic kidney disease (PKD1, polycystin), Huntington's disease (HDD, Huntingtin), Neurofibromatosis type I (tumor suppressor gene NF1, NF1), myotonic dystrophy (DM, myotonin), tuberous sclerosis (TSC1, Tubererin), Achondroplasia (FGFR3, fibroblast growth factor receptor), Fragile X syndrome (FMR1, RNA-binding protein), Duchenne muscular dystrophy (DMD, dystrophin), hemophilia A (F8C, blood clotting factor VIII), Lesch-Nyhan syndrome (HPRT1, hypoxanthine-guanine ribosyltransferase 1), and adrenoleukodystrophy (ABCD1).

[00209] Illustrative examples of cancers that may be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to: B-cell cancer, e.g., multiple myeloma, melanomas, breast cancer, lung cancer (such as non-small cell lung carcinoma or NSCLC), bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical, uterine, or endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel, or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, hematologic tissue cancer, adenocarcinomas, inflammatory myofibroblastic tumors, gastrointestinal stromal tumor (GIST), colon cancer, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), soft tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioarcoma, lymphangiosarcoma, lymphangioendothelioarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gland carcinoma sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, head and neck cancer, small cell cancer, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familial hypereosinophilic, chronic eosinophilic leukemia, neuroendocrine cancers, carcinoid tumors, and similar.

[00210] In one embodiment, the genetic lesion is a lesion annotated in the Cosmic database (lesions and sequence data are available online and can be downloaded from the Cancer Gene Census section of the Cosmic website) or a lesion annotated in The Cancer Genome Atlas (lesions and sequence data are available online and can be downloaded from The Cancer Genome Atlas website).

[00211] Illustrative examples of genes harboring one or more genetic lesions associated with cancer that can be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to, ABCB1, ABCC2, ABCC4, ABCG2, ABL1, ABL2, AKT1, AKT2, AKT3, ALDH4A1, ALK, APC, AR, ARAF, ARFRP1, ARID1A, ATM, ATR, AURKA, AURKB, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL6, BRAF, BRCA1, BRCA2, Clorf144, CARD11, CBL, CCND1, CCND2, CCND3, CCNE1, CDH1, CDH2, CDH20, CDH5, CDK4, CDK6, CDK8, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CRKL, CRLF2, CTNNB1, CYP1B1, CYP2C19, CYP2C8, CYP2D6, CYP3A4, CYP3A5, DNMT3A, DOT1L, DPYD, EGFR, EPHA3, EPHA5, EPHA6, EPHA7, EPHB1, EPHB4, EPHB6, EPHX1, ERBB2, ERBB3, ERBB4, ERCC2, ERG, ESR1, ESR2, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FANCA, FBXW7, FCGR3A, FGFR1, FGFR2, FGFR3, FGFR4, FLT1, FLT3, FLT4, FOXP4, GATA1, GNA11, GNAQ, GNAS, GPR124, GSTP1, GUCY1A2, HOXA3, HRAS, HSP90AA1, IDH1, IDH2, IGF1R, IGF2R, IKBKE, IKZF1, INHBA, IRS2, ITPA, JAK1, JAK2, JAK3, JUN, KDR, KIT, KRAS, LRP1B, LRP2, LTK, MAN1B1, MAP2K1, MAP2K2, MAP2K4, MCL1, MDM2, MDM4, MEN1, MET, MITF, MLH1, MLL, MPL, MRE11A, MSH2, MSH6, MTHFR, MTOR, MUTYH, MYC, MYCL1, MYCN, NF1, NF2, NKX2-1, NOTCH1, NPM1, NQO1, NRAS, NRP2, NTRK1, NTRK3, PAK3, PAX5, PDGFRA, PDGFRB, PIK3CA, PIK3R1, PKHD1, PLCG1, PRKDC, PTCH1, PTEN, PTPN11, PTPRD, RAF1, RARA, RB1, RET, RICTOR, RPTOR, RUNX1, SLC19A1, SLC22A2, SLCO1B3, SMAD2, SMAD3, SMAD4, SMARCA4, SMARCB1, SMO, SOD2, SOX10, SOX2, SRC, STK11, SULT1A1, TBX22, TET2, TGFBR2, TMPRSS2, TNFRSF14, TOP1, TP53, TPMT, TSC1, TSC2, TYMS, UGT1A1, UMPS, USP9X, VHL, and WT1.

[00212] In particular embodiments, the genetic lesion comprises a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, a copy number alteration, or a gene fusion.

[00213] In one embodiment, the genetic lesion is a genetic fusion that fuses the 3' coding region of the ALK gene to another gene.

[00214] In one embodiment, the genetic lesion is a genetic fusion that fuses the 3' coding region of the ALK gene with the EML4 gene.

[00215] Illustrative examples of conditions suitable for fetal testing that may be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to: Down Syndrome (Trisomy 21), Edwards Syndrome (Trisomy 18), Patau Syndrome (Trisomy 13), Klinefelter Syndrome (XXY), Triple X Syndrome, XYY Syndrome, Trisomy 8, Trisomy 16, Turner Syndrome (XO), Robertsonian Translocation, DiGeorge Syndrome, and Wolf-Hirschhorn Syndrome.

[00216] Illustrative examples of alleles suitable for paternity testing that can be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to, 16 or more of: D20S1082, D6S474, D12ATA63, D22S1045, D10S1248, D1S1677, D11S4463, D4S2364, D9S1122, D2S1776, D10S1425, D3S3053, D5S2500, D1S1627, D3S4529, D2S441, D17S974, D6S1017, D4S2408, D9S2157, Amelogenin, D17S1301,D1GATA113, D18S853, D20S482 and D14S1434.

[00217] Illustrative examples of genes suitable for predicting response to drug treatment that can be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to, one or more of the following gene sequences: ABCB1 (ATP-binding cassette, subfamily B (MDR/TAP), member 1), ACE (angiotensin I converting enzyme), ADH1A (alcohol dehydrogenase 1A (class I), alpha polypeptide), ADH1B (alcohol dehydrogenase IB (class I), beta polypeptide), ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ADRB1 (adrenergic, beta-1, receptor), ADRB2 (adrenergic, beta-2, receptor, surface), AHR (aryl hydrocarbon receptor), ALDH1A1 (aldehyde dehydrogenase family 1, member A1), ALOX5 (arachidonate 5-lipoxygenase), BRCA1 (breast cancer 1, early onset), COMT (catechol-O-methyltransferase), CYP2A6 (cytochrome P450 family 2, subfamily A, polypeptide 6), CYP2B6 (cytochrome P450 family 2, subfamily B, polypeptide 6), CYP2C9 (cytochrome P450 family 2, subfamily C, polypeptide 9), CYP2C19 (cytochrome P450 family 2, subfamily C, polypeptide 19), CYP2D6 (cytochrome P450 family 2, subfamily D, polypeptide 6), CYP2J2 (cytochrome P450 family 2, subfamily J, polypeptide 2), CYP3A4 (cytochrome P450 family 3, subfamily A, polypeptide 4), CYP3A5 (cytochrome P450 family 3, subfamily A, polypeptide 5), DPYD (dihydropyrimidine dehydrogenase), DRD2 (dopamine D2 receptor), F5 (coagulation factor V), GSTP1 (glutathione S-transferase 1I), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), KCNH2 (potassium voltage-gated channel, subfamily H (EAG-related), member 2), KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11), MTHFR (5,10-methylenetetrahydrofolate (NADPH) reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), P2RY1 (P2Y purinergic receptor, G-protein coupled, 1), P2RY12 (P2Y purinergic receptor, G-protein coupled, 12), PTGIS (prostaglandin I2 (prostacyclin) synthase), SCN5A (sodium channel, voltage-gated, type V, alpha (long QT syndrome 3)), SLC19A1 (solute transporter family 19 (folate transporter), member 1), SLCO1B1 (solute carrier organic anion transporter, member 1B1), SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), TPMT (thiopurine S-methyltransferase), TYMS (thymidylate synthetase), UGT1A1 (UDP glucuronosyltransferase family 1, polypeptide A1), VDR (vitamin D receptor (1,25-dihydroxyvitamin D3)), VKORC1 (vitamin K epoxide reductase complex, subunit 1).

[00218] Illustrative examples of medical conditions that may be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to: stroke, transient ischemic attack, traumatic brain injury, heart disease, heart attack, angina, atherosclerosis, and high blood pressure.

[00219] Illustrative examples of pathogens that may be screened for with the compositions and methods contemplated herein include, but are not limited to: bacteria fungi and viruses.

[00220] Illustrative examples of bacterial species that may be screened with the compositions and methods contemplated herein include, but are not limited to: a Mycobacterium spp., a Pneumococcus spp., an Escherichia spp., a Campylobacter spp., a Corynebacterium spp., a Clostridium spp., a Streptococcus spp., a Staphylococcus spp., a Pseudomonas spp., a Shigella spp., a Treponema spp., or a Salmonella spp.

[00221] Illustrative examples of fungal species that may be screened for with the compositions and methods contemplated herein include, but are not limited to: an Aspergillis spp., a Blastomyces spp., a Candida spp., a Coccicioides spp., a Cryptococcus spp., dermatophytes, a Tinea spp., a Trichophyton spp., a Microsporum spp., a Fusarium spp., a Histoplasma spp., a Mucoromycotina spp., a Pneumocystis spp., a Sporothrix spp., an Exserophilum spp., or a Cladosporium spp.

[00222] Illustrative examples of viruses that may be screened for with the compositions and methods contemplated herein include, but are not limited to: Influenza A, such as H1N1, H1N2, H3N2, and H5N1 (avian flu), Influenza B, Influenza C, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Rotavirus, any virus of the Norwalk group of viruses, enteric adenoviruses, parvoviruses, dengue virus, smallpox, mononegavirales, lyssaviruses such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2, and Australian bat virus, Ephemoviruses, Vesiculoviruses, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Barr virus (EBV), human herpesviruses (HHV), human herpesvirus types 6 and 8, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV), and Rous sarcoma virus (RSV), HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), immunodeficiency virus bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV), papillomavirus, murine gammaherpesvirus, Arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia virus-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing viruses, Rift Valley fever virus, Filoviridae (filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing viruses, and Paramyxoviridae such as Hendra and Nipah viruses, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, and any virus that causes encephalitis.

[00223] Illustrative examples of genes suitable for monitoring an organ transplant in a transplant recipient that can be detected, identified, predicted, diagnosed, or monitored with the compositions and methods contemplated herein include, but are not limited to, one or more of the following genes: HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, and HLA-DQ.

[00224] In particular embodiments, a bioinformatics analysis is used to quantify the number of genome equivalents analyzed in the cfDNA clone library; detect genetic variants at a target genetic locus; detect mutations within a target genetic locus; detect genetic fusions within a target genetic locus; or measure copy number fluctuations within a target genetic locus.

F. COMPLEMENTARY DIAGNOSIS

[00225] In various embodiments, a complementary diagnostic for a genetic disease is provided, comprising: isolating or obtaining genomic DNA from a biological sample of a subject; treating the DNA with one or more end repair enzymes to generate end-repaired DNA; ligating one or more adapters to each end of the end-repaired DNA to generate a DNA library; amplifying the DNA library to generate a DNA clone library; determining the number of genome equivalents in the DNA clone library; and performing a quantitative genetic analysis of one or more biomarkers associated with the genetic disease in the DNA clone library, wherein detection or failure to detect at least one or more biomarkers indicates whether the subject should be treated for the genetic disease. In some embodiments, the DNA is cfDNA. In particular embodiments, the DNA is cellular DNA.

[00226] As used herein, the term "companion diagnostic" refers to a diagnostic test that is linked to a particular anticancer therapy. In a particular embodiment, the diagnostic methods comprise detecting the genetic lesion in a biomarker associated with a biological sample, thus allowing the immediate identification of whether or not patients should be treated with the anticancer therapy.

[00227] Anticancer therapy includes, but is not limited to, surgery, radiation, chemotherapeutics, anticancer drugs, and immunomodulators.

[00228] Illustrative examples of anticancer drugs include, but are not limited to: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine retum; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin and its pegylated formulations, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptomycin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamin, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as phrolininic acid; aceglaton; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofirane; spirogermanium; tenuazonic acid; triaziquone; 2, 2',2”-trichlorohydrylamine; urethane; vindesine; dacarbazine; mannomone; mitobronitol; mitolactol; pipobromane; gamma-cytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomitin (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (alitretinoin); ONTAK™ (denileukin diftitox); esperamycins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included in this definition are antihormonal agents that act to regulate or inhibit hormonal action in cancers, such as antiestrogens, including for example tamoxifen, raloxifene, aromatase inhibitors 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifen, keoxifen, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[00229] Illustrative examples of immunomodulators include, but are not limited to: cyclosporine, tacrolimus, tresperimus, pimecrolimus, sirolimus, verolimus, laflunimus, laquinimod, and imiquimod, as well as analogs, derivatives, salts, ions, and complexes thereof.

[00230] In some embodiments, an anticancer drug may include a poly-ADP ribose polymerase (PARP) inhibitor. Illustrative examples of PARP inhibitors include, but are not limited to, olaparib (AZD-2281), rucaparib (AG014699 or PF-01367338, niraparib (MK-4827), talazoparib (BMN-673) veliparib (ABT-888), CEP 9722, E7016, BGB-290, 3-aminobenzamide.

[00231] All publications, patent applications, and issued patents cited in this specification are incorporated herein by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference. In particular, the entire contents of PCT International Publication WO 2016/028316 are specifically incorporated by reference.

[00232] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce essentially similar results.

EXAMPLES Example 1: Copy number analysis of samples containing mixtures of fragmented genomic DNA

[00233] Meticulous mixtures of fragmented genomic DNA were generated containing DNA derived from immortalized human ΔATM or ΔBRCA2 samples added to a fragmented wild-type human gDNA sample. The advantage of this type of sample is that the composition can be carefully controlled and the availability of the sample is essentially unlimited.

[00234] Wild-type human female genomic DNA was purified from whole blood samples donated by a healthy volunteer. Genomic DNA isolated from an immortalized cell harboring a heterozygous deletion covering the entire ATM gene (NA09596, ΔATM) and a separate sample containing a heterozygous deletion of BRCA2 (NA02718, ΔBRCA2) were obtained from the Coriell repository. Importantly, these samples appeared to have largely normal ploidy throughout the remainder of their genomes. The ΔATM sample was derived from a male donor and therefore also hemizygous for the X-linked AR gene copy number. Cell-free DNA (cfDNA) was obtained from plasma samples from healthy donors of either female or male origin. For library construction, genomic DNA was sonicated to a 200 bp setting with a Covaris instrument, then further size selected using a “double-sided” DNA bead purification. The input DNA samples in the library are shown in FIG. 7.

[00235] Appropriate combinations of fragmented samples and cfDNA were mixed to defined percentages, end-repaired, and converted to genomic libraries. Approximately 500 ng of each library was combined into pools of eight samples and hybridized to the copy number loss (CNL) prostate probe pool containing 2304 DNA probes. After sample processing, each pool of eight samples was sequenced on an Illumina NextSeq NGS instrument to a depth of ~480 million pass-filter reads; this corresponds to 60 million reads/sample. Approximately 95% of the reads had legitimate sample ID identifiers and aligned to the human reference genome, and of these, ~98% mapped to the intended target loci. The overall sequencing depth, measured as the number of reads per genome input per probe (calculated as on-target reads (60 million) divided by the average genome depth (2500) and divided by the probe count (2400)) was approximately 10 reads per genome per probe. A graphical representation of the copy number loss analysis is shown in Fig. 1. Copy number perturbations are highlighted by arrows. (Sample 1, 5% male DNA in female DNA; sample 2, 5% ΔATM DNA (male) in female DNA; sample 3, 5% ΔBRCA2 DNA (female) in female DNA; sample 4, pure female DNA).

[00236] The CNL caller identifies redundant reads and condenses them into a single consensus read that is then quantified at each probe site. This information was further condensed into gene-by-gene copy number averages. Finally, a statistical significance was assigned to the deviations detected in each CNL measurement; this is shown graphically as the log10 P-value of statistical significance.

[00237] FIG. 8 shows copy number plots for the AR (FIG. 8B) and ATM (FIG. 8C) genes in fragmented and mixed genomic libraries. Because the ΔATM sample is male, the AR gene (X-linked, hemizygous) and the ATM gene exhibited CNL behavior. As anticipated, the magnitude of the measured copy variation was modest. The statistical analysis shown in FIG. 9B demonstrates that the observed copy fluctuation was statistically significant. Furthermore, very little significant fluctuation was observed in the remaining genes that were predicted to exhibit uniform copy characteristics. These values correlated well with the predicted frequencies for the various genomic mixtures. FIG. 10 shows that statistically significant copy fluctuation was also readily observed in samples that were primarily cfDNA with minor hints of either cfDNA from the opposite sex or minor additions of fragmented gDNA. These values correlated well with the predicted frequencies for the various genomic mixtures. The results observed with fragmented gDNA and cfDNA were comparable, demonstrating the integrity of the assay and suggesting that this integrity will translate to clinical samples.

[00238] These data demonstrate the assay system's ability to detect subtle gene copy number changes down to allele frequencies of less than 2%. While the focus of the examples demonstrated is on copy number loss, the technology is equally suitable for detecting copy number gains, including increases in gene copy number that occur through chromosome arm duplications and focal amplifications. This assay also maintains the ability to detect other types of genomic variants, including SNVs, indels, and gene fusions (chromosomal rearrangements). Importantly, these data demonstrate that the method can be applied to genomic DNA derived from plasma, but also to genomic DNA derived from other sources, such as tissues and other bodily sources.

Example 2: cfDNA copy number analysis of healthy donors and a cancer patient

[00239] The following example illustrates how molecular features added during genomic library construction and post-hybridization processing are used to generate copy number analysis. DNA was extracted from the plasma of sixteen healthy donors and one patient with castration-resistant prostate cancer using the Qiagen Circulating Nucleic Acid Extraction Kit (Qiagen, Hilden, Germany). Double-stranded DNA yield was quantified using a Qubit fluorometer (Thermo Fisher, Waltham, MA) and the corresponding hsDNA quantification kit. Size analysis was performed using gel electrophoresis on 2% agarose gels with PCR markers as size standards (New England Biolabs, Ipswich, MA). Approximately 40-100 ng of cfDNA, depending on the cfDNA yield of the sample, was used for library construction.

[00240] The basic features of library construction are illustrated in FIG. 11A-11C. The cfDNA was first dephosphorylated and then repaired to blunt ends in a two-step process. Short 10 nt anchor sequences consisting of a phosphorylated linker strand and an inert partner strand were then ligated to the cfDNA. The eight oligonucleotides used to create the set of four anchor sequences are shown in Table 1. Table 1: Ligation Anchor Oligonucleotides *[3-d(A, C, G, or T)-Q] indicates a modified base in which the hydroxyl group resides at the 2' position of the ribose ring**/5Phos/ indicates the chemical addition of a 5' phosphate group to the base's 5' position

[00241] The adapter structures were completed by the addition of full-length adapter sequences that ligated to the anchor sequence. Thirty-two sets of adapter sequences, each consisting of 240 members, are shown in FIG. 12 - FIG. 22. These adapters were ligated to cfDNA and extended through the concerted actions of polynucleotide kinase, DNA polymerase, and DNA ligase to generate genomic libraries. As a pre-sequencing quality control step, the resulting genomic libraries were quantified by qPCR for depth of coverage. The genomic libraries were then amplified and hybridized to probe sets targeting specific genes (FIG. 11B). Following hybridization, probe primer extension was used to copy the captured genomic sequences and the information encoded in the attached adapter (FIG. 11C). An example of post-sequencing analysis using standard state-of-the-art analysis software is shown in FIG. 11D. This analysis was performed on a sequencing run that contained 32 samples (28 cancer patient samples and 4 wild-type controls) and displays the overall distribution of the sequencing reads.

[00242] A central feature of the targeted hybrid capture platform described herein is that it provides multiple types of genomic information. An essential function of the capture probes is to provide mutation detection across target regions at a high depth of coverage. This function is governed by the sequence context, density, and placement of the capture probes and is illustrated in FIG. 23 with the TP53 gene (TP53 probe sequences are shown in Table 2 below). Of equal importance, the targeted hybrid capture platform assay generated a readout of equal depth of coverage in regions where no significant mutations were detected. This data is critical to clinicians and patients as it adds statistical significance in cases where no deleterious mutations were detected. Table 2: TP53 Probes

[00243] Ligation of the capture probe with captured genomic sequence (FIG. 11C) also facilitated measurement of genomic depth at each probe location. The number of unique reads associated with each capture probe used in the experiment was measured (FIG. 24). The data shown in FIG. 24 were derived from a sequencing run in which 16 healthy donor cfDNA samples were analyzed. The depth of unique reads found in each sample at a probe location in the TP53 gene was calculated (Raw unique read counts shown in FIG. 24A). Each sample comprised a unique library depth, as reflected in the broad sample-to-sample distribution of unique reads. The overall average of the unique read depth across all 2596 capture probes in the experiment was also calculated (FIG. 24B). Significantly, the normalization of read depth observed at the unique probe location shown in FIG. 24C analysis of the global single-read depth measured for all probes revealed a uniform density of normalized single-reads. These data indicate that the capture performance of a specific probe chosen for analysis was uniform from sample to sample and proportional to the genomic depth of each individual library.

[00244] This same normalization function was applied to the 45 TP53-specific probes shown in FIG. 23 (normalization data shown in FIG. 25). Whereas FIG. 23 shows the aggregate contribution of all probes to the sequencing depth of the TP53 coding regions, FIG. 25 shows the normalized depth recovered by each individual probe. The normalized depth recovered by each individual probe was generally consistent from sample to sample for any given probe, but somewhat variable when one probe was compared to another. Several factors determined the differences in post-normalization capture depths observed between probes, the most significant being the placement of probes relative to each other and the proximity of probes to genomic repeat regions. Not all probes exhibited uniform capture behavior; two probes whose capture performance was not consistent are highlighted by arrows in FIG. 25. However, these data indicate that these probes are rare and easily identified. As such, they can be excluded from downstream copy number analysis.

[00245] The uniform capture performance exhibited by the 45 TP53 targeting probes in FIG. 25 is a general characteristic of the target hybrid capture platform described herein. In FIG. 26, the average capture depth for each probe in a panel of 2596 capture probes was calculated for all 16 normal cfDNA libraries that were profiled in this experiment. The average was then compared individually to three representative samples using scatterplot analysis. Each point represents a different probe, and its position on the graph is a comparison of the average on the y-axis and the individual sample on the y-axis. The tight diagonal distribution of most probes reflected the highly correlated unique read capture performance of most probes (R2 correlation > 0.95 for all three graphs). Importantly, the consistency of probe-by-probe sequencing depth supports the use of the target hybrid capture platform in copy number measurement.

[00246] With respect to copy number, the most straightforward treatment of the probe data is to further normalize the adjusted genomic depth values that occur on autosomal chromosomes to a diploid mean value of “2”. The same is true for probe values that occur in females for X-linked loci. For both X-linked and Y-linked regions in normal males, the mean copy values are appropriately adjusted to “1”. This numerical transformation was applied to a set of chromosomal control probes (239 probes targeting selected loci on all 22 autosomal chromosomes, Table 3), a set of 199 probes targeting the X-linked AR gene, and the 45 TP53-specific probes considered in detail above (FIGs. 27A and 27B). Each dot represents the value of an individual probe. With the exception of infrequent “noisy” probes, the vast majority of individual probe counts in regions considered diploid had values that were approximately “2.” Probes for the AR gene in a healthy male fluctuated with a mean value close to the expected “1.” Table 3: Chromosome Control Probes

Significantly, when the same analysis was applied to cfDNA collected from the blood plasma fraction of a patient with castration-resistant prostate cancer using healthy samples as normalization controls, three prominent features emerged (FIG. 27C). First, all control probes exhibited noisy counting behavior. Second, the counts of all AR probes were significantly elevated from a normal value of “1” to an amplified value of approximately “5.” AR gene amplification is consistently observed in patients with advanced prostate cancer. Third, the counts of the TP53 probe, while more tightly clustered, have a mean value much closer to “1” than the expected value of “2.” This likely reflected inactivation of one or both TP53 alleles by copy number loss in the circulating DNA fraction derived from tumor tissue.

[00248] These data indicated that the methods of the present invention encompass three important aspects of karyotyping. Namely, the methods described herein detect widespread chromosomal aneuploidy, copy increases of specific target genes, and copy losses in the same specific target genes. These results further indicate that the methods and platforms described herein may guide the use of precision therapies, since all three genomic anomalies occur frequently in cancer.

[00249] The generalized chromosomal aneuploidy for samples from patients with castration-resistant prostate cancer (blue dots) relative to a healthy control (brown dots) was measured (FIG. 28). In this analysis, the approximate ploidy for all 239 control probes used in the experiment was ordered according to their chromosomal targets. For some chromosomes (e.g., chromosome 1 and chromosome 22), a similar ploidy value of "2" was observed between the patient and control samples. In other cases, deviation between the two samples was observed. The degree of information about global genomic ploidy provided by these experiments was limited by the number and density of the control probes used. However, these data indicate that a denser probe panel, covering all chromosome segments with uniform density, can be utilized—in conjunction with the additional unique features of the present invention. Such analyses will provide a higher resolution, genome-wide measurement of chromosome copy number.

[00250] These data further highlight the capabilities of the present invention as a guide to precision therapy. For example, tumors that have genomic deficiencies in homologous recombination repair often exhibit highly destabilized chromosome ploidies, and patients with such tumors are good candidates for inhibitors of the PARP enzyme complex (see Popova et al., Genome Biol. 2009;10(11):R128). Unlike most sequencing assays that seek to genotype a tumor, the assays described herein utilize sequencing to detect destabilized chromosome ploidy as a tumor phenotype, even if the causal mutations driving this phenotype remain hidden from targeted analysis.

The ability to detect gene loss in the altered DNA of solid tumors is particularly significant. Mutation and deletion of tumor suppressor genes is a frequent event in cancer genomes; furthermore, individuals with germline loss of tumor suppressor genes are particularly vulnerable to developing cancer later in life. The diagnostic value of a liquid biopsy copy number loss (CNL) assay is directly proportional to its sensitivity. To determine the lower limit of detection for the invention described herein, the immortalized lines described in Example 1 were systematically diluted into the "genome-in-a-bottle" reference cell line, NA12878. One line had a single-copy deletion (monoallelic loss) of ATM, the other a single-copy deletion of BRCA2. The experiment included four pure NA12878 control samples and eight spike samples containing 16% of each monoallelic deletion line (FIG. 29). For reporting purposes, this corresponds to a minor allele frequency of 8% biallelic loss. Average values for all gene-specific probes and two additional, non-deleted control genes are shown in FIG. 29. Copy loss of ATM and BRCA2 was confined to peak samples only. Further computational processing of the data revealed reliable copy loss of biallelic deletions up to 2% minor allele frequencies. This sensitivity indicated that the present invention did not require specialized considerations to routinely include copy loss calls in standard blood-based genotyping assays.

[00252] These data demonstrate the use of probe-specific genomic capture data for copy number analysis, including copy number gain and copy number loss, of targeted genomic loci. Additionally, the invention described herein has been demonstrated to have the sensitive ability to detect single nucleotide variants, insertions, and deletions, ranging from single nucleotides to many thousands of base pairs, and gene fusions resulting from chromosomal rearrangement by aberrant mutational processes. (See PCT Publication WO 2016/028316; and US Patent Publication 2014-0274731). All of these mutational processes can contribute to the transformation of normal tissue into neoplastic cancers, and as precision therapies continue to emerge, accurate diagnosis of these diseased genomic signatures will become an increasingly indispensable feature of precision medicine.

Claims (30)

1. A method for performing a genetic analysis on a target DNA region from a test sample comprising a plurality of genomic DNA fragments, wherein the method comprises: (i) generating a genomic DNA library by contacting the test sample with a set of adapters, wherein each adapter of the set of adapters comprises a sample tag region selected from a set of unique sample tag regions, wherein the set is selected from a plurality of sets, and wherein the selected set is unique to the test sample; wherein the genomic DNA library comprises a plurality of DNA library fragments and at least two unique sample tag regions; wherein each of the DNA library fragments comprises a genomic DNA fragment from the test sample ligated to an adapter; (ii) contacting the genomic DNA library with a plurality of capture probes that specifically bind to the target DNA region, thereby forming complexes between the capture probes and DNA library fragments comprising the target DNA region; and(iii) performing a quantitative genetic analysis of the genomic DNA fragments comprising the target DNA region; wherein the adapter is a DNA polynucleotide comprising: (iv) an amplification region, (v) a sample tag region, (vi) a unique molecule identifier (UMI) multiplier, and (vii) an anchor region; wherein the amplification region comprises a polynucleotide sequence capable of serving as a primer recognition site for PCR amplification; wherein the sample tag region identifies a unique DNA library fragment and identifies the test sample; wherein the UMI multiplier is adjacent to or contained within the sample tag region; wherein the sample tag region and the UMI multiplier create a UMI that represents or identifies the test sample and uniquely identifies the DNA library fragment; and wherein the anchor region comprises a polynucleotide sequence that is capable of binding to the genomic DNA fragment. 2. The method of claim 1, wherein the method identifies a genetic alteration indicative of a disease state selected from a single nucleotide variant (SNV), an insertion less than 40 nucleotides in length, a deletion of a region of DNA less than 40 nucleotides in length, and/or a copy number change. 3. The method of claim 1 or 2, wherein the test sample is a tissue biopsy, optionally wherein the tissue biopsy is taken from a tumor or tissue suspected of being a tumor. 4. The method of any one of claims 1 to 3, wherein the genomic DNA fragments are cell-free DNA (cfDNA) or cellular DNA. 5. Method according to claim 1 or 2, characterized in that the genomic DNA fragments are cfDNA isolated from the test sample; and in which the test sample is a biological sample selected from the group consisting of: amniotic fluid, blood, plasma, serum, semen, lymphatic fluid, cerebrospinal fluid, ocular fluid, urine, saliva, feces, mucus and sweat. 6. Method according to any one of claims 1 to 4, characterized in that the genomic DNA fragments are obtained by the steps comprising (i) isolating cellular DNA from the test sample; (ii) fragmenting the cellular DNA to obtain the genomic DNA fragments. 7. The method of claim 6, wherein step (ii) is performed by contacting the cellular DNA with at least one digestion enzyme or by applying mechanical stress to the cellular DNA, optionally where the mechanical stress is applied by sonicating the cellular DNA. 8. The method of any one of claims 1 to 7, wherein the amplification region is 10 to 50, 20 to 30, or 25 nucleotides in length. 9. The method of any one of claims 1 to 8, wherein the sample tag region is 5 to 50, 5 to 15, or 8 nucleotides in length. 10. The method of any one of claims 1 to 9, wherein the UMI multiplier is 1 to 5 nucleotides in length, or wherein the UMI multiplier is 3 nucleotides in length and comprises one of 64 possible nucleotide sequences. 11. The method of any one of claims 1 to 10, wherein the anchor region is 1 to 50, 5 to 25, or 10 nucleotides in length. 12. The method of any one of claims 1 to 11, wherein step (a) comprises ligating a first adapter to one end of a test sample genomic DNA fragment and ligating a second adapter to the other end of the genomic DNA fragment; optionally wherein the genomic DNA fragment is end-repaired prior to ligating the first and second adapters. 13. The method of any one of claims 1 to 12, wherein the amplification region of each adapter of the adapter set is identical to the amplification region of all other adapters of the adapter set. 14. The method of any one of claims 1 to 13, wherein the set of sample tag regions comprises 2 to 1,000, 50 to 500, 100 to 400, 200 to 300, or 240 single sample tag region sequences. 15. The method of any one of claims 1 to 14, wherein each sample tag region of the set of sample tag regions is 8 nucleotides in length. 16. The method of any one of claims 1 to 15, wherein each single sample tag region sequence is distinct from any other single sample tag region sequence by a Hamming distance of at least two. 17. The method of any one of claims 1 to 16, wherein the anchor region of each adapter of the adapter set comprises one of four sequences, and wherein each sample tag region of a given sequence is paired with only one of the four anchor regions of a given sequence. 18. The method of any one of claims 1 to 17, wherein the amplification region of each adapter of the set of adapters comprises an identical nucleotide sequence; wherein each sample tag region of the set of sample tag regions is 8 nucleotides in length, wherein each unique sample tag region sequence is distinct from any other unique sample tag region sequence by a Hamming distance of at least two, wherein the UMI multiplier of each adapter of the set of adapters is three nucleotides in length, and wherein the UMI multiplier of a given sequence is paired with a sample tag region of a given sequence, wherein the anchor region of each adapter of the set of adapters comprises one of four nucleotide sequences, and wherein each sample tag region of a given sequence is paired with only one of four anchor regions of a given sequence. 19. The method of any one of claims 1 to 18, wherein the step of ligating each of the first and second adapters to the genomic DNA fragment comprises: (i) ligating an oligonucleotide comprising at least a portion of an anchor region to the genomic DNA fragment, wherein the oligonucleotide is part of a DNA duplex comprising a 5' phosphorylated linker strand duplexed with a partner strand, wherein the partner strand is blocked from binding to the genomic DNA fragment by chemical modification at its 3' end, and wherein the linker strand is ligated to the genomic DNA fragment; and(ii) contacting the genomic DNA fragment ligated to the linker strand with a DNA oligonucleotide encoding a full-length adapter sequence, a T4 polynucleotide kinase, a Taq DNA ligase, and a full-length Bst polymerase under conditions suitable for DNA ligation; thereby ligating each of the first and second adapters to the genomic DNA fragment. 20. The method of any one of claims 1 to 19, wherein the target DNA region is analyzed for a change in copy number. 21. Method according to any one of claims 1 to 20, characterized in that step (c) comprises purifying the complexes formed between the capture probes and the DNA library fragments comprising the target DNA region. 22. The method of claim 21, wherein step (c) comprises performing primer extension and/or amplification of DNA library fragments comprising the target DNA region. 23. The method of claim 21 or 22, wherein step (c) comprises sequencing DNA from fragments of the DNA library comprising the target DNA region to generate a plurality of sequencing reads. 24. The method of any one of claims 1 to 23, wherein the genomic analysis comprises determining a copy number change in a DNA region of interest, and wherein step (c) comprises: (i) determining a copy number of the region of interest present in the genomic DNA library derived from the test sample, and (ii) comparing the copy number determined in step (i) with a copy number of the region of interest present in a genomic DNA library derived from a reference sample, wherein the reference sample comprises a known copy number of the target DNA region. 25. The method of claim 24, wherein determining the copy number of the region of interest comprises sequencing DNA from DNA library fragments comprising the target DNA region to generate a plurality of sequencing reads, wherein each sequencing read comprises a unique molecular identification element (UMIE); wherein the UMIE comprises sequencing information of the adapter and at least a portion of the genomic DNA fragment sequence; wherein sequencing reads comprising identical UMIEs are identified as a unique genomic sequence (UGS). 26. The method of claim 24 or 25, further comprising determining a gross genomic depth (RGD) for each of the capture probes contacted with the genomic DNA library. 27. The method of claim 26, wherein determining the RGD comprises determining the average number of UGSs associated with each capture probe sequence within a group of sample replicates; optionally wherein capture probes associated with a highly variable number of UGSs are identified as noisy probes and are removed from further calculations. 28. The method of claim 27, further comprising calculating an RGD for a sample, comprising calculating a numerical average of all RGDs for all capture probes in the sample; optionally wherein RGD values for noisy probes are not included in the calculation of an RGD for a sample. 29. The method of any one of claims 26 to 28, wherein the RGDs for the capture probes are normalized across all samples in an experimental group by converting the RGD for each capture probe to a probe-specific normalized read count comprising: (i) multiplying each capture probe RGD in a sample by a normalization constant, wherein the normalization constant comprises any real number; and (ii) dividing the product of (i) by the RGD calculated for the corresponding sample; or (iii) dividing the product of (i) by an average RGD calculated from a subset of probes; optionally wherein the subset of probes is a set of control probes. 30. The method of claim 29, wherein the probe-specific normalized read counts are converted to a copy number value comprising: (i) multiplying the probe-specific normalized read counts of probes targeting autosomal and/or X-linked regions by 2 in samples derived from females; (ii) multiplying the probe-specific normalized read counts of probes targeting Y-linked and/or X-linked regions by 1 in samples derived from males; (iii) averaging the products of (i) and/or (ii) across all samples in an experiment; and (iv) dividing the product of (i) and/or (ii) by the average of (iii); optionally wherein the approximate copy number values for all probes targeting a particular gene are averaged.