GB2564847A

GB2564847A - Knowledgebase for non-invasive prenatal genetic screening and diagnosis

Info

Publication number: GB2564847A
Application number: GB1711536.1A
Authority: GB
Inventors: Lench Nick; Hurles Matt; McGonigle John; Martin Alan; Drury Suzie
Original assignee: Congenica Ltd
Current assignee: Congenica Ltd
Priority date: 2017-07-18
Filing date: 2017-07-18
Publication date: 2019-01-30
Also published as: GB201711536D0

Abstract

Described is a prenatal screening system. The prenatal screening system includes a wet-laboratory arrangement for processing a blood sample (e.g. maternal blood sample) to determine cell-free DNA (cfDNA) readout data from the blood sample and a data processing arrangement for processing the cell-free DNA readout data with reference to information stored in a database arrangement to generate a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities. The prenatal screening system is operable to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data when generating the risk score. The data processing arrangement can concentrate its resources on testing certain selective portions of a given DNA molecule depending upon secondary indications, such as abnormalities in parental DNA sequence, or abnormalities identified in ultrasound scans. Associated methods and a computer program are also claimed.

Description

The present disclosure relates to prenatal screening systems and methods; for example, the present disclosure relates to prenatal screening systems and methods that utilize a targeted assay approach. Furthermore, the present disclosure is concerned with computer program products comprising a non-transitory computer-readable storage medium having computerreadable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute aforesaid methods of prenatal screening using the prenatal screening systems.

BACKGROUND

Zygote formation and associated subsequent fetal (foetal) development is a complex biological process that does not always occur without defects arising. It is of great societal benefit that such defects are detected reliably, for example as early as possible, during fetal growth.

Antenatal or prenatal screening is provided to pregnant women to prevent or treat potential health problems that may occur during pregnancy. Such problems may affect both a given mother and/or her fetus; moreover, the problems may be determined by factors such as lifestyle, environment or genetics. For example, radioactive contamination during pregnancy can give rise to corresponding birth defects (for example, Chernobyl, Fukushima Dai'ichi) and such birth defects are potentially passed down through generations of human beings. However, of particular importance are fetal abnormalities that are genetic in origin. These genetic abnormalities may be caused by mutations inherited from one or both parents, or may arise de novo] such de novo abnormalities can arise spontaneously and stochastically. The nature of such mutations can range extensively from changes in single nucleotides (namely 'rungs' of DNA molecule) to the presence of additional whole chromosomes within a genome of a fetus. Of particular clinical significance are the chromosomal disorders known as 'aneuploidies' that occur when there is an abnormal number of chromosomes (e.g. Down's syndrome); the human genome normally has 46 chromosomes, but aneuploidy can result in, for example, an occurrence of 45 or 47 chromosomes. Many chromosomal disorders are incompatible with life or result in multiple congenital anomalies for the new born child.

Prenatal screening for fetal chromosomal abnormalities during pregnancy is widely available through public and private healthcare providers. This screening is normally carried out during a first trimester of pregnancy (10 to 14 weeks after conception) and typically involves performing a maternal blood test on a given mother combined with an ultrasound scan of a fetus of the given mother; this is known as the Combined Test. Levels of human chorionic gonadotrophin (hCG) and pregnancy-associated plasma protein (PAPP-A) are measured along with a nuchal translucency (NT) scan; once other medical factors have been taken into account (e.g. maternal age), a risk-score is provided at a conclusion of the aforesaid 'Combined Test'. If a given pregnancy of a given mother is categorised as being 'high-risk', an invasive diagnostic procedure (for example, involving chorionic villus sampling, an amniocentesis, an cordocentesis) is offered to the given mother to confirm or rule out Down's syndrome (associated with the trisomy chromosome 21 - T21), Edwards's syndrome (associated with the trisomy chromosome 18 - T18) and Patau syndrome (associated with the trisomy chromosome 13 - T13). Pregnant women are also offered a second ultrasound scan at 18 to 21 weeks (after conception) to check for structural fetal anomalies, such as cardiac malformations, brain malformations and skeletal abnormalities. This second scan can be used:

(i) to direct antenatal treatments;

(ii) to identify anomalies that require early intervention following delivery; or (iii) to enable follow-on diagnostic testing and pregnancy management. Invasive tests, such as chorionic villus sampling, an amniocentesis and a cordocentesis carry a 1% chance of a miscarriage of a fetus.

Over recent past years, non-invasive techniques (without having an associated risk of miscarriage) have been developed for diagnosing fetal chromosomal anomalies, wherein the non-invasive techniques rely upon a presence of circulating cell-free fetal DNA in a given pregnant mother's blood. The testing of cell-free fetal DNA (cffDNA) has now entered routine clinical practice for non-invasive prenatal testing (NIPT) for aneuploidy (associated with chromosomes T21, T18, T13, for example) and has broad application as a replacement for the aforementioned 'Combined Test'. The anomalies that can be tested by NIPT are increasing as methods are developed for the identification of sub-chromosomal rearrangements such as 22qll.2/DiGeorge syndrome and other microdeletion syndromes. However, the non-invasive tests are presently susceptible to provide a false positive rate for these anomalies that is considered to be too high to offer the noninvasive test on a screening basis; therefore, presently, the non-invasive test is only offered if there is an accompanying clinical indication such as a congenital heart defect. NIPT is classified as 'testing' rather than 'diagnosis' as the cffDNA which is measured is derived from a placenta of a given pregnant mother, rather than from a fetus of the mother, meaning that false positives can occur due to confined placental mosaicism. For this reason, it is recommended that positive NIPT results are confirmed by an invasive test, for example, amniocentesis, as aforementioned.

Non-invasive prenatal diagnosis (NIPD) is generally classified as a diagnostic assay, wherein a subsequent invasive assay is not required to confirm results from the NIPD. The use of NIPD is more limited than NIPT, and is used for fetuses at risk of single gene disorders (for example, inherited and 'de novo’ mutations, as aforementioned) or those with a suspicion of a genetic disorder on a fetal ultrasound scanning that has been performed on a given fetus.

Accurately reconstructing a given child's genetics from circulating cffDNA is a technically exceptionally challenging task. A child's genetics must be recapitulated (reconstructed) from small fragments of circulating DNA that occur as only a small sub-population of the cell-free DNA present in the mother's plasma, which is itself at relatively low concentrations in a maternal blood sample. Coupled with this small sub-population, next generation sequencing library preparation methods for analysing this cellfree DNA material requires PCR amplification. PCR amplification introduces errors into a given sample being thereby amplified, wherein a frequency of the errors can be greater than a total number of foetal reads within the sample, resulting in a true variation in a given fetal genome being obscured or false positives obtained; such errors are a major technical problem that the present disclosure seeks to address. Even for techniques involving PCRfree approaches, the final sequencing step requires copies of the original DNA molecule to be made, which can also introduce errors. Such sources of error are particularly relevant when trying to identify aforementioned 'de novo' variants which have occurred in a given fetus.

In order to account for these significant (namely, 'not insignificant') sources of error, it is important that identified genetic variants are required to pass extremely stringent quality thresholds, that are employed in methods employed when implementing aforementioned NIPT and associated analyses. When investigating variants across large tracks (portions) of the genome, it is potentially required to employ unconventionally high sequencing depths in order to achieve a cell-free DNA, and thus as a byproduct foetal, coverage level that is tractable with such analyses. These very high sequencing depth methods, while being very informative in terms of what can be achieved by NGPS, are generally impractical (namely, too costly and too slow) to implement in a contemporary clinical setting providing NIPT to a general public.

Reducing the technical limitations surrounding NGPS is an area of great importance. The development of methods to handle sources of error, or to empower analysis by targeting investigations towards one or more likely genes of interest, is potentially susceptible to improving an ability to screen accurately for disease-causing mutations using realistic sequence depths when reading collected DNA samples. Thus, a functionality to collect and synthesise information pertinent to analyses, and moreover to learn from earlier analyses that have been performed, is susceptible to improving a utility and an effectiveness of NGPS.

Current approaches attempt to reduce error through the use of one or more thresholds, set at a value relative to the probability of mutational change in the genome (New et al., 2014). Such use of one or more thresholds provides a base filter method requiring a very high coverage of a majority of genetic nucleic acid base sites, whereby the likelihood that each individual base has been called accurately is determined, using a strict threshold for a number of times a base must have been observed before it is accepted as a 'true' call (Yu et al., 2014). Such a method filters out much of the erroneous variation, thereby allowing a much more confident estimation of real from non-real variance to be computed, but requires an incredibly high sequence depth for this method to be tenable for practical use in a clinical environment delivering a service to the public.

In order to filter at the level of the variant (for example, giving rise to a genetic abnormality), a dynamic cut off range has been earlier used in order to attempt to identify one or more 'de novo' variations (Chan et al., 2016). Thus, a method has been developed to distinguish between 'de novo' mutations present in a given fetus (foetus) and sequencing errors. Such distinguishing is achieved by calculating a probability of a same given variant being observed as many times as a current variant purely due to sequencing error and applying a cut off relative to this probability (Chan etat., 2016; Yu et al., 2014); as such, the distinguishing is achieved by taking a plurality of sequencing reads and performing a correlation to remove stochastic noise, and to reinforce systematic variations that are indicative of genetic problems.

Other known methods attempt to improve confidence in DNA nucleic acid base reads being analysed by using a property that, reads derived from circulating cell-free DNA are far more likely to 'stack' at positions in the genome than non-cell free DNA (Chan et al., 2016), sharing both their start and end positions with other read; such stacking arises, for example, on account of apoptotic processes that occur within the fetus during fetal growth. Such a property of 'stacking' is important, because current quality assurance methods in genetic pipelines are designed to remove reads that share the same start and end position as they are assumed to come from PCR. duplication of the same read (Chandrananda et al., 2015).

SUMMARY

The present disclosure seeks to provide an improved prenatal screening system that is capable of storing information relating to a capture area of a next generation prenatal screening assay.

Moreover, the present disclosure seeks to provide an improved method of using a prenatal screening system that is capable of storing information relating to a capture area of a next generation prenatal screening assay.

In a first aspect, embodiments of the present disclosure provide a prenatal screening system, wherein the prenatal screening system includes a wetlaboratory arrangement for processing a blood sample to determine cell-free DNA readout data from the blood sample, and a data processing arrangement for processing the cell-free DNA readout data with reference to information stored in a database arrangement to generate a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities, characterized in that the prenatal screening system is operable to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data when generating the risk score.

The present disclosure is of the advantage in that use of such secondary data describing one of the members of the genetic source material (for modifying data processing performed in the data processing arrangement and for processing selective regions of the cell-free DNA readout data when generating the risk score) provides a harmonized repository for information pertaining to targeted assay approach to next generation prenatal screening.

Embodiments of the disclosure are advantageous in terms of providing a rapid, simple, low cost, patient specific and highly efficient method and system for performing next generation prenatal screening. Moreover, the method and system are helpful in making possible next generation prenatal screening at an earlier time in pregnancy and also reducing diagnosis time.

Embodiments of the present disclosure employ, for example, a 'NGPS knowledgebase' which is operable to function as a repository for information pertaining to one or more targeted assay approaches that are employed to implement NGPS. The 'NGPS knowledgebase' optionally includes a database in which to store information regarding a given patient, one or more regions of the human genome included in an assay being used to execute a test or diagnosis, an indication of sequencing error rate of a given run used to implement such a test or diagnosis, as well as algorithms to allow the 'NGPS knowledgebase' to be updated and associated models to be re-evaluated. By such a method, the 'NGPS knowledgebase' is beneficially operable to inform, and to be informed, by NGPS analyses that are performed in conjunction with access being made to the 'NGPS knowledgebase'.

Optionally, the blood sample is a maternal blood sample, and the cell-free DNA readout data is determined from fragments of DNA present in a plasma fraction of the blood sample. More optionally, the wet-laboratory arrangement is operable to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data.

Optionally, the selective regions are determined by accessing one or more databases of the database arrangement, wherein the prenatal screening system is operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations. More optionally, the secondary data is determined by non-invasive procedures, and the subsequent fetal investigation involves executing one or more invasive sampling of tissue or liquids in respect of the fetus.

Optionally, the one or more database is implemented as a NGPS knowledgebase, wherein the NGPS knowledgebase includes metrics sourced from, and the ability to perform, analyses that are recalibrated in operation to include and incorporate information that is specific to genetic origins of the sample. More optionally, the NGPS knowledgebase includes phenotypic information that is employed in analyses of data performed by the data processing arrangement when generating the risk score.

In a second aspect, embodiments of the present disclosure provide a method of using a prenatal screening system, wherein the prenatal screening system includes a wet-laboratory arrangement for processing a blood sample to determine cell-free DNA readout data from the blood sample, and a data processing arrangement for processing the cell-free DNA readout data with reference to information stored in a database arrangement to generate a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities, characterized in that the method includes operating the prenatal screening system to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data when generating the risk score.

Optionally, the blood sample is a maternal blood sample, and the method includes determining the cell-free DNA readout data from fragments of DNA present in a plasma fraction of the blood sample. More optionally, the method includes operating the wet-laboratory arrangement (20) to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data.

Optionally, the method includes determining the selective regions by accessing one or more databases of the database arrangement, wherein the prenatal screening system is operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations. More optionally, the method includes determining the secondary data by non-invasive procedures, and determining the subsequent fetal investigation by executing one or more invasive sampling of tissue or liquids in respect of the fetus.

Optionally, the method includes implementing the one or more database as a NGPS knowledgebase, wherein the NGPS knowledgebase includes metrics sourced from, and the ability to perform, analyses that are recalibrated in operation to include and incorporate information that relates to the genetic origin of the source. More optionally, the method includes arranging for the NGPS knowledgebase to include phenotypic information that is employed in analyses of data performed by the data processing arrangement when generating the risk score.

In a third aspect, embodiments of the present disclosure provide a computer program product comprising a non-transitory computer-readable storage medium having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute the aforementioned method.

Additional aspects, advantages, features and objects of the present disclosure would be made apparent from the drawings and the detailed description of the illustrative embodiments construed in conjunction with the appended claims that follow.

It will be appreciated that features of the present disclosure are susceptible to being combined in various combinations without departing from the scope of the present disclosure as defined by the appended claims.

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A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

BRIEF DESCRIPTION OF DRAWINGS

The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the present disclosure is not limited to specific methods and apparatus disclosed herein. Moreover, those in the art will understand that the drawings are not to scale. Wherever possible, like elements have been indicated by identical numbers.

Embodiments of the present disclosure will be more fully understood from examples described herein below and the accompanying drawings, which is given by way of illustration only, and thus are not limitative of the present invention, and wherein:

FIG. 1 is a schematic illustration of a next generation prenatal screening system, in accordance with an embodiment of the present disclosure; and

FIG. 2 is an illustration of steps of a method of operating the system of FIG. 1 for providing next generation prenatal screening pursuant to the present disclosure.

In the accompanying diagrams, an underlined number is employed to represent an item over which the underlined number is positioned or an item to which the underlined number is adjacent. A non-underlined number relates to an item identified by a line linking the non-underlined number to the item. When a number is non-underlined and accompanied by an associated arrow, the non-underlined number is used to identify a general item at which the arrow is pointing.

LIST OF ABBREVIATIONS

Abbreviation	Meaning
NGPS	Next Generation Prenatal Screening
RMD	Relative Mutation Dosage
RHDO	Relative Haplotype Dosage
PCR	Polymerase Chain Reaction
NT	Nuchal Translucency
hCG	Human Chorionic Gonadotrophin
PAPP-A	Pregnancy-Associated Plasma Protein
cffDNA	Cell-Free Fetal DNA
NIPT	Non-Invasive Prenatal Testing
CfDNA	Cell-Free DNA

DEFINITIONS

As used herein, the following terms shall have the following meanings:

As used herein, the term 'data processing arrangement' refers to a process and/or system that can be embodied in software that determines the biological significance of acquired data (i.e., ultimate results of an assay). For example, a data processing arrangement can determine an amount of each nucleotide sequence species based upon data that is collected. A data processing arrangement also may control an instrument and/or a data collection system based upon results determined. A data processing and a data collection arrangement often are integrated and provide feedback to operate data acquisition by the instrument, and hence provide assay-based judging methods provided herein.

As used herein, the term 'database arrangement' refers to a nucleic acid databases known in the art including, for example, GenBank®, dbEST®, dbSTS®, EMBL® (European Molecular Biology Laboratory) and DDBJ® (DNA Databank of Japan). BLAST® or similar tools can be used to search the identified sequences against a sequence database.

As used herein, the term 'genetic information' refers to information related to nucleic acids, altered nucleotide sequence, chromosomes, segments of chromosomes, polymorphic regions, translocated regions, the like or combinations of the foregoing. Furthermore the nucleic acids may include, but are not limited to, DNA, cDNA, RNA, mRNA, t RNA and rRNA. Moreover, the genetic information may include information related to mutations, copy number variations, transversions, translocations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, abnormal changes in nucleic acid methylation infection or cancer.

As used herein, the term 'cell-free DNA' refers to DNA that is not within a cell. In one embodiment, cell free DNA includes DNA circulating in blood. In another embodiment, cell free DNA includes DNA existing outside a cell. In yet another embodiment, cell free DNA includes DNA existing outside a cell as well as DNA present in a blood sample after such blood sample has undergone partial or gentle cell lysing.

As used herein, the term 'cell-free fetal DNA' refers to DNA that originated from the fetus and not the mother and is not within a cell. In one embodiment, cell-free fetal DNA includes fetal DNA circulating in maternal blood. In another embodiment, cell-free fetal DNA includes fetal DNA existing outside of a cell, for example a fetal cell. In yet another embodiment, cell-free fetal DNA includes fetal DNA existing outside of a cell as well as fetal DNA present in maternal blood sample after such blood sample has undergone partial or gentle cell lysing. Herein, the term 'cell-free fetal DNA' also refers to small DNA fragments (i.e. about <300 base pairs) circulating in maternal plasma, in other terms it is the excluding DNA contained in fetal cells that may circulate in the maternal plasma.

As used herein, the terms 'maternal sample' or 'maternal blood sample' refers to the sample obtained from a female who is pregnant, the sample may include, but is not limited to, plasma, serum, peripheral blood and urine. Typically, the sample is a maternal plasma sample, although other tissue sources that contain both maternal and fetal DNA are optionally used. Maternal plasma can be obtained from a peripheral whole blood sample from a pregnant woman and the plasma can be obtained by standard methods. A volume of 3 ml to 5 ml of plasma is sufficient to provide suitable DNA material for analysis. The cell free DNA can be extracted from the sample using standard techniques, non-limiting examples of which include a Qiasymphony protocol (Qiagen®) suitable for free fetal DNA isolation or any other automated or manual extraction method suitable for cell free DNA isolation.

As used herein, the term 'biological characteristics' refers to the genetic variations, abnormalities, irregularities or mutations which range extensively from changes in single nucleotides to the presence of additional whole chromosomes or abnormal number of chromosomes. The chromosomal abnormality is a structural abnormality, including, but not limited to, copy number changes including microdeletions and microduplications, insertions, translocations, inversions and small-size mutations including point mutations and mutational signatures.

As used herein, the term 'wet-laboratory arrangement' refers to a facility, clinic and/or a setup of: instruments, equipment and/or devices used for extraction, collection, processing and/or analysis of body fluid samples; instruments, equipment and/or devices used for extraction, collection, processing and/or analysis of genetic material; instruments, equipment and/or devices used for amplification, enrichment and/or processing of genetic material received from the body fluid samples; instruments, equipment and/or devices used for extraction and/or analysis of the genetic information received from the amplified genetic material. Herein the instruments, equipment and/or devices may include, but is not limited to, centrifuge, ELISA®, spectrophotometer, PCR., RT- PCR, High-ThroughputScreening (HTS) system, Microarray system, Ultrasound, genetic analyser, deoxyribonucleic acid (DNA) sequencer and SNP analyser.

The wet-laboratory arrangement is operable to monitor and/or scan a fetus, for example using ultrasonic scanning apparatus providing animated images of the fetus (ultrasound scanner'). Herein, the wet-laboratory arrangement may include equipment, instruments and/or devices for scanning the fetus. Such equipment, instruments and/or devices include ultrasound scanners (as aforementioned), presymptomatic genetic testing and/or combined tests.

DETAILED DESCRIPTION

Practical implementation of the embodiments of the present disclosure are described in further detail below; these embodiments are operable to employ, unless otherwise indicated, conventional methods of diagnostics, molecular biology, cell biology, biochemistry and immunology within the skill of the art. Such techniques are explained fully in the literature, for example contemporary academic research literature pertaining to pregnancy and genetic material processing.

It will be appreciated that certain features of the present invention, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely various features of the invention, which are for brevity, described in the context of a single embodiment, may also be provided separately and/or in any suitable sub-combination.

The following detailed description illustrates embodiments of the present disclosure and ways in which they can be implemented. Although some modes of carrying out the present disclosure has been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

In overview, the aforementioned known approaches, when performing an analysis of a maternal blood sample for testing or diagnostic purposes, do not include a multitude of information relevant in determining risk of a given child having a disease. Specifically, the multitude of information includes, but is not limited to, patient data, data regarding the exact genes being screened, sequence run information.

The NGPS knowledgebase, employed in the present disclosure, may function as a repository for information pertaining to one or more targeted assay approaches that may be employed for implementation of NGPS. The 'NGPS knowledgebase' optionally may include a database to store information regarding a given patient. Specifically, the information may include data relating to one or more regions of the human genome included in an assay, indication of sequencing error rate of a given run, and algorithms to allow the 'NGPS knowledgebase' to be updated and associated models to be reevaluated. Beneficially, the 'NGPS knowledgebase' may inform, and be informed by, NGPS analyses that are performed in conjunction therewith. Specifically, such information exchange may allow improvement and evolution of'NGPS knowledgebase'.

The aforementioned 'NGPS knowledgebase' may be composed of a plurality of parts. A first part Pl, considered to be foremost, concerns construction of a database arrangement, for example including one or more databases, containing information directly relevant to a screening process being performed for the given person, for example for a pregnant mother. Other parts of the 'NGPS knowledgebase' include:

(a) a second part P2 concerning a genomic capture area database;

(b) a third part P3 concerning a patient information database; and (c) a fourth part P4 concerning a sequencing run database.

Furthermore, the parts Pl to P4 may store information; the 'NGPS knowledgebase' may include one or more algorithms that may be built to extract information, to populate the aforementioned one or more databases, and to convert the information into computationally useable weighting or confidence parameters. In addition, the 'NGPS knowledgebase' may include one or more algorithms that are operable to update and remodel existing data set information with every new data set of additional information. Therefore, the 'NGPS knowledgebase' may be operable to employ artificial intelligence (Al) to learn from these databases. For example, an artificial intelligence (Al) engine may include a hierarchical layered configuration of computer-implemented neural networks that are operable to provide pseudo-analogue variable state machines for decision making purposes.

Furthermore, the components of the 'NGPS knowledgebase' may include genomic capture area database; sequencing information database; and patient information database.

The genomic capture area database may contain information relating to a capture area of an NGPS assay employed when implementing embodiments of the present disclosure. Furthermore, the genomic capture area database is concerned with only the regions, of the genome, that are on target for the screening assay. The genomic capture area database may include information indicative of:

(i) a likelihood of erroneous calling in these regions of the genome; and (ii) a likelihood of mutation at the genetic level as well as the known deleterious variants and their associated phenotype (HPO terms).

The sequencing information database may contain information about each sample including library preparation quality scores, a degree to which sequencing errors have been observed within the sequence data for sequencing run executed for each sample, an estimated fetal fraction for each sample, and an estimated percentage coverage of the genome by cellfree DNA for each sample. Such a store of information enables both an investigation of patterns in sequencing results obtained, that may not be related to associated underlying genetics, and the potential to learn from challenges due to sequencing technology limitations or artefacts.

Generally, performing analyses in an absence of valuable collected phenotypic data on patient phenotype leads to a sub-optimal performance of analyses as all regions of the genome may be considered with equal weight, even though this may not be logically sound reasoning; in contradistinction, embodiments of the present disclosure utilize phenotypic data on patient phenotype. In an exemplary embodiment, in an event of information regarding an abnormal scan result, the event may lend evidence to sequencing information that may be present, but not in sufficient depth to be highlighted. Furthermore, by utilizing pieces of information in combination, there may be provided an improvement in sensitivity and detective power when performing prenatal screening, beneficially leading to a reduction in the rate of false negatives. Moreover, the patent information database may help to reduce the rate of false positives, such as including the age of the father in analyses as this is known to have an impact on the mutational rate in the germ line and thus the number of 'de novo' mutations that are expected to be encountered.

Embodiments of the present disclosure may include an algorithm to update and restructure information in the aforementioned one or more databases of the NGPS system, for example in a recursive or iterative manner.

Thus, in embodiments of the present disclosure, computing new error scores and re-evaluating computational assumptions in light of new data supplied to the one or more aforementioned databases is an important essential of the 'NGPS knowledgebase'.

In FIG. 1, there is shown an illustration of a prenatal screening system 100, in accordance with an embodiment of the present disclosure. The prenatal screening system 100 includes a wet-laboratory arrangement 102 and a data processing arrangement 104. The data processing arrangement 104 further includes a database arrangement 106. The wet-laboratory arrangement 102 of the prenatal screening system 100 is operable to exchange instructions and data with the data processing arrangement 104.

Generally, the wet-laboratory arrangement 102 of the prenatal screening system 100 is operable to process a maternal blood sample for determining cell free DNA data from the blood sample. Furthermore, the data processing arrangement 104 of the prenatal screening system 100 is operable to process the cell free DNA readout data with reference to information stored in the database arrangement 106 to generate a risk score. The risk score generated with reference to information stored in the database arrangement 106 is indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities. Moreover, the prenatal screening system 100 is operable to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement 104 for processing selective regions of the cell-free DNA readout data while generating the risk score.

In an embodiment, the wet-laboratory arrangement 102 may include apparatuses for taking maternal blood samples, ultrasound scanning apparatuses for fetal imaging, PCR. sequencing apparatuses, centrifuges, gel electrophoresis DNA sequencing apparatuses, microscopes and so forth. For example, the wet-laboratory arrangement 102 may include apparatus manufactured by Illumina Inc. for performing gene sequencing tasks. The data processing arrangement 104 includes a database arrangement 106 including a plurality of databases, as aforementioned. The data processing arrangement 104 also includes data communication to networks such as the Internet®, for example for accessing various external databases associated with university research departments and hospitals. For example, the external databases may include but not limited to GenBank®, dbEST®, dbSTS®, EMBL® (European Molecular Biology Laboratory) and DDBJ® (DNA Databank of Japan). BLAST® or similar tools can be used to search the identified sequences against a sequence database.

In an embodiment, the blood sample processed in the wet-laboratory arrangement 102 may be a maternal blood sample. In this embodiment, the cell-free DNA readout data may be determined from fragments of DNA present in a plasma fraction of the maternal blood sample. Furthermore, in this embodiment, the wet-laboratory arrangement 104 may be operable to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data. In this exemplary embodiment, the wet-laboratory arrangement 102 may include a PCR. or RT-PCR for amplifying the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA to the data processing arrangement 104 for accessing genetic information in the database arrangement 106.

According to an embodiment, the selective regions may be determined by accessing one or more databases of the database arrangement 106. In this embodiment, the database arrangement 106 may include, but not limited to, GenBank®, dbEST®, dbSTS®, EMBL® (European Molecular Biology Laboratory) and DDBJ® (DNA Databank of Japan). BLAST® or similar tools that can be used to search the identified sequences against a sequence database. Furthermore, in this embodiment, the prenatal screening system 100 may be operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations.

In yet another embodiment, the secondary data may be determined by noninvasive procedures. In such embodiment, the subsequent fetal investigation involves executing one or more invasive sampling of tissue or liquids in respect of the fetus. In this embodiment, non-invasive procedures may include, but not limited to, maternal blood test and/or ultrasonic scan of a fetus.

In another embodiment, the one or more database may be implemented as a NGPS knowledgebase. Furthermore, analyses of the NGPS knowledgebase may be recalibrated to include and incorporate information that is specific to a donor of the blood sample. In this embodiment, the NGPS knowledgebase may include phenotypic information that may be employed in data analyses performed by the data processing arrangement 100 while generating the risk score.

In an exemplary embodiment, the data processing arrangement 106 may concentrate, namely employs its computational resources, on testing certain selective portions of a given DNA molecule depending upon secondary indications, such as abnormalities in parental DNA sequence, abnormalities identified in ultrasound scans, and so forth. Moreover, the data processing arrangement 104 is operable to update and evolve recursively information included in the one or more databases depending upon test results obtained from using the prenatal screening system 100 on maternal blood samples, and from information, for example tests undertaken by third parties, obtained from external databases.

Beneficially, the prenatal screening system 100 may employ one or more the aforementioned parts Pl to P4. Optionally, the prenatal screening system 100 may not be limited to use the parts Pl to P4, and may access other external databases.

In another exemplary embodiment, the prenatal screening system 100 may optionally employ an artificial intelligence (Al) engine implemented using an array of RISC processors (for example proprietary ARM Cortex® processors) with associated data memory for implementing many hundred million pseudo-analogue variable state machines arranged in a hierarchical manner for providing data processing and data analysis within the data processing arrangement 106; thereby, machine deduction processes can be implemented, for example based upon use of black box neural network analogue variable state machines.

In FIG. 2, there is shown a flow chart of method 200 of using a prenatal screening system (such as prenatal screening system 100 of FIG. 1), in accordance with an embodiment of the present disclosure. At a step 202, the flow chart initiates. At the step 202, a blood sample is processed to determine cell-free DNA readout data from the blood sample. At a step 204, the cell-free DNA readout data is processed with reference to information stored in a database arrangement. At a step 206, a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities is generated. At a step 208, data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data is processed using secondary data describing a donor of the blood sample.

In an embodiment, the blood sample processed in the wet-laboratory arrangement may be a maternal blood sample. In this embodiment, the cell-free DNA readout data may be determined from fragments of DNA present in a plasma fraction of the maternal blood sample. Further in this embodiment, the method 200 may include using wet-laboratory arrangement to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data. In this exemplary embodiment, the method 200 may include using a PCR or RT-PCR for amplifying the free fetal DNA fragments for providing a plurality of copies of the free fetal DNA to the data processing arrangement for accessing genetic information in the database arrangement.

In another embodiment, the method 200 may include determining the selective regions by accessing one or more databases of the database arrangement, wherein the prenatal screening system may be operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations. In this embodiment, the method 200 may include determining the secondary data by non-invasive procedures, and determining the subsequent fetal investigation by executing one or more invasive sampling of tissue or liquids in respect of the fetus. In this embodiment, by non-invasive procedures may include but not limited to maternal blood test and/or ultrasonic scan of a fetus.

In yet another embodiment, the method 200 may include implementing the one or more database as a NGPS knowledgebase, wherein the NGPS knowledgebase includes metrics sourced from, and the ability to perform analyses that are recalibrated in operation to include and incorporate information that is specific to a donor of the blood sample. In this embodiment, the method 200 may include arranging the NGPS knowledgebase to include phenotypic information that may be employed in analyses of data performed by the data processing arrangement when generating the risk score.

Beneficially, the method 200 may include using the prenatal screening system to employ one or more the aforementioned parts Pl to P4, for example a plurality of the parts Pl to P4. Optionally, the prenatal screening system may not be limited to use these parts Pl to P4, and can access other external databases.

Optionally, the aforementioned method 200 of using the prenatal screening system is implemented by using a computer program product comprising a non-transitory computer-readable storage medium having computerreadable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware.

Modifications to embodiments described in the foregoing are possible without departing from the scope of the invention as defined by the accompanying claims. Expressions such as including, comprising, incorporating, consisting of, have, is used to describe and claim the present invention are intended to be construed in a non-exclusive manner, namely allowing for items, components or elements not explicitly described also to be present. Reference to the singular is also to be construed to relate to the plural. Numerals included within parentheses in the accompanying claims are 5 intended to assist understanding of the claims and should not be construed in any way to limit subject matter claimed by these claims.

REFERENCES [1] Chan, K.C.A., Jiang, P., Sun, K., Cheng, Y.K.Y., Tong, Y.K., Cheng,

S.H., Wong, A.I.C., Hudecova, I., Leung, T.Y., Chiu, R.W.K., et al. (2016).

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends. Proc. Natl. Acad. Sci. U. S. A. 201615800.

[2] Chandrananda, D., Thorne, N.P., and Bahlo, M. (2015). Highresolution characterization of sequence signatures due to non-random io cleavage of cell-free DNA. BMC Med. Genomics 8, 29.

[3] New, M.I., Tong, Y.K., Yuen, T., Jiang, P., Pina, C., Chan, K.C.A., Khattab, A., Liao, G.J.W., Yau, M., Kim, S.-M., et al. (2014). Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. J. Clin. Endocrinol. Metab. 99, E1022-30.

is [4] Yu, S.C.Y., Chan, K.C.A., Zheng, Y.W.L., Jiang, P., Liao, G.J.W., Sun, H., Akolekar, R., Leung, T.Y., Go, A.T.J.I., van Vugt, J.M.G., et al. (2014). Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proc. Natl. Acad. Sci. U. S. A. Ill, pp 8583-8588.

Claims

1. A prenatal screening system (100), wherein the prenatal screening system (102) includes a wet-laboratory arrangement (102) for processing a blood sample to determine cell-free DNA readout data from the blood sample, and a data processing arrangement (104) for processing the cellfree DNA readout data with reference to information stored in a database arrangement (106) to generate a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities, characterized in that the prenatal screening system (100) is operable to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data when generating the risk score.

2. A prenatal screening system (100) of claim 1, characterized in that the blood sample is a maternal blood sample, and the cell-free DNA readout data is determined from fragments of DNA present in a plasma fraction of the blood sample.

3. A prenatal screening system (100) of claim 2, characterized in that the wet-laboratory arrangement (102) is operable to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data.

4. A prenatal screening system (100) of claim 1, 2 or 3, characterized in that the selective regions are determined by accessing one or more databases of the database arrangement (106), wherein the prenatal screening system (100) is operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations.

5. A prenatal screening system (100) of claim 4, characterized in that the secondary data is determined by non-invasive procedures, and the subsequent fetal investigation involves executing one or more invasive sampling of tissue or liquids in respect of the fetus.

6. A prenatal screening system (100) of any one of the preceding claims, characterized in that the one or more database are implemented as a NGPS knowledgebase, wherein the NGPS knowledgebase includes metrics sourced from, and the ability to perform, analyses that are recalibrated in operation to include and incorporate information that is specific to a donor of the blood sample.

7. A prenatal screening system (100) of claim 6, characterized in that the NGPS knowledgebase includes phenotypic information that is employed in analyses of data performed by the data processing arrangement (104) when generating the risk score.

8. A method of using a prenatal screening system (100), wherein the prenatal screening system (102) includes a wet-laboratory arrangement (102) for processing a blood sample to determine cell-free DNA readout data from the blood sample, and a data processing arrangement (104) for processing the cell-free DNA readout data with reference to information stored in a database arrangement (106) to generate a risk score indicative of whether or not there are genetic abnormalities in the blood sample that are indicative of fetal abnormalities, characterized in that the method includes operating the prenatal screening system (100) to use secondary data describing a donor of the blood sample for modifying data processing performed in the data processing arrangement for processing selective regions of the cell-free DNA readout data when generating the risk score.

9. A method of claim 8, characterized in that the blood sample is a maternal blood sample, and the method includes determining the cell-free DNA readout data from fragments of DNA present in a plasma fraction of the blood sample.

10. A method of claim 9, characterized in that the method includes operating the wet-laboratory arrangement (102) to amplify the fragments of DNA to provide amplified DNA for nucleic acid base sequencing or readout to generate the cell-free DNA readout data.

11. A method of claim 8, 9 or 10, characterized in that the method includes determining the selective regions by accessing one or more databases of the database arrangement (106), wherein the prenatal screening system (100) is operable to update the one or more databases recursively or iteratively depending upon a determined accuracy of the risk score to one or more subsequent fetal investigations.

12. A method of claim 11, characterized in that the method includes determining the secondary data by non-invasive procedures, and determining the subsequent fetal investigation by executing one or more invasive sampling of tissue or liquids in respect of the fetus.

13. A method of any one of claims 8 to 12, characterized in that the method includes implementing the one or more database as a NGPS knowledgebase, wherein the NGPS knowledgebase includes metrics sourced from, and the ability to perform, analyses that are recalibrated in operation to include and incorporate information that is specific to a donor of the blood sample.

14. A method of claim 13, characterized in that the method includes arranging for the NGPS knowledgebase to include phenotypic information that is employed in analyses of data performed by the data processing arrangement (104) when generating the risk score.

5

15. A computer program product comprising a non-transitory computerreadable storage medium having computer-readable instructions stored thereon, the computer-readable instructions being executable by a computerized device comprising processing hardware to execute a method as claimed in any one of claims 8 or 14.