WO2014143994A2

WO2014143994A2 - Methods and compositions for evaluating genetic markers

Info

Publication number: WO2014143994A2
Application number: PCT/US2014/028212
Authority: WO
Inventors: Gregory Porreca; Mark UMBARGER
Original assignee: Good Start Genetics, Inc.
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18
Also published as: EP2971114A4; CA2907177A1; EP2971114A2; WO2014143994A3

Abstract

Aspects of the invention relates to methods and compositions that are useful to reduce bias and increase the reproducibility of multiplex analysis of genetic loci. In some configurations, predetermined preparative steps and/or nucleic acid sequence analysis techniques are usd in multiplex analyses for a plurality of genetic loci in a plurality of samples.

Description

METHODS AND COMPOSITIONS FOR EVALUATING GENETIC MARKERS

RELATED APPLICATIONS

The present application claims the benefit of and priority to U.S. non-provisional application serial number 13/934,093, filed July 2, 2013, which claims the benefit of and priority to U.S. provisional application serial number 61/789,164, filed March 15, 2013, the content of each of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 13, 2014, is named GSGE_002_03WO_Sequence_Listing.txt and is 1,921,826 bytes in size. FIELD OF INVENTION

The invention relates to methods and compositions for determining genotypes in patient samples.

BACKGROUND OF THE INVENTION

Information about the genotype of a subject is becoming more important and relevant for a range of healthcare decisions as the genetic basis for many diseases, disorders, and

physiological characteristics is further elucidated. Medical advice is increasingly personalized, with individual decisions and recommendations being based on specific genetic information. Information about the type and number of alleles at one or more genetic loci impacts disease risk, prognosis, therapeutic options, and genetic counseling amongst other healthcare considerations.

For cost-effective and reliable medical and reproductive counseling on a large scale, it is important to be able correctly and unambiguously identify the allelic status for many different genetic loci in many subjects.

Numerous technologies have been developed for detecting and analyzing nucleic acid sequences from biological samples. These technologies can be used to genotype subjects and determine the allelic status of any locus of interest. However, they are not sufficiently robust and cost-effective to be scaled up for reliable high throughput analysis of many genetic loci in large numbers of patients. The frequency of incorrect or ambiguous calls is too high for current technology to manage large numbers of patient samples without involving expensive and time- consuming steps to resolve uncertainties and provide confidence in the information output.

SUMMARY OF THE INVENTION

Aspects of the invention relate to preparative and analytical methods and compositions for evaluating genotypes, and in particular, for determining the allelic identity (or identities in a diploid organism) of one or more genetic loci in a subject.

Aspects of the invention are based, in part, on the identification of different sources of ambiguity and error in genetic analyses, and, in part, on the identification of one or more approaches to avoid, reduce, recognize, and/or resolve these errors and ambiguities at different stages in a genetic analysis.

According to aspects of the invention, certain types of genetic information can be under- represented or over-represented in a genetic analysis due to a combination of stochastic variation and systematic bias in any of the preparative stages (e.g., capture, amplification, etc.), determining stages (e.g., allele- specific detection, sequencing, etc.), data interpretation stages (e.g., determining whether the assay information is sufficient to identify a subject as homozygous or heterozygous), and/or other stages.

According to aspects of the invention, error or ambiguity may be apparent in a genetic analysis, but not readily resolved without running additional samples or more expensive assays (e.g., array-based assays may report no-calls due to noisy/low signal). According to further aspects of the invention, error or ambiguity may not be accounted for in a genetic analysis and incorrect base calls may be made even when the evidence for them is limited and/or not statistically significant (e.g., next- generation sequencing technologies may report base calls even if the evidence for them is not statistically significant). According to further aspects of the invention error or ambiguity may be problematic for a multi-step genetic analysis because it is apparent but not readily resolved in one or more steps of the analysis and not apparent or accounted for in other steps of the analysis. In some embodiments, sources of error and ambiguity in one or more steps can be addressed by capturing and/or interrogating each target locus of interest with one or more sets of overlapping probes that are designed to overcome any systematic bias or stochastic effects that may impact the complexity and/or fidelity of the genetic information that is generated.

In some embodiments, sources of error and ambiguity in one or more steps can be addressed by capturing and/or interrogating each target locus of interest with at least one set of probes, wherein different probes are labeled with different identifiers that can be used to track the assay reactions and determine whether certain types of genetic information are under-represented or over-represented in the information that is generated.

In some embodiments, errors and ambiguities associated with the analysis of regions containing large numbers of sequence repeats are addressed by systematically analyzing frequencies of certain nucleic acids at particular stages in an assay (e.g., at a to capture, sequencing, or detection stage). It should be appreciated that such techniques may be particularly useful in the context of a standardized protocol that is designed to allow many different loci to be evaluated in parallel without requiring different assay procedures for each locus. In some embodiments, the use of a single detection modality (e.g., sequencing) to assay multiple types of genetic lesions (e.g., point mutations, insertions/deletions, length polymorphisms) is

advantageous in the clinical setting. In some embodiments of the invention, methods are provided that facilitate the use of multiple sample preparation steps in parallel, coupled with multiple analytical processes following sequence detection. Thus, in some embodiments of the invention, an improved workflow is provided that reduces error and uncertainty when

simultaneously assaying different types of genetic lesions across multiple loci in multiple patients.

In some embodiments, aspects of the invention provide methods for overcoming preparative and/or analytical bias by combining two or more techniques, each having a different bias (e.g., a known bias towards under-representation or over-representation of one or more types of sequences), and using the resulting data to determine a genetic call for a subject with greater confidence.

It should be appreciated that in some embodiments, aspects of the invention relate to multiplex diagnostic methods. In some embodiments, multiplex diagnostic methods comprise capturing a plurality of genetic loci in parallel (e.g., one or more genetic loci from Table 1). In some embodiments, the genetic loci possess one or more polymorphisms (e.g., one or more polymorphisms from Table 2) the genotypes of which correspond to disease causing alleles. Accordingly, in some embodiments, the disclosure provides methods for assessing multiple heritable disorders in parallel. In some embodiments, methods are provided for diagnosing multiple heritable disorders in parallel at a pre-implantation, prenatal, perinatal, or postnatal stage. In some embodiments, the disclosure provides methods for analyzing multiple genetic loci (e.g., a plurality of target nucleic acids selected from Table 1) from a patient sample, such as a blood, pre-implantation embryo, chorionic villus or amniotic fluid sample, or other sample (e.g., other biological fluid or tissue sample such as a biopsy sample) as aspects of the invention are not limited in this respect.

Other samples may include tumor tissue or circulating tumor cells. In some embodiments, a patient sample (e.g., a tumor tissue or cell sample) is mosaic for one or more mutations of interest, and thus, may require higher sensitivity than is needed for a germline mutation analysis. In some embodiments, a sample comprises cells from a non-host organism (e.g., bacterial or viral infections in a human subject) or a sample for environmental monitoring (e.g., bacterial, viral, fungal composition of a soil, water, or air sample).

Accordingly, in some embodiments, aspects of the methods disclosed herein relate to genotyping a polymorphism of a target nucleic acid. In some embodiments, the genotyping may comprise determining that one or more alleles of the target nucleic acid are heterozygous or homozygous. In further embodiments, the genotyping may comprise determining the sequence of a polymorphism and comparing that sequence to a control sequence that is indicative of a disease risk. In some embodiments, the polymorphism is selected from a locus in Table 1 or Table 2. However, it should be appreciated that any locus associated with a disease or condition of interest may be used.

In some embodiments, a diagnosis, prognosis, or disease risk assessment is provided to a subject based on a genotype determined for that subject at one or more genetic loci (e.g., based on the analysis of a biological sample obtained from that subject). In some embodiments, an assessment is provided to a couple, based on their respective genotypes at one or more genetic loci, of the risk of their having one or more children having a genotype associated with a disease or condition (e.g., a homozygous or heterozygous genotype associated with a disease or condition). In some embodiments, a subject or a couple may seek genetic or reproductive counseling in connection with a genotype determined according to embodiments of the invention. In some embodiments, genetic information from a tumor or circulating tumor cells is used to determine prognosis and guide selection of appropriate drugs/treatments.

It should be appreciated that any of the methods or compositions described herein may be used in combination with any of the medical evaluations associated with one or more genetic loci as described herein.

In some embodiments, aspects of the invention provide effective methods for overcoming challenges associated with systematic errors (bias) and/or stochastic effects in multiplex genomic capture and/or analysis (including sequencing analysis). In some embodiments, aspects of the invention are useful to avoid, reduce and/or account for variability in one or more sampling and/or analytical steps. For example, in some embodiments, variability in target nucleic acid representation and unequal sampling of heterozygous alleles in pools of captured target nucleic acids can be overcome.

Accordingly, in some embodiments, the disclosure provides methods that reduce variability in the detection of target nucleic acids in multiplex capture methods. In other embodiments, methods improve allelic representation in a capture pool and, thus, improve variant detection outcomes. In certain embodiments, the disclosure provides preparative methods for capturing target nucleic acids (e.g., genetic loci) that involve the use of different sets of multiple probes (e.g., molecular inversion probes MIPs) that capture overlapping regions of a target nucleic acid to achieve a more uniform representation of the target nucleic acids in a capture pool compared with methods of the prior art. In other embodiments, methods reduce bias, or the risk of bias, associated with large scale parallel capture of genetic loci, e.g., for diagnostic purposes. In other embodiments, methods are provided for increasing reproducibility (e.g., by reducing the effect of polymorphisms on target nucleic acid capture) in the detection of a plurality of genetic loci in parallel. In further embodiments, methods are provided for reducing the effect of probe synthesis and/or probe amplification variability on the analysis of a plurality of genetic loci in parallel.

According to some aspects, methods of analyzing a plurality of genetic loci are provided. In some embodiments, the methods comprise contacting each of a plurality of target nucleic acids with a probe set, wherein each probe set comprises a plurality of different probes, each probe having a central region flanked by a 5' region and a 3' region that are complementary to nucleic acids flanking the same strand of one of a plurality of subregions of the target nucleic acid, wherein the subregions of the target nucleic acid are different, and wherein each subregion overlaps with at least one other subregion, isolating a plurality of nucleic acids each having a nucleic acid sequence of a different subregion for each of the plurality of target nucleic acids, and analyzing the isolated nucleic acids.

In other embodiments, methods comprise contacting each of a plurality of target nucleic acids with a probe set, wherein each probe set comprises a plurality of different probes, each probe having a central region flanked by a 5' region and a 3' region that are complementary to nucleic acids flanking the same strand of one of a plurality of subregions of the target nucleic acid, wherein the subregions of the target nucleic acid are different, and wherein a portion of the 5' region and a portion of the 3' region of a probe have, respectively, the sequence of the 5' region and the sequence of the 3' region of a different probe, isolating a plurality of nucleic acids each having a nucleic acid sequence of a different subregion for each of the plurality of target nucleic acids, and analyzing the isolated nucleic acids.

In certain aspects, methods of the invention involve analyzing one or more genes with one or more molecular inversion probes provided in Appendix A. Particularly, those molecular inversion probes are used to capture various targets or subregions thereof on a gene selected from the group consisting of ABCC8, ASPA, BCKDHA, BCKDHB, BLM, CFTR, CLRN1, DLD, FANCC, G6PC, HEXA, IKBKAP, MCOLN1, PCDH15, and SMPD1. In certain applications, a set of two or more molecular inversion probes provided in Appendix A may be used to tile across different, but overlapping sub-regions of one or more genes so that one or more targets on the one or more genes are captured by at least two molecular inversion probes of the set. The number of molecular inversion probes used in a set for tile capture depends on the amount of overlapping coverage one desires for a certain target. In certain embodiments, a portion of one or more genes is captured using one or more molecular inversion probes in

Appendix A. One or more molecular inversion probes of Appendix A may also be chosen to capture particular regions of interest, such as coding or noncoding regions, of a gene. In addition, one or more molecular inversion probes may be chosen to capture regions specific to certain diseases. The diseases may include, for example, Familial hyperinsulinism, Canavan disease, Maple syrup urine disease type la/lb, Bloom syndrome, Cystic fibrosis, Usher syndrome type IDA, Dihydrolipoamide dehydrogenase deficiency, Fanconi anemia group C, Glycogen storage disease type la, Tay-Sachs disease, Familial dysautonomia, Mucolipidosis type IV, Usher syndrome type IF, Niemann-Pick disease type A/B.

Aspects of the disclosure are based, in part, on the discovery of methods for overcoming problems associated with systematic and random errors (bias) in genome capture, amplification and sequencing methods, namely high variability in the capture and amplification of nucleic acids and disproportionate representation of heterozygous alleles in sequencing libraries.

Accordingly, in some embodiments, the disclosure provides methods that reduce errors associated with the variability in the capture and amplification of nucleic acids. In other embodiments, the methods improve allelic representation in sequencing libraries and, thus, improve variant detection outcomes. In certain embodiments, the disclosure provides preparative methods for capturing target nucleic acids (e.g., genetic loci) that involve the use of differentiator tag sequences to uniquely tag individual nucleic acid molecules. In some embodiments, the differentiator tag sequence permit the detection of bias based on the occurrence of combinations of differentiator tag and target sequences observed in a sequencing reaction. In other

embodiments, the methods reduce errors caused by bias, or the risk of bias, associated with the capture, amplification and sequencing of genetic loci, e.g., for diagnostic purposes.

Aspects of the invention relate to providing sequence tags (referred to as differentiator tags) that are useful to determine whether target nucleic acid sequences identified in an assay are from independently isolated target nucleic acids or from multiple copies of the same target nucleic acid molecule (e.g., due to bias in a preparative step, for example, amplification). This information can be used to help analyze a threshold number of independently isolated target nucleic acids from a biological sample in order to obtain sequence information that is reliable and can be used to make a genotype conclusion (e.g., call) with a desired degree of confidence. This information also can be used to detect bias in one or more nucleic acid preparative steps.

In some embodiments, the methods disclosed herein are useful for any application where reduction of bias, e.g., associated with genomic isolation, amplification, sequencing, is important. For example, detection of cancer mutations in a heterogeneous tissue sample, detection of mutations in maternally-circulating fetal DNA, and detection of mutations in cells isolated during a preimplantation genetic diagnostic procedure.

Accordingly, in some aspects, methods of genotyping a subject are provided. In some embodiments, the methods comprise determining the sequence of at least a threshold number of independently isolated nucleic acids, wherein the sequence of each isolated nucleic acid comprises a target nucleic acid sequence and a differentiator tag sequence, wherein the threshold number is a number of unique combinations of target nucleic acid and differentiator tag sequences, wherein the isolated nucleic acids are identified as independently isolated if they comprise unique combinations of target nucleic acid and differentiator tag sequences, and wherein the target nucleic acid sequence is the sequence of a genomic locus of a subject.

In some embodiments, the isolated nucleic acids are products of a circularization selection-based preparative method, e.g., molecular inversion probe capture products. In other embodiments, the isolated nucleic acids are products of an amplification-based preparative methods. In other embodiments, the isolated nucleic acids are products of hybridization-based preparative methods.

Circularization selection-based preparative methods selectively convert regions of interest (target nucleic acids) into a covalently-closed circular molecule which is then isolated typically by removal (usually enzymatic, e.g. with exonuclease) of any non-circularized linear nucleic acid. Oligonucleotide probes (e.g., molecular inversion probes) are designed which have ends that flank the region of interest (target nucleic acid) and, optionally, primer sites, e.g., sequencing primer sites. The probes are allowed to hybridize to the genomic target, and enzymes are used to first (optionally) fill in any gap between probe ends and second ligate the probe closed. Following circularization, any remaining (non-target) linear nucleic acid is typically removed, resulting in isolation (capture) of target nucleic acid. Circularization selection-based preparative methods include molecular inversion probe capture reactions and 'selector' capture reactions. In some embodiments, molecular inversion probe capture of a target nucleic acid is indicative of the presence of a polymorphism in the target nucleic acid.

In amplification-based (e.g., PCR-based or LCR-based, etc.) preparative methods, genomic loci (target nucleic acids) are isolated directly by means of a polymerase chain reaction or ligase chain reaction (or other amplification method) that selectively amplifies each locus using one or more oligonucleotide primers. It is to be understood that primers will be sufficiently complementary to the target sequence to hybridize with and prime amplification of the target nucleic acid. Any one of a variety of art known methods may be utilized for primer design and synthesis. One or more of the primers may be perfectly complementary to the target sequence. Degenerate primers may also be used. Primers may also include additional nucleic acids that are not complementary to target sequences but that facilitate downstream applications, including for example restriction sites and differentiator tag sequences. Amplification-based methods include amplification of a single target nucleic acid and multiplex amplification (amplification of multiple target nucleic acids in parallel).

Hybridization-based preparative methods involve selectively immobilizing target nucleic acids for further manipulation. It is to be understood that one or more oligonucleotides

(immobilization oligonucleotides), which comprise differentiator tag sequences, and which may be from 15 to 170 nucleotides in length, are used which hybridize along the length of a target region of a genetic locus to immobilize it. In some embodiments, immobilization

oligonucleotides, are either immobilized before hybridization is performed (e.g.,

Roche/Nimblegen 'sequence capture'), or are prepared such that they include a moiety (e.g. biotin) which can be used to selectively immobilize the target nucleic acid after hybridization by binding to e.g., streptavidin-coated microbeads (e.g. Agilent 'SureSelect').

It should be appreciated that any of the circularization, amplification, and/or

hybridization based methods described herein may be used in connection with one or more of the tiling/staggering, tagging, size-detection, and/or sensitivity enhancing algorithms described herein.

In some embodiments, the methods disclosed herein comprise determining the sequence of molecular inversion probe capture products, each comprising a molecular inversion probe and a target nucleic acid, wherein the sequence of the molecular inversion probe comprises a differentiator tag sequence and, optionally, a primer sequence, and wherein the target nucleic acid is a captured genomic locus of a subject, and genotyping the subject at the captured genomic locus based on the sequence of at least a threshold number of unique combinations of target nucleic acid and differentiator tag sequences of molecular inversion probe capture products.

In some embodiments, the methods disclosed herein comprise obtaining molecular inversion probe capture products, each comprising a molecular inversion probe and a target nucleic acid, wherein the sequence of the molecular inversion probe comprises a differentiator tag sequence and, optionally, a primer sequence, wherein the target nucleic acid is a captured genomic locus of the subject, amplifying the molecular inversion probe capture products, and genotyping the subject by determining, for each target nucleic acid, the sequence of at least a threshold number of unique combinations of target nucleic acid and differentiator tag sequence of molecular inversion probe capture products. In certain embodiments, obtaining comprises capturing target nucleic acids from a genomic sample of the subject with molecular inversion probes, each comprising a unique differentiator tag sequence. In specific embodiments, capturing is performed under conditions wherein the likelihood of obtaining two or more molecular inversion probe capture products with identical combinations of target and differentiator tag sequences is equal to or less than a predetermined value, optionally wherein the predetermined value is about 0.05.

In one embodiment, the threshold number for a specific target nucleic acid sequence is selected based on a desired statistical confidence for the genotype. In some embodiments, the methods further comprising determining a statistical confidence for the genotype based on the number of unique combinations of target nucleic acid and differentiator tag sequences.

According to some aspects, methods of analyzing a plurality of genetic loci are provided. In some embodiments, the methods comprise obtaining a plurality of molecular inversion probe capture products each comprising a molecular inversion probe and a target nucleic acid, wherein the sequence of the molecular inversion probe comprises a differentiator tag sequence and, optionally, a primer sequence (e.g., a sequence that is complementary to the sequence of a nucleic acid that is used as a primer for sequencing or other extension reaction), amplifying the plurality of molecular inversion probe capture products, determining numbers of occurrence of combinations of target nucleic acid and differentiator tag sequence of molecular inversion probe capture products in the amplified plurality, and if the number of occurrence of a specific combination of target nucleic acid sequence and differentiator tag sequence exceeds a predetermined value, detecting bias in the amplification of the molecular inversion probe comprising the specific combination. In some embodiments, the methods further comprise genotyping target sequences in the plurality, wherein the genotyping comprises correcting for bias, if detected.

In some embodiments, the target nucleic acid is a gene (or portion thereof) selected from Table 1. In some embodiments, the genotyping comprises determining the sequence of a target nucleic acid (e.g., a polymorphic sequence) at one or more (both) alleles of a genome (a diploid genome) of a subject. In certain embodiments, the genotyping comprises determining the sequence of a target nucleic acid at both alleles of a diploid genome of a subject, wherein in the target nucleic acid comprises, or consists of, a sequence of Table 1, Table 2, or other locus of interest.

In some embodiments, aspects of the invention provide methods and compositions for identifying nucleic acid insertions or deletions in genomic regions of interest without

determining the nucleotide sequences of these regions. Aspects of the invention are particularly useful for detecting nucleic acid insertions or deletions in genomic regions containing nucleic acid sequence repeats (e.g., di- or tri-nucleotide repeats). However, the invention is not limited to analyzing nucleic acid repeats and may be used to detect insertions or deletions in any target nucleic acid of interest. Aspects of the invention are particularly useful for analyzing multiple loci in a multiplex assay.

In some embodiments, aspects of the invention relate to determining whether an amount of target nucleic acid that is captured in a genomic capture assay is higher or lower than expected. In some embodiments, a statistically significant deviation from an expected amount (e.g., higher or lower) is indicative of the presence of a nucleic acid insertion or deletion in the genomic region of interest. In some embodiments, the amount is a number of nucleic acid molecules that are captured. In some embodiments, the amount is a number of independently captured nucleic acid molecules in a sample. It should be appreciated that the captured nucleic acids may be literally captured from a sample, or their sequences may be captured without actually capturing the original nucleic acids in the sample. For example, nucleic acid sequences may be captured in an assay that involves a template-based extension of nucleic acids having the region of interest, in the sample.

Aspects of the invention are based on the recognition that the efficiency of certain capture techniques is affected by the length of the nucleic acid being captured. Accordingly, an increase or decrease in the length of a target nucleic acid (e.g., due to an insertion or deletion of a repeated sequence) can alter the capture efficiency of that nucleic acid. In some embodiments, a difference in the capture efficiency (e.g., a statistically significant difference in the capture efficiency) of a target nucleic acid is indicative of an insertion or deletion in the target nucleic acid. It should be appreciated that the capture efficiency for a target nucleic acid may be evaluated based on an amount of captured nucleic acid (e.g., number of captured nucleic acid molecules) relative to a control amount (e.g., based on an amount of control nucleic acid that is captured). However, the invention is not limited in this respect and other techniques for evaluating capture efficiency also may be used.

According to aspects of the invention, evaluating the capture efficiency as opposed to determining the sequence of the entire repeat region reduces errors associated with sequencing through repeat regions. Repeat sequences often give rise to stutters or skips in sequencing reactions that make it very difficult to accurately determine the number of repeats in a target region without running multiple sequencing reactions under different conditions and carefully analyzing the results. Such procedures are cumbersome and not readily scalable in a manner that is consistent with high throughput analyses of target nucleic acids. In some embodiments, repeat regions may be longer than the length of the individual sequence read, making length

determination on the basis of a single read impossible. For example, when using next-generation sequencing the repeat regions may be longer than the length of the individual sequence read, making length determination on the basis of a single read impossible. Accordingly, aspects of the invention are useful to increase the sensitivity of detecting insertions or deletions in target regions, particularly target regions containing repeated sequences.

In some embodiments, aspects of the invention relate to capturing genomic nucleic acid sequences using a molecular inversion probe (e.g., MIP or Padlock probe) technique, and determining whether the amount (e.g., number) of captured sequences is higher or lower than expected. In some embodiments, the amount (e.g., number) of captured sequences is compared to an amount (e.g., number) of sequences captured in a control assay. The control assay may involve analyzing a control sample that contains a nucleic acid from the same genetic locus having a known sequence length (e.g., a known number of nucleic acid repeats). However, a control may involve analyzing a second (e.g., different) genetic locus that is not expected to contain any insertions or deletions. The second genetic locus may be analyzed in the same sample as the locus being interrogated or in a different sample where its length has been previously determined. The second genetic locus may be a locus that is not characterized by the presence of nucleic acid repeats (and thus not expected to contain insertions or deletions of the repeat sequence).

In some embodiments, a target nucleic acid region that is being evaluated may be determined by the identity of the targeting arms of a probe that is designed to capture the target region (or sequence thereof). For example, the targeting arms of a MIP probe may be designed to be complementary (e.g., sufficiently complementary for selective hybridization and/or polymerase extension and/or ligation) to genomic regions flanking a target region suspected of containing an insertion or deletion. It should be appreciated that two targeting arms may be designed to be complementary (e.g., sufficiently complementary for selective hybridization and/or polymerase extension and/or ligation) to the two flanking regions that are immediately adjacent (e.g., immediately 5' and 3', respectively) to a region of a sequence repeat on one strand of a genomic nucleic acid. However, one or both targeting arms may be designed to hybridize several bases (e.g., 1-5, 5-10, 10-25, 25-50, or more) upstream or downstream from the repeat region in such a way that the captured sequence includes a region of unique genomic sequence that on one or both sides of the repeat region. This unique region can then be used to identify the captured target (e.g., based on sequence or hybridization information).

In some embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different loci may be interrogated in parallel in a single assay (e.g., in a multiplex assay). In some

embodiments, the ratio of captured nucleic acids for each locus may be used to determine whether a nucleic acid insertion or deletion is present in one locus relative to the other. For example, the ratio may be compared to a control ratio that is representative of the two loci when neither one has an insertion or deletion relative to control sequences (e.g., sequences that are normal or known to be associated with healthy phenotypes for those loci). However, the amount of captured nucleic acids may be compared to any suitable control as discussed herein.

The locus of a captured sequence may be identified by determining a portion of unique sequence 5' and/or 3' to the repeat region in the target nucleic acid suspected of containing a deletion or insertion. This does not require sequencing the captured repeat region itself.

However, some or all of the repeat region also could be sequenced as aspects of the invention are not limited in this respect.

Aspects of the invention may be combined with one or more sequence-based assays (e.g.,

SNP detection assays), for example in a multiplex format, to determine the genotype of one or more regions of a subject.

In some embodiments, methods of detecting a polymorphism in a nucleic acid in a biological sample are provided. In some embodiments, the methods comprise evaluating the efficiency of capture at one or more loci and determining whether one or both alleles at that locus contain an insertion or deletion relative to a control locus (e.g., a locus indicative of a length of repeat sequence that is associated with a healthy phenotype).

Accordingly, aspects of the invention relate to methods for determining whether a target nucleic acid has an abnormal length by evaluating the capture efficiency of a target nucleic acid in a biological sample from a subject, wherein a capture efficiency that is different from a reference capture efficiency is indicative of the presence, in the biological sample, of a target nucleic acid having an abnormal length. It should be appreciated that the term "abnormal" is a relative term based on a comparison to a "normal" length. In some embodiments, a normal length is a length that is associated with a normal (e.g., healthy or non-carrier phenotype).

Accordingly, an abnormal length is a length that is either shorter or longer than the normal length. In some embodiments, the presence of an abnormal length is indicative of an increased risk that the locus is associated with a disease or a disease carrier phenotype. In some embodiments, the abnormal length is indicative that the subject is either has a disease or condition or is a carrier of a disease or condition (e.g., associated with the locus). However, it should be appreciated that the description of embodiments relating to detecting the presence of an abnormal length also support detecting the presence of a length that is different from an expected or control length.

In some embodiments, aspects of the invention relate to estimating the length of a target nucleic acid (e.g., of a sub-target region within a target nucleic acid). In some embodiments, aspects of the invention relate to methods for estimating the length of a target nucleic acid by contacting the target nucleic acid with a plurality of detection probes under conditions that permit hybridization of the detection probes to the target nucleic acid, wherein each detection probe is a polynucleotide that comprises a first arm that hybridizes to a first region of the target nucleic acid and a second arm that hybridizes to a second region of the target nucleic acid, wherein the first and second regions are on a common strand of the target nucleic acid, and wherein the nucleotide sequence of the target between the 5' end of the first region and the 3' end of the second region is the nucleotide sequence of a sub-target nucleic acid; and capturing a plurality of sub-target nucleic acids that are hybridized with the plurality of detection probes; and measuring the frequency of occurrence of a sub-target nucleic acid in the plurality of sub-target nucleic acids, wherein the frequency of occurrence of the sub-target nucleic acid in the plurality of sub-target nucleic acids is indicative of the length of the sub-target nucleic acid. It should be appreciated that methods for estimating a nucleic acid length may involve comparing a capture efficiency for a target nucleic acid region to two or more reference efficiencies for known nucleic acid lengths in order to determine whether the target nucleic acid region is smaller, intermediate, or larger in size than the known control lengths. In some embodiments, a series of nucleic acids of known different lengths may be used to provide a calibration curve for evaluating the length of a target nucleic acid region of interest.

In some embodiments, the capture efficiency of a target region suspected of having a deletion or insertion is determined by comparing the capture efficiency to a reference indicative of a normal capture efficiency. In some embodiments, the capture efficiency is lower than the reference capture efficiency. In some embodiments, the subject is identified as having an insertion in the target region. In some embodiments, the capture efficiency is higher than the reference capture efficiency. In some embodiments, the subject is identified as having a deletion in the target region. In some embodiments, the subject is identified as being heterozygous for the insertion. In some embodiments, the subject is identified as being heterozygous for the deletion.

In some embodiments of any of the methods described herein (e.g., tiling/staggering, tagging, size-detection, and/or sensitivity enhancement) aspects of the invention relate to capturing a sub-target nucleic acid (or a sequence of a sub-target nucleic acid). In some embodiments, a molecular inversion probe technique is used. In some embodiments, a molecular inversion probe is a single linear strand of nucleic acid that comprises a first targeting arm at its 5' end and a second targeting arm at its 3' end, wherein the first targeting arm is capable of specifically hybridizing to a first region flanking one end of the sub-target nucleic acid, and wherein the second targeting arm is capable of specifically hybridizing to a second region flanking the other end of the sub-target nucleic acid on the same strand of the target nucleic acid. In some embodiments, the first and second targeting arms are between about 10 and about 100 nucleotides long. In some embodiments, the first and second targeting arms are about 10-20, 20- 30, 30-40, or 40-50 nucleotides long. In some embodiments, the first and second targeting arms are about 20 nucleotides long. In some embodiments, the first and second targeting arms have the same length. In some embodiments, the first and second targeting arms have different lengths. In some embodiments, each pair of first and second targeting arms in a set of probes has the same length. Accordingly, if one of the targeting arms is longer, the other one is

correspondingly shorter. This allows for a quality control step in some embodiments to confirm that all captured probe/target sequence products have the same length after a multiplexed plurality of capture reactions. In some embodiments, a set of probes may be designed to have the same length if the intervening region is varied to accommodate any differences in the length of either one or both of the first and second targeting arms.

In some embodiments, the hybridization Tms of the first and second targeting arms are similar. In some embodiments, the hybridization Tms of the first and second targeting arms are within 2-5° C. of each other. In some embodiments, the hybridization Tms of the first and second targeting arms are identical. In some embodiments, the hybridization Tms of the first and second targeting arms are close to empirically-determined optima but not necessarily identical.

In some embodiments, the first and second targeting arms of a molecular inversion probe have different Tms. For example, the Tm of the first targeting arm (at the 5' end of the molecular inversion probe) may be higher than the Tm of the second targeting arm (at the 3' end of the molecular inversion probe). According to aspects of the invention, and without wishing to be bound by theory, a relatively high Tm for the first targeting arm may help avoid or prevent the first targeting arm from being displaced after hybridization by the extension product of the 3' end of the second targeting arm. It should be appreciated that a reference to the Tm of a targeting arm as used herein relates to the Tm of hybridization of the targeting arm to a nucleic acid having the complementary sequence (e.g., the region of the target nucleic acid that has a sequence that is complementary to the sequence of the targeting arm). It also should be appreciated that the Tms of the targeting arms described herein may be calculated using any appropriate method. For example, in some embodiments an experimental method (e.g., a gel shift assay, a hybridization assay, a melting curve analysis, for example in a PCR machine with a SYBR dye by stepping through a temperature ramp while monitoring signal level from an intercalating dye, for example, bound to a double-stranded DNA, etc.) may be used to determine one or more Tms empirically. In some embodiments, an optimal Tm may be determined by evaluating the number of products formed (e.g., for each of a plurality of MIP probes), and determining the optimal Tm as the center point in a histogram of Tm for all targeting arms. In some embodiments, a predictive algorithm may be used to determine a Tm theoretically. In some embodiments, a relatively simple predictive algorithm may be used based on the number of G/C and A/T base pairs when the sequence is hybridized to its target and/or the length of the hybridized product (e.g., for example, 64.9+41*([G+C]-16.4)/(A+T+G+C), see for example, Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Nucleic Acids Res 6:3543-3557). In some embodiments, a more complex algorithm may be used to account for the effects of base stacking entropy and enthalpy, ion concentration, and primer concentration (see, for example, SantaLucia J (1998), Proc Natl Acad Sci USA, 95: 1460-5). In some embodiments an algorithm may use modified parameters (e.g., nearest-neighbor parameters for basepair entropy/enthalpy values). It should be appreciated that any suitable algorithm may be used as aspects of the invention are not limited in this respect. However, it also should be appreciated that different methodologies may results in different calculated or predicted Tms for the same sequences. Accordingly, in some embodiments, the same empirical and/or theoretical method is used to determine the Tms of different sequences for a set of probes to avoid a negative impact of any systematic difference in the Tm determination or prediction when designing a set of probes with predetermined similarities or differences for different Tms.

In some embodiments, the Tm of the first targeting arm may be about 1° C, about 2° C, about 3° C, about 4° C, about 5° C, or more than about 5° C. higher than the Tm of the second targeting arm. In some embodiments, each probe in a plurality of probes (e.g., each probe in a set of 5-10, each probe in a set of at least 10, each probe in a set of 10-50, each probe in a set of 50- 100, each probe in a set of 100-500, each probe in a set of 500-1,000, each probe in a set of 1,000-1,500, each probe in a set of 1,500-2,000, each probe in a set of 2,000-3,000, 3,000-5,000, 5,000-10,000 or each probe in a set of at least 5,000 different probes) has a unique first targeting arm (e.g., they all have different sequences) and a unique second targeting arm (e.g., they all have different sequences). In some embodiments, for at least 10% of the probes (e.g., at least 25%, 25%-50%, 50%-75%, 75%-90%, 90%-95% or over 95%, or all of the probes) the first targeting arm has a Tm for its complementary sequence that is higher (e.g., about 1° C, about 2° C, about 3° C, about 4° C, about 5° C, or more than about 5° C. higher) than the Tm of the second targeting arm for its complementary sequence. In some embodiments, each of the first targeting arms have similar or identical Tms for their respective complementary sequences and each of the second targeting arms have similar or identical Tms for their respective

complementary sequences (and the first targeting arms have higher Tms than the second targeting arms). For example, in some embodiments, the Tm of the first arm(s) may be about 58° C. and the Tm of the second arm(s) may be about 56° C. In some embodiments, the Tm of the first arm(s) may be about 68° C, and the Tm of the second arm(s) may be about 65° C. It should be appreciated that in some embodiments the similarity (e.g., within a range of 1° C, 2° C, 3° C, 4° C, 5° C.) or identity of the Tms for the different targeting arms should be based either on empirical data for each arm or based on the same predictive algorithm for each arm (e.g., Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Nucleic Acids Res 6:3543-3557, SantaLucia J (1998), Proc Natl Acad Sci USA, 95: 1460-5, or other algorithm).

In some embodiments, the Tm of the first targeting arm of a molecular inversion probe (at the 5' end of the molecular inversion probe) is selected to be sufficiently stable to prevent displacement of the first targeting arm from its complementary sequence on a target nucleic acid. In some embodiments, the Tm of the first targeting arm is 50-55° C, at least 55° C, 55-60° C, at least 60° C, 60-65° C, at least 65° C, at least 70° C, at least 75° C, or at least 80° C. As discussed above, it should be appreciated that the for a particular targeting arm may be determined empirically or theoretically. Different theoretical models may be used to determine a Tm and it should be appreciated that the predicted Tm for a particular sequence may be different depending on the algorithm used for the prediction. In some embodiments, each probe in a plurality of probes (e.g., each probe in a set of 5-10, each probe in a set of at least 10, each probe in a set of 10-50, each probe in a set of 50-100, each probe in a set of 100-500, or each probe in a set of at least 500 different probes) has a different first targeting arm (e.g., different sequences) but each different first targeting arm has a similar or identical Tm for its complementary sequence on a target nucleic acid. It should be appreciated that in some embodiments the similarity (e.g., within a range of 1 C, 2 C, 3 C, 4 C, 5 C) or identity of the Tms for the different targeting arms should be based either on empirical data for each arm or based on the same predictive algorithm for each arm (e.g., Wallace, R. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., and Itakura, K. (1979) Nucleic Acids Res 6:3543-3557, SantaLucia J (1998), Proc Natl Acad Sci USA, 95: 1460-5, or other algorithm).

In some embodiments, the sub-target nucleic acid contains a nucleic acid repeat. In some embodiments, the nucleic acid repeat is a dinucleotide or trinucleotide repeat. In some embodiments, the sub-target nucleic acid contains 10-100 copies of the nucleic acid repeat in the absence of an abnormal increase or decrease in nucleic acid repeats. In some embodiments, the sub-target nucleic acid is a region of the Fragile-X locus that contains a nucleic acid repeat. In some embodiments, one or both targeting arms hybridize to a region on the target nucleic acid that is immediately adjacent to a region of nucleic acid repeats. In some embodiments, one or both targeting arms hybridize to a region on the target nucleic acid that is separated from a region of nucleic acid repeats by a region that does not contain any nucleic acid repeats. In some embodiments, the molecular inversion probe further comprises a primer-binding region that can be used to sequence the captured sub-target nucleic acid and optionally the first and/or second targeting arm.

In some embodiments, aspects of the invention relate to evaluating the length of a plurality of different target nucleic acids in a biological sample. In some embodiments, the plurality of target nucleic acids are analyzed using a plurality of different molecular inversion probes. In some embodiments, each different molecular inversion probe comprises a different pair of first and second targeting arms at each of the 3' and 5' ends. In some embodiments, each different molecular inversion probe comprises the same primer-binding sequence.

In some embodiments, aspects of the invention relate to analyzing nucleic acid from a biological sample obtained from a subject. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a tissue sample, specific cell population, tumor sample, circulating tumor cells, or environmental sample. In some embodiments, the biological sample is a single cell. In some embodiments, nucleic acids are analyzed in biological samples obtained from a plurality of different subjects. In some embodiments, nucleic acids from a biological sample are analyzed in multiplex reactions. It should be appreciated that a biological sample contains a plurality of copies of a genome derived from a plurality of cells in the sample. Accordingly, a sample may contain a plurality of independent copies of a target nucleic acid region of interest, the capture efficiency of which can be used to evaluate its size as described herein.

In some embodiments, aspects of the invention relate to evaluating a nucleic acid capture efficiency by determining an amount of target nucleic acid that is captured (e.g., an amount of sub-target nucleic acid sequences that are captured). In some embodiments, the amount of target nucleic acid that is captured is determined by determining a number of independently captured target nucleic acid molecules (e.g., the amount of independently captured molecules that have the sequence of the sub-target region). In some embodiments, the amount of target nucleic acid that is captured is compared to a reference amount of captured nucleic acid. In some embodiments, the reference amount is determined by determining a number of independently captured molecules of a reference nucleic acid. In some embodiments, the reference nucleic acid is a nucleic acid of a different locus in the biological sample that is not suspected of containing a deletion or insertion. In some embodiments, the reference nucleic acid is a nucleic acid of known size and amount that is added to the capture reaction. As described herein, a number of independently captured nucleic acid sequences can be determined by contacting a nucleic acid sample with a preparation of a probe (e.g., a MIP probe as described herein). It should be appreciated that the preparation may comprise a plurality of copies of the same probe and accordingly a plurality of independent copies of the target region may be captured by different probe molecules. The number of probe molecules that actually capture a sequence can be evaluated by determining an amount or number of captured molecules using any suitable technique. This number is a reflection of both the number of target molecules in the sample and the efficiency of capture of those target molecules, which in turn is related to the size of the target molecules as described herein. Accordingly, the capture efficiency can be evaluated by controlling for the abundance of the target nucleic acid, for example by comparing the number or amount of captured target molecules to an appropriate control (e.g., a known size and amount of control nucleic acid, or a different locus that should be present in the same amount in the biological sample and is not expected to contain any insertions or deletions). It should be appreciated that other factors may affect the capture efficiency of a particular target nucleic acid region (e.g., the sequence of the region, the GC content, the presence of secondary structures, etc.). However, these factors also can be accounted for by using appropriate controls (e.g., known sequences having similar properties, the same sequences, other genomic sequences expected to be present in the biological sample at the same frequency, etc., or any combination thereof).

In some embodiments, aspects of the invention relate to identifying a subject as having an insertion or deletion in one or more alleles of a genetic locus if the capture efficiency for that genetic locus is statistically significantly different than a reference capture efficiency.

It should be appreciated that hybridization conditions used for any of the capture techniques described herein (e.g., MIP capture techniques) can be based on known hybridization buffers and conditions.

In some embodiments, the methods disclosed herein are useful for any application where the detection of deletions or insertions is important. In some embodiments, aspects of the invention relate to basing a nucleic acid sequence analysis on results from two or more different nucleic acid preparatory techniques that have different systematic biases in the types of nucleic acids that they sample. According to the invention, different techniques have different sequence biases that are systematic and not simply due to stochastic effects during nucleic acid capture or amplification. Accordingly, the degree of oversampling required to overcome variations in nucleic acid preparation needs to be sufficient to overcome the biases (e.g., an oversampling of 2-5 fold, 5-10 fold, 5-15 fold, 15-20 fold, 20-30 fold, 30-50 fold, or intermediate to higher fold).

According to some embodiments, different techniques have different characteristic or systematic biases. For example, one technique may bias a sample analysis towards one particular allele at a genetic locus of interest, whereas a different technique would bias the sample analysis towards a different allele at the same locus. Accordingly, the same sample may be identified as being different depending on the type of technique that is used to prepare nucleic acid for sequence analysis. This effectively represents a sensitivity limitation, because each technique has different relative sensitivities for polymorphic sequences of interest.

According to aspects of the invention, the sensitivity of a nucleic acid analysis can be increased by combining the sequences from different nucleic acid preparative steps and using the combined sequence information for a diagnostic assay (e.g., for a making a call as to whether a subject is homozygous or heterozygous at a genetic locus of interest).

In some embodiments, the invention provides a method of increasing the sensitivity of a nucleic acid detection assay by obtaining a first preparation of a target to nucleic acid using a first preparative method on a biological sample, obtaining a second preparation of a target nucleic acid using a second preparative method on the biological sample, assaying the sequences obtained in both first and second nucleic acid preparations, and using the sequence information from both first and second nucleic acid preparations to determine the genotype of the target nucleic acid in the biological sample, wherein the first and second preparative methods have different systematic sequence biases. In some embodiments, the first and second nucleic acid preparations are combined prior to performing a sequence assay. In some embodiments, separate sequence assays are performed on the first and second nucleic acid preparations and the sequence information from both assays are combined to determine the genotype of the target nucleic acid in the biological sample. In some embodiments, the first preparative method is an amplification- based, a hybridization-based, or a circular probe-based preparative method. In some embodiments, the second method is an amplification-based, a hybridization-based, or a circular probe-based preparative method. In some embodiments, the first and second methods are of different types (e.g., only one of them is an amplification-based, a hybridization-based, or a circular probe-based preparative method, and the other one is one or the other two types of method). Accordingly, in some embodiments the second preparative method is an amplification- based, a hybridization-based, or a circular probe-based preparative method, provided that the second method is different from the first method. However, in some embodiments, both methods may be of the same type, provided they are different methods (e.g., both are amplification based or hybridization-based, but are different types of amplification or hybridization methods, e.g., with different relative biases).

In amplification-based (e.g., PCR-based or LCR-based, etc.) preparative methods, genomic loci (target nucleic acids) are isolated directly by means of a polymerase chain reaction or ligase chain reaction (or other amplification method) that selectively amplifies each locus using a pair of oligonucleotide primers. It is to be understood that primers will be sufficiently complementary to the target sequence to hybridize with and prime amplification of the target nucleic acid. Any one of a variety of art known methods may be utilized for primer design and synthesis. One or both of the primers may be perfectly complementary to the target sequence. Degenerate primers may also be used. Primers may also include additional nucleic acids that are not complementary to target sequences but that facilitate downstream applications, including for example restriction sites and identifier sequences (e.g., source sequences). PCR based methods may include amplification of a single target nucleic acid and multiplex amplification

(amplification of multiple target nucleic acids in parallel).

Hybridization-based preparative may methods involve selectively immobilizing target nucleic acids for further manipulation. It is to be understood that one or more oligonucleotides (immobilization oligonucleotides), which in some embodiments may be from 10 to 200 nucleotides in length, are used which hybridize along the length of a target region of a genetic locus to immobilize it. In some embodiments, immobilization oligonucleotides are either immobilized before hybridization is performed (e.g., Roche/Nimblegen 'sequence capture'), or are prepared such that they include a moiety (e.g., biotin) which can be used to selectively immobilize the target nucleic acid after hybridization by binding to e.g., streptavidin-coated microbeads (e.g., Agilent 'SureSelect').

Circularization selection-based preparative methods selectively convert each region of interest into a covalently-closed circular molecule which is then isolated by removal (usually enzymatic, e.g., with exonuclease) of any non-circularized linear nucleic acid. Oligonucleotide probes are designed which have ends that flank the region of interest. The probes are allowed to hybridize to the genomic target, and enzymes are used to first (optionally) fill in any gap between probe ends and second ligate the probe closed. In some embodiments, following circularization, any remaining (non-target) linear nucleic acid can be removed, resulting in isolation (capture) of target nucleic acid. Circularization selection-based preparative methods include molecular inversion probe capture reactions and 'selector' capture reactions. However, other techniques may be used as aspects of the invention are not limited in this respect. In some embodiments, molecular inversion probe capture of a target nucleic acid is indicative of the presence of a polymorphism in the target nucleic acid.

A variety of methods may be used to evaluate and compare bias profiles of each preparative technique. Next- generation sequencing may be used to quantitatively measure the abundance of each isolated target nucleic acid obtained from a certain preparative method. This abundance may be compared to a control abundance value (e.g., a known starting abundance of the target nucleic acid) and/or with an abundance determined through the use of an alternative preparative method. For example, a set of target nucleic acids may be isolated by one or more of the three preparative methods; the target nucleic acid may be observed x times using the amplification technique, y times using the hybridization enrichment technique, and z times using the circularization selection technique. A pairwise correlation coefficient may be computed between each abundance value (e.g., x and y, x and z, and y and z) to assess bias in nucleic acid isolation between pairs of preparative methods. Since the mechanisms of isolation are different in each approach, the abundances will usually be different and largely uncorrelated with each other.

In some embodiments, the invention provides a method of obtaining a nucleic acid preparation that is representative of a target nucleic acid in a biological sample by obtaining a first preparation of a target nucleic acid using a first preparative method on a biological sample, obtaining a second preparation of a target nucleic acid using a second preparative method on the biological sample, and combining the first and second nucleic acid preparations to obtain a combined preparation that is representative of the target nucleic acid in the biological sample. In some embodiments of any of the methods described herein, a third preparation of the target nucleic acid is obtained using a third preparative method that is different from the first and second preparative methods, wherein the first, second, and third preparative methods all have different systematic sequence biases. In some embodiments of any of the methods described herein, the different preparative methods are used for a plurality of different loci in the biological sample to increase the sensitivity of a multiplex nucleic acid analysis. In some embodiments, the target nucleic acid has a sequence of a gene selected from Table 1.

However, it should be appreciated that a genotyping method of the invention may include several steps, each of which independently may involve one or more different preparative techniques described herein. In some embodiments, a nucleic acid preparation may be obtained using one or more (e.g., 2, 3, 4, 5, or more) different techniques described herein (e.g., amplification, hybridization capture, circular probe capture, etc., or any combination thereof) and the nucleic acid preparation may be analyzed using one or more different techniques (e.g., amplification, hybridization capture, circular probe capture, etc., or any combination thereof) that are selected independently of the techniques used for the initial preparation.

In some embodiments, aspects of the invention also provide compositions, kits, devices, and analytical methods for increasing the sensitivity of nucleic acid assays. Aspects of the invention are particularly useful for increasing the confidence level of genotyping analyses. However, aspects of the invention may be used in the context of any suitable nucleic acid analysis, for example, but not limited to, a nucleic acid analysis that is designed to determine whether more than one sequence variant is present in a sample.

In some embodiments, aspects of the invention relate to a plurality of nucleic acid probes (e.g., 10-50, 50-100, 100-250, 250-500, 500-1,000, 1,000-2,000, 2,000-5,000, 5,000-7,500, 7,500-10,000, or lower, higher, or intermediate number of different probes). In some

embodiments, each probe or each of a subset of probes (e.g., 10-25%, 25-50%, 50-75%, 75-90%, or 90-99%) has a different first targeting arm. In some embodiments, each probe or each probe of a subset of probes (e.g., 10-25%, 25-50%, 50-75%, 75-90%, or 90-99%) has a different second targeting arm. In some embodiments, the first and second targeting arms are separated by the same intervening sequence. In some embodiments, the first and second targeting arms are complementary to target nucleic acid sequences that are separated by the same or a similar length (e.g., number of nucleic acids, for example, 0-25, 25-50, 50-100, 100-250, 250-500, 500-1,000, 1,000-2,500 or longer or intermediate number of nucleotides) on their respective target nucleic acids (e.g., genomic loci). In some embodiments, each probe or a subset of probes (e.g., 10-25%, 25-50%, 50-75%, 75-90%, or 90-99%) includes a first primer binding sequence. In some embodiments, the primer binding sequence is the same (e.g., it can be used to prime sequencing or other extension reaction). In some embodiments, each probe or a subset of probes (e.g., 10- 25%, 25-50%, 50-75%, 75-90%, or 90-99%) includes a unique identifier sequence tag (e.g., that is predetermined and can be used to distinguish each probe).

In some embodiments, the methods disclosed herein are useful for any application where sensitivity is important. For example, detection of cancer mutations in a heterogenous tissue sample, detection of mutations in maternally-circulating fetal DNA, and detection of mutations in cells isolated during a preimplantation genetic diagnostic procedure.

According to some aspects of the invention, methods of detecting a polymorphism in a nucleic acid in a biological sample are provided. In some embodiments, the methods comprise obtaining a nucleic acid preparation using a preparative method (e.g., any of the preparative methods disclosed herein) on a biological sample, and performing a molecular inversion probe capture reaction on the nucleic acid preparation, wherein a molecular inversion probe capture (e.g., using a mutation-detection MIP) of a target nucleic acid of the nucleic acid preparation is indicative of the presence of a mutation (polymorphism) in the target nucleic acid, optionally wherein the polymorphism is selected from Table 2.

According to some aspects of the invention, methods of genotyping a nucleic acid in a biological sample are provided. In some embodiments, the methods comprise obtaining a nucleic acid preparation using a preparative method on a biological sample, sequencing a target nucleic acid of the nucleic acid preparation, and performing a molecular inversion probe capture reaction on the biological sample, wherein a molecular inversion probe capture of the target nucleic acid in the biological sample is indicative of the presence of a polymorphism in the target nucleic acid, genotyping the target nucleic acid based on the results of the sequencing and the capture reaction.

In some embodiments of the methods disclosed herein, the target nucleic acid has a sequence of a gene selected from Table 1. It should be appreciated that any one or more embodiments described herein may be used for evaluating multiple genetic markers in parallel. Accordingly, in some embodiments, aspects of the invention relate to determining the presence of one or more markers (e.g., one or more alleles) at multiple different genetic loci in parallel. Accordingly, the risk or presence of multiple heritable disorders may be evaluated in parallel. In some embodiments, the risk of having offspring with one or more heritable disorders may be evaluated. In some embodiments, an evaluation may be performed on a biological sample of a parent or a child (e.g., at a pre- implantation, prenatal, perinatal, or postnatal stage). In some embodiments, the disclosure provides methods for analyzing multiple genetic loci (e.g., a plurality of target nucleic acids selected from Table 1 or 2) from a patient sample, such as a blood, pre-implantation embryo, chorionic villus or amniotic fluid sample. A patient or subject may be a human. However, aspects of the invention are not limited to humans and may be applied to other species (e.g., mammals, birds, reptiles, other vertebrates or invertebrates) as aspects of the invention are not limited in this respect. A subject or patient may be male or female. In some embodiments, in connection with reproductive genetic counseling, samples from a male and female member of a couple may be analyzed. In some embodiments, for example, in connection with an animal breeding program, samples from a plurality of male and female subjects may be analyzed to determine compatible or optimal breeding partners or strategies for particular traits or to avoid one or more diseases or conditions. Accordingly, reproductive risks may be determined and/or reproductive recommendations may be provided based on information derived from one or more embodiments of the invention.

However, it should be appreciated that aspects of the invention may be used in connection with any medical evaluation where the presence of one or more alleles at a genetic locus of interest is relevant to a medical determination (e.g., risk or detection of disease, disease prognosis, therapy selection, therapy monitoring, etc.). Further aspects of the invention may be used in connection with detection, in tumor tissue or circulating tumor cells, of mutations in cellular pathways that cause cancer or predict efficacy of treatment regimens, or with detection and identification of pathogenic organisms in the environment or a sample obtained from a subject, e.g., a human subject.

These and other aspects of the invention are described in more detail in the following description and non-limiting examples and drawings. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a non-limiting embodiment of a tiled probe layout;

FIG. 2 illustrates a non-limiting embodiment of a staggered probe layout;

FIG. 3 illustrates a non-limiting embodiment of an alternating staggered probe layout; FIG. 4, panels a), b), and c) depict various non-limiting methods for combining differentiator tag sequence and target sequences (NNNN depicts a differentiator tag sequence);

FIG. 5 depicts a non-limiting method for genotyping based on target and differentiator tag sequences;

FIG. 6 depicts non-limiting results of a simulation of a MIP capture reaction;

FIG. 7 depicts a non-limiting graph of sequencing coverage;

FIG. 8 illustrates that shorter sequences are captured with higher efficiency that longer sequences using MIPs;

FIG. 9 illustrates a non-limiting scheme of padlock (MIP) capture of a region that includes both repetitive regions (thick wavy line) and the adjacent unique sequence (thick strait line);

FIG. 10 illustrates a non-limiting hypothetical relationship between target gap size and the relative number of reads of the repetitive region;

FIG. 11 A depicts MIP capture of FMR1 repeat regions from a diploid genome;

FIG. 1 IB depicts preparative methods for biallelic resolution of FMR1 repeat region lengths in a diploid genome using MIP capture probes and unique differentiator tags;

FIG. 11C depicts an analysis of FMR1 repeat region lengths in a diploid genome;

FIG. 12 is a schematic of an embodiment of an algorithm of the invention;

FIG. 13 illustrates a non-limiting example of a graph of per-target abundance with MIP capture; and,

FIG. 14 shows a non-limiting a graph of correlation between two MIP capture reactions.

FIGS. 15A-B show a SNaPshot validation of a putative Sanger variant call. FIG. 15A discloses "GM17080" sequences as SEQ ID NO: 6328, 6329, and 6328 and FIG. 15B discloses the "GM 17074" sequences as SEQ ID NO: 6328, 6328, and 6328, all respectively, in order of appearance. FIGS. 16A-16D depict skewed allelic fractions in aneuploid cell line GM18540. FIG. 16A depicts an IGV view of NGS data from GM 18540 for the genotype call of interest (shown between vertical lines) (figure 16A discloses SEQ ID NO: 6330-6331). FIG. 16B depicts bidirectional Sanger data for the variant-containing region. FIG. 16C depicts a histogram of allele ratios for all non-reference genotype calls in chromosome 11 derived from wholegenome shotgun sequencing (WGSS) of GM18540 and control sample GM18537. FIG. 16D depicts genome- wide relative coverage for GM 18540. WGSS coverage data for each of the

25 autosomes was binned into 50 Kb intervals and the log-ratio of the per-sample mean normalized values was plotted versus chromosome position. Dashed vertical lines denote chromosome boundaries; within a chromosome the ratios are arranged according to

genomic position.

FIGS. 17A-D depict detection of previously-uncharacterized mutations in samples from individuals affected with cystic fibrosis. FIG. 17A depicts IGV of heterozygous splice site mutation c.3368-2A>T in sample GM 12960 (figure 17A discloses SEQ ID NO: 6332-6333). FIG. 17B depicts IGV of heterozygous premature stop codon mutation Rl 158X in sample GM18802 (figure 17B discloses SEQ ID NO: 6334-6335). FIG. 17C depicts Sanger data confirming existence of mutation c.3368-2A>T in sample GM12960 (figure 17C discloses SEQ ID NO: 6336 and 6336). FIG. 17D depicts Sanger data confirming existence of mutation R1158X in sample GM18802 (figure 17D discloses SEQ ID NO: 6337 and 6337).

FIGS. 18A-E depict next-generation DNA sequencing workflow according to certain embodiments. FIG. 18B discloses (top panel) SEQ ID NO: 6338-6349, (left panel) SEQ ID NO: 6338-6343, and (right panel) SEQ ID NO: 6344-6349, all respectively, in order of appearance. FIG. 18C discloses SEQ ID NO: 6350-6356, 6353, 6352, 6357, and 6357, respectively, in order of appearance. FIG. 18D discloses (left panel) SEQ ID NO: 6352, 6358, 6350, 6352, 6358, 6350, 6359, and 6359, and (right panel) SEQ ID NO: 6360, 6361, 6355, 6360, 6361, 6355,

6362, and 6363, all respectively, in order of appearance. FIG. 18E discloses (left panel) SEQ ID NO: 6358, 6352, and 6350, (right panel) SEQ ID NO: 6360, 6361, and 6355, and (bottom panel) SEQ ID NO: 6364 and 6364, all respectively, in order of appearance.

FIGS. 19A-D depict data from genotyping by assembly template alignment (GATA). GATA correctly genotypes insertions and deletions that are undetectable by the Alignment Only method. Read from top to bottom, each panel provides tracks for cumulative depth of coverage (vertical grey bars); representative MIP alignments (horizontal grey bars) with mismatches (letters), insertions (black bars), and gaps (dashed lines); chromatogram; reference DNA andamino acid sequence for FIG. 19A heterozygous BLM c.2207_2212delinsTAGATTC in sample GM04408 as well as several alleles in the first exon of SMPDl (FIG. 19A discloses SEQ ID NO: 6365 and 6366) including FIG. 19B a heterozygous 18bp deletion in sample GM20342 (minus strand) (FIG.19B discloses SEQ ID NO: 6367 and 6368), FIG. 19C a heterozygous 12bp insertion and homozygous substitution in sample GM17282 (plus strand) (FIG. 19C discloses SEQ ID NO: 6369 and 6370), and FIG. 19D compound heterozygous 6 and 12 bp deletions in sample GM00502 (minus strand) (FIG. 19D discloses SEQ ID NO: 6369 and 6370).

Chromatogram trace offsets corresponding to specific heterozygous insertion and deletion patterns are indicated with slanted lines color coded by reference base. For

clarity offsets are shown for FIGS. 19C-D only.

FIGS. 20A-1, 20A-2, 20A-3, 20B-1, 20B-2 and 20B-3 show NGS detection of allele dropout in Sanger reactions. FIG. 20A-1 discloses SEQ ID NO: 6371, 6372, and 6372, FIG. 20A-B depicts SEQ ID NO: 6371, 6371, 6372, and FIG. 20A-3 disclosesSEQ ID NO: 6373 and 6374, all respectively, in order of appearance. FIG. 20B-1 discloses SEQ ID NO: 6371, 6372, and 6372, FIG. 20B-2 discloses SEQ ID NO: 6371, 6371, 6372, and FIG. 20B-3 discloses SEQ ID NO: 6373 and 6374, all respectively, in order of appearance.

FIG. 21 diagrams use of methods of the invention to validate a genotyping by assembly- templated alignment (GATA) technique.

FIG. 22 illustrates obtaining sequence reads and inserting a simulated mutation.

FIG. 23 shows standard analysis of sequence reads for comparison to GATA.

FIG. 24 shows analysis by GATA. DETAILED DESCRIPTION

Aspects of the invention relate to preparative and analytical methods and compositions for evaluating genotypes, and in particular, for determining the allelic identity (or identities in a diploid organism) of one or more genetic loci in a subject. Aspects of the invention are based, in part, on the identification of different sources of ambiguity and error in genetic analyses, and, in part, on the identification of one or more approaches to avoid, reduce, recognize, and/or resolve these errors and ambiguities at different stages in a genetic analysis. Aspects of the invention relate to methods and compositions for addressing bias and/or stochastic variation associated with one or more preparative and/or analytical steps of a nucleic acid evaluation technology. In some embodiments, preparative methods can be adapted to avoid or reduce the risk of bias skewing the results of a genetic analysis. In some embodiments, analytical methods can be adapted to recognize and correct for data variations that may give rise to misinterpretation (e.g., incorrect calls such as homozygous when the subject is actually heterozygous or heterozygous when the subject is actually homozygous). Methods of the invention may be used for any type of mutation, for example a single base change (e.g., insertion, deletion, transversion or transition, etc.), a multiple base insertion, deletion, duplication, inversion, and/or any other change or combination thereof.

In some embodiments, additional or alternative techniques may be used to address loci characterized by multiple repeats of a core sequence where the length of the repeat is longer than a typical sequencing read thereby making it difficult to determine whether a deletion or duplication of one or more core sequence units has occurred based solely on a sequence read. In some embodiments, increased confidence in an assay result may be obtained by i) selecting two or more different preparative and/or analytical techniques that have different biases (e.g., known to have different biases), ii) evaluating a patient sample using the two or more different techniques, iii) comparing the results from the two or more different techniques, and/or iv) determining whether the results are consistent for the two or more different techniques. In some embodiments, if determining in step (iv) indicates that the results are consistent (e.g., the same) then increased confidence in the assay result is obtained. In other embodiments, if determining in step (iv) indicates that the results are inconsistent (e.g., that the results are ambiguous) then one or more additional preparative and/or analytical techniques, which have a different bias (e.g., known to have a different bias) compared with the two or more different preparative and/or analytical techniques selected in step (i), are used to evaluate the patient sample, and the results of the one or more additional preparative and/or analytical techniques are compared with the results from step (ii) to resolve the inconsistency.

In some embodiments, two or more independent samples may be obtained from a subject and independently analyzed. In some embodiments, two or more independent samples are obtained at approximately the same time point. In some embodiments, two or more independent samples are obtained at multiple different time points. In some embodiments, the use of two or more independent sample facilitates the elimination, normalization, and/or quantification of stochastic measurement noise. It is to be appreciated that two or more independent samples may be obtained in connection with any of the methods disclosed herein, including, for example, methods for pathogen profiling in a human or other animal subjects, monitoring tumor progression/regression, analyzing circulating tumor cells, analyzing fetal cells in maternal circulation, and analyzing/monitoring/profiling of environmental pathogens.

In some embodiments, one or more of the techniques described herein may be combined in a single assay protocol for evaluating multiple patient samples in parallel.

It should be appreciated that aspects of the invention may be useful for high throughput, cost- effective, yet reliable, genotyping of multiple patient samples (e.g., in parallel, for example in multiplex reactions). In some embodiments, aspects of the invention are useful to reduce the error frequency in a multiplex analysis. Certain embodiments may be particularly useful where multiple reactions (e.g., multiple loci and/or multiple patient samples) are being processed. For example, 10-25, 25-50, 50-75, 75-100 or more loci may be evaluated for each subject out of any number of subject samples that may be processed in parallel (e.g., 1-25, 25-50, 50-100, 100-500, 500-1,000, 1,000-2,500, 2,500-5,000 or more or intermediate numbers of patient samples). It should be appreciated that different embodiments of the invention may involve conducting two or more target capture reactions and/or two or more patient sample analyses in parallel in a single multiplex reaction. For example, in some embodiments a plurality of capture reactions (e.g., using different capture probes for different target loci) may be performed in a single multiplex reaction on a single patient sample. In some embodiments, a plurality of captured nucleic acids from each one of a plurality of patient samples may be combined in a single multiplex analysis reaction. In some embodiments, samples from different subjects are tagged with subject- specific (e.g., patient- specific) tags (e.g., unique sequence tags) so that the information from each product can be assigned to an identified subject. In some embodiments, each of the different capture probes used for each patient sample have a common patient-specific tag. In some embodiments, the capture probes do not have patient- specific tags, but the captured products from each subject may be amplified using one or a pair of amplification primers that are labeled with a patient- specific tag. Other techniques for associating a patient-specific tag with the captured product from a single patient sample may be used as aspects of the invention are not limited in this respect. It should be appreciated that patient- specific tags as used herein may refer to unique tags that are assigned to identify patients in a particular assay. The same tags may be used in a separate multiplex analysis with a different set of patient samples (e.g., from different patients) each of which is assigned one of the tags. In some embodiments, different sets of unique tags may be used in sequential (e.g., alternating) multiplex reactions in order to reduce the risk of contamination from one assay to the next and allow contamination to be detected on the basis of the presence of tags that are not expected to be present in a particular assay.

Embodiments of the invention may be used for any of a number of different settings: reproductive settings, disease screening, identifying subjects having cancer, identifying subjects having increased risk for a disease, stratifying a population of subjects according to one or more of a number of factors, for example responsiveness to a particular drug, lack or not of an adverse reaction (or risk therefore) to a particular drug, and/or providing information for medical records (e.g., homozygosity, heterozygosity at one or more loci). It should be appreciated that the invention is not limited to genomic analysis of patient samples. For example, aspects of the invention may be useful for high throughput genetic analysis of environment samples to detect pathogens.

In some embodiments, the methods disclosed herein are useful for diagnosis of one or more heritable disorders. In some embodiments, a heritable disorder that may be diagnosed with the methods disclosed herein is a genetic disorder that is prevalent in the Ashkenazi Jewish population. In some embodiments, the heritable disorders are selected from: 21-Hydroxylase- Defiocient Congenital Adrenal Hyperplasia; ABCC8-Related Hyperinsulinism; Alpha- Thalassemia, includes Constant Spring, & MR associated; Arylsulfatase A Deficiency- Metyachromatic Leukodystrophy; Biotimidase Deficiency-Holocarboxylase Synthetase

Deficiency; Bloom's Syndrome; Canavan Disease; CFTR-Related Disorders-cystic fibrosis; Citrullinemia Type I; Combined MMA & Homocystinuria-dblC; Dystrophinopathies (DMD & BMD); Familial Dysautonomia; Fanconi Anemia-FANCC; Galactosemia-Ci.assical:

Galactokinase Defiency & Galactose Epimerase Deficiency; Gaucher Disease; GJB2-Related DFNB 1 Nonsyndromic Hearing Loss and Deafness; Glutaric acidemia Type 1 ;

Hemoglobinopathies beta-chain disorders; Glycogen Storage Disease Type 1A; Maple Syrup Urine Disease; Types 1A, IB, 2, 3; Medium Chain Acyl-Coenzyme A; Dehydrogenase

Deficiency-MCADD; Methylmalonic Acidemia; Mucolipidosis TV; Nemaline Myopathy;

Nieman-Pick Type A- Acid Sphingomyelinase Deficiency; Non-Ketotic Hyperglycinemia- Glycine Encephalopathy; Ornithine Transcarbamylase Deficiency; PKU Phenylalanine Hydroxylase Deficiency; Propionic Acidemia; Short Chain Acyl-CoA Dehydrogenase Deficiency-SCADD; Smith-Lemli-Opitz Syndrome; Spinal Muscular Atrophy (SMNl)-SMA; Tay Sachs-HexA Deficiency; Usher Synbdrome-Type I (Type IB, Type IC, Type ID, Type IF, Type IG); X-Linked Mental Retardation ARX-Related Disorders; X-Linked Mental Retardation with Cerebellar Cypoplasia and sistinctive Facial Appearance; X-Linked Mental Retardation; includes 9, 21, 30, 46, 58, 63, 88, 89; X-linked mental retardation: FMl-Related Disorders- FRXA, Fragile X MR; X-linked SMR: Renpenning Syndrome 1; Zellweger Spectrum

disorders— Peroxisomal Bifunctional Enzyme Deficiencies including Zellweger, NALD, and/or infantile Refsums. However, all of these, subsets of these, other genes, or combinations thereof may be used.

According to some aspects, the disclosure relates to multiplex diagnostic methods. In some embodiments, multiplex diagnostic methods comprise capturing a plurality of genetic loci in parallel (e.g., a genetic locus of Table 1). In some embodiments, genetic loci possess one or more polymorphisms (e.g., a polymorphism of Table 2) the genotypes of which correspond to disease causing alleles. Accordingly, in some embodiments, the disclosure provides methods for assessing multiple heritable disorders in parallel.

In some embodiments, methods are provided for diagnosing multiple heritable disorders in parallel at a pre-implantation, prenatal, perinatal, or postnatal stage. In some embodiments, the disclosure provides methods for analyzing multiple genetic loci (e.g., a plurality of target nucleic acids selected from Table 1) from a patient sample, such as a blood, pre-implantation embryo, chorionic villus or amniotic fluid sample. A patient or subject may be a human. However, aspects of the invention are not limited to humans and may be applied to other species (e.g., mammals, birds, reptiles, other vertebrates or invertebrates) as aspects of the invention are not limited in this respect. A subject or patient may be male or female. In some embodiments, in connection with reproductive genetic counseling, samples from a male and female member of a couple may be analyzed. In some embodiments, for example, in connection with an animal breeding program, samples from a plurality of male and female subjects may be analyzed to determine compatible or optimal breeding partners or strategies for particular traits or to avoid one or more diseases or conditions.

However, it should be appreciated that any other diseases may be studied and/or risk factors for diseases or disorders including, but not limited to allergies, responsiveness to treatment, cancer tumor profiling for treatment and prognosis, monitoring and identification of patient infections, and monitoring of environmental pathogens.

1. Reducing Representational Bias in Multiplex Amplification Reactions:

In some embodiments, aspects of the invention relate to methods that reduce bias and increase reproducibility in multiplex detection of genetic loci, e.g., for diagnostic purposes. Molecular inversion probe technology is used to detect or amplify particular nucleic acid sequences in potentially complex mixtures. Use of molecular inversion probes has been demonstrated for detection of single nucleotide polymorphisms (Hardenbol et al. 2005 Genome Res 15:269-75) and for preparative amplification of large sets of exons (Porreca et al. 2007 Nat Methods 4:931-6, Krishnakumar et al. 2008 Proc Natl Acad Sci USA 105:9296-301). One of the main benefits of the method is in its capacity for a high degree of multiplexing, because generally thousands of targets may be captured in a single reaction containing thousands of probes. However, challenges associated with, for example, amplification efficiency (See, e.g., Turner E H, et al., Nat. Methods. 2009 Apr. 6: 1-2.) have limited the practical utility of the method in research and diagnostic settings.

Aspects of the disclosure are based, in part, on the discovery of effective methods for overcoming challenges associated with systematic errors (bias) in multiplex genomic capture and sequencing methods, namely high variability in target nucleic acid representation and unequal sampling of heterozygous alleles in pools of captured target nucleic acids (e.g., isolated from a biological sample). Accordingly, in some embodiments, the disclosure provides methods that reduce variability in the detection of target nucleic acids in multiplex capture methods. In other embodiments, methods improve allelic representation in a capture pool and, thus, improve variant detection outcomes. In certain embodiments, the disclosure provides preparative methods for capturing target nucleic acids (e.g., genetic loci) that involve the use of different sets of multiple probes (e.g., molecular inversion probes MIPs) that capture overlapping regions of a target nucleic acid to achieve a more uniform representation of the target nucleic acids in a capture pool compared with methods of the prior art. In other embodiments, methods reduce bias, or the risk of bias, associated with large scale parallel capture of genetic loci, e.g., for diagnostic purposes. In other embodiments, methods are provided for increasing reproducibility (e.g., by reducing the effect of polymorphisms on target nucleic acid capture) in the detection of a plurality of genetic loci in parallel. In further embodiments, methods are provided for reducing the effect of probe synthesis and/or probe amplification variability on the analysis of a plurality of genetic loci in parallel.

In some aspects, the disclosure provides probe sets that comprise a plurality of different probes. As used herein, a 'probe' is a nucleic acid having a central region flanked by a 5' region and a 3' region that are complementary to nucleic acids flanking the same strand of a target nucleic acid or subregion thereof. An exemplary probe is a molecular inversion probe (MIP). A 'target nucleic acid' may be a genetic locus. Exemplary genetic loci are disclosed herein in Table 1 (RefSeqGene Column).

While probes have been typically designed to meet certain constraints (e.g. melting temperature, G/C content, etc.) known to partially affect capture/amplification efficiency (Ball et al (2009) Nat Biotech 27:361-8 AND Deng et al (2009) Nat Biotech 27:353-60), a set of constraints which is sufficient to ensure either largely uniform or highly reproducible

capture/amplification efficiency has not previously been achieved. As disclosed herein, uniformity and reproducibility can be increased by designing multiple probes per target, such that each base in the target is captured by more than one probe. In some embodiments, the disclosure provides multiple MIPs per target to be captured, where each MIP in a set designed for a given target nucleic acid has a central region and a 5' region and 3' region ('targeting arms') which hybridize to (at least partially) different nucleic acids in the target nucleic acid

(immediately flanking a subregion of the target nucleic acid). Thus, differences in efficiency between different targeting arms and fill-in sequences may be averaged across multiple MIPs for a single target, which results in more uniform and reproducible capture efficiency.

In some embodiments, the methods involve designing a single probe for each target (a target can be as small as a single base or as large as a kilobase or more of contiguous sequence). It may be preferable, in some cases, to design probes to capture molecules (e.g., target nucleic acids or subregions thereof) having lengths in the range of 1-200 bp (as used herein, a by refers to a base pair on a double-stranded nucleic acid— however, where lengths are indicated in bps, it should be appreciated that single-stranded nucleic acids having the same number of bases, as opposed to base pairs, in length also are contemplated by the invention). However, probe design is not so limited. For example, probes can be designed to capture targets having lengths in the range of up to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, or more bps, in some cases. It is to be appreciated that the length of a capture molecule (e.g., a target nucleic acid or subregion thereof) is selected based upon multiple considerations. For example, where analysis of a target involves sequencing, e.g., with a next- generation sequencer, the target length should typically match the sequencing read-length so that shotgun library construction is not necessary. However, it should be appreciated that captured nucleic acids may be sequenced using any suitable sequencing technique as aspects of the invention are not limited in this respect.

It is also to be appreciated that some target nucleic acids are too large to be captured with one probe. Consequently, it may be necessary to capture multiple subregions of a target nucleic acid in order to analyze the full target.

In some embodiments, a subregion of a target nucleic acid is at least 1 bp. In other embodiments, a subregion of a target nucleic acid is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp or more. In other embodiments, a subregion of a target nucleic acid has a length that is up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more percent of a target nucleic acid length.

The skilled artisan will also appreciate that consideration is made, in the design of MIPs, for the relationship between probe length and target length. In some embodiments, MIPs are designed such that they are several hundred basepairs (e.g., up to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp or more) longer than corresponding target (e.g., subregion of a target nucleic acid, target nucleic acid).

In some embodiments, lengths of subregions of a target nucleic acid may differ. For example, if a target nucleic acid contains regions for which probe hybridization is not possible or inefficient, it may be necessary to use probes that capture subregions of one or more different lengths in order to avoid hybridization with problematic nucleic acids and capture nucleic acids that encompass a complete target nucleic acid.

Aspects of the invention involve using multiple probes, e.g., MIPs, to amplify each target nucleic acid. In some embodiments, the set of probes for a given target can be designed to 'tile' across the target, capturing the target as a series of shorter sub-targets. In some embodiments, where a set of probes for a given target is designed to 'tile' across the target, some probes in the set capture flanking non-target sequence). Alternately, the set can be designed to 'stagger' the exact positions of the hybridization regions flanking the target, capturing the full target (and in some cases capturing flanking non-target sequence) with multiple probes having different targeting arms, obviating the need for tiling. The particular approach chosen will depend on the nature of the target set. For example, if small regions are to be captured, a staggered-end approach might be appropriate, whereas if longer regions are desired, tiling might be chosen. In all cases, the amount of bias-tolerance for probes targeting pathological loci can be adjusted ('dialed in') by changing the number of different MIPs used to capture a given molecule.

In some embodiments, the 'coverage factor', or number of probes used to capture a basepair in a molecule, is an important parameter to specify. Different numbers of probes per target are indicated depending on whether one is using the tiling approach (see, e.g., FIG. 1) or one of the staggered approaches (see, e.g., FIG. 2 or 3).

FIG. 1 illustrates a non-limiting embodiment of a tiled probe layout showing ten captured sub-targets tiled across a single target. Each position in the target is covered by three sub-targets such that MIP performance per base pair is averaged across three probes.

FIG. 2 illustrates a non-limiting embodiment of a staggered probe layout showing the targets captured by a set of three MIPs. Each MIP captures the full target, shown in black, plus (in some cases) additional extra- target sequence, shown in gray, such that the targeting arms of each MIP fall on different sequence. Each position in the target is covered by three sub-targets such that MIP performance per basepair is averaged across three probes. Targeting arms land immediately adjacent to the black or gray regions shown. It should be appreciated that in some embodiments, the targeting arms (not shown) can be designed so that they do not overlap with each other.

FIG. 3 illustrates a non-limiting embodiment of an alternating staggered probe layout showing the targets captured by a set of three MIPs. Each MIP captures the full target, shown in black, plus (in some cases) additional extra-target sequence, shown in gray, such that the targeting arms of each MIP fall on different sequence. Each position in the target is covered by three sub-targets such that MIP performance per basepair is averaged across three probes.

Targeting arms land immediately adjacent to the black or gray regions shown.

It should be appreciated that for any of the layouts, the targeting arms on adjacent tiled or staggered probes may be designed to either overlap, not overlap, or overlap for only a subset of the probes.

In certain embodiments for any of the layouts, a coverage factor of about 3 to to about 10 is used. However, the methods are not so limited and coverage factors of up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more may be used. It is to be appreciated that the coverage factor selected may depend the probe layout being employed. For example, in the tiling approach, for a desired coverage factor, the number of probes per target is typically a function of target length, sub- target length, and spacing between adjacent sub-target start locations (step size). For example, for a desired coverage factor of 3, a 200 bp target with a start- site separation of 20 bp and sub- target length of 60 bp may be encompassed with 12 MIPs (FIG. 1). Thus, a specific coverage factor may be achieved by varying the number of probes per target nucleic acid and the length of the molecules captured. In the staggered approach, a fixed-length target nucleic acid is captured as several subregions or as 'super- targets', which are molecules comprising the target nucleic acid and additional flanking nucleic acids, which may be of varying lengths. For example, a target of 50 bp can be captured at a coverage factor of 3 with 3 probes in either a 'staggered' (FIG. 2) or 'alternating staggered' configuration (FIG. 3).

The coverage factor will be driven by the extent to which detection bias is tolerable. In some cases, where the bias tolerance is small, it may be desirable to target more subregions of target nucleic acid with, perhaps, higher coverage factors. In some embodiments, the coverage factor is up to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In some embodiments, when a tiled probe layout is used, when the target length is greater than 1 bp and when a step size (distance between the 5 '-end of a target and the 5' end of its adjacent target) is less than the length of a target or subregion thereof, it is possible to compute probe number for a particular target based on target length (T), sub-target length (S), and coverage factor (C), such that probe number=T/(S/C)+(C-l).

In some aspects, the disclosure provides methods to increase the uniformity of amplification efficiency when multiple molecules are amplified in parallel; methods to increase the reproducibility of amplification efficiency; methods to reduce the contribution of targeting probe variability to amplification efficiency; methods to reduce the effect on a given target nucleic acid of polymorphisms in probe hybridization regions; and/or methods to simplify downstream workflows when multiplex amplification by MIPs is used as a preparative step for analysis by nucleic acid sequencing.

Polymorphisms in the target nucleic acid under the regions flanking a target can interfere with hybridization, polymerase fill-in, and/or ligation. Furthermore, this may occur for only one allele, resulting in allelic drop-out, which ultimately decreases downstream sequencing accuracy. In some embodiments, using a set of MIPs having multiple hybridization sites for the capture of any given target, the probability of loss from polymorphism is substantially decreased because not all targeting arms in the set of MIPs will cover the location of the mutation.

Probes for MIP capture reactions may be synthesized on programmable microarrays because of the large number of sequences required. Because of the low synthesis yields of these methods, a subsequent amplification step is required to produce sufficient probe for the MIP amplification reaction. The combination of multiplex oligonucleotide synthesis and pooled amplification results in uneven synthesis error rates and representational biases. By synthesizing multiple probes for each target, variation from these sources may be averaged out because not all probes for a given target will have the same error rates and biases.

Multiplex amplification strategies disclosed herein may be used analytically, as in detection of SNPs, or preparatively, often for next- generation sequencing or other sequencing techniques. In the preparative setting, the output of an amplification reaction is generally the input to a shotgun library protocol, which then becomes the input to the sequencing platform. The shotgun library is necessary in part because next- generation sequencing yields reads significantly shorter than amplicons such as exons. In addition to the bias-reduction afforded by the multi-tiled approach described here, tiling also obviates the need for shotgun library preparation. Since the length of the capture molecule can be specified when the probes, e.g., MIPs, are designed, it can be chosen to match the readlength of the sequencer. In this way, reads can 'walk' across an exon by virtue of the start position of each capture molecule in the probe set for that exon.

Exemplary molecular inversion probes are provided in Appendix A. These molecular inversion probes are designed to capture targets or sub-regions thereof on one or more genes listed in Table 5 (provided in Example 8). In certain applications, the molecular inversion probes provided in Appendix A may be used to tile-capture targets or sub-regions thereof on the one or more genes provided in Table 5. In particular applications, two or more of the molecular inversion probes of Appendix A tile across different, but overlapping sub-regions of one or more genes listed in Table 5 so that a target on the gene is capture by both of the two or more molecular inversion probes, as exemplified in Figure 1.

In certain embodiments, the molecular inversion probes of Appendix A that are chosen for tile-capture a target depends on the desired amount of overlapping coverage for the target. In one example, two or more molecular inversion probes of Appendix A, being in directly ascending SEQ ID NO: order and corresponding to a target nucleic acid, will tile across the target nucleic acid with a period of 25 base pairs such that every genomic position of the target nucleic acid is capture by multiple probes with orthogonal targeting arm sequences. If less coverage is desired for a target nucleic acid, one may select, for example, every other molecular inversion probes of Appendix A in ascending order that correspond to that target.

The first and second targeting arms of the molecular inversion probes are designed to hybridize to nucleotides upstream and downstream of a capture region of a gene (i.e. the targeting arms flank the region to be captured). The capture region may be a target nucleic acid or a sub-region thereof. Appendix B lists the capture regions of the genes that correspond to the molecular inversion probes listed in Appendix A. Appendix A also specifies the upstream and downstream regions of the capture regions corresponding to each targeting arm of the molecular inversion probes. The upstream and downstream regions of the capture region are between the start position and the end position coordinates, which are relative to the Human Genome 18 (HG 18).

The molecular inversion probes of Appendix A include a central region flanked by a 5' first targeting arm (i.e. ligation arm or left arm) and a 3' second targeting arm (i.e. extension arm or right arm). The targeting arm sequences are shown in lowercase letters and the central region sequence is shown in uppercase letters. The 5' first targeting arm and the 3' second targeting arm of the molecular inversion probes provided in Appendix A include a total of 40 nucleotides, and are designed to flank 130 bp capture regions. Some of the molecular inversion probes listed in Appendix A are designed to capture the coding regions of the genes, whereas others are designed to capture non-coding regions of the genes. The genes listed in Table 5 corresponded to diseases, and as such, the molecular inversion probes listed in Appendix A can be utilized to analyze one or more of the diseases provided in table 5. The molecular inversion probes provided in Appendix A are described in more detail in Example 8.

While all of the molecular inversion probes provided in Appendix A may be used in a single assay to comprehensively examine several or all of the genomic regions of the genes provided in Table 5, one may also select one or more molecular inversion probes provided in Appendex A to evaluate one or more targets present in one gene or a combination of the genes provided in Table 5. For example, one may choose to only examine the coding regions of one or more of the genes listed in Table 5, and therefore use the one or more of the molecular inversion probes designed to capture those regions. In another example, one may choose to only examine the non-coding regions of one or more gene listed in Table 5, and therefore use the one or more molecular inversion probes designed to capture those regions. In another example, one may choose to only examine a portion of or the entirety of a gene listed in Table 5, and therefore use the one or more molecular inversion probes design to capture the portion of or the entirety of that gene. In another example, one may choose to examine nucleic acid regions specific to one or more diseases listed in Table 5, and therefore use the one or more nucleic acids corresponding to those diseases. In yet another example, one may chose to examine a portion or entirety of two or more of the genes listed in Table 5, and there uses the moleculre inversion probes specific to those genes. In yet another example, one may chose to only examine certain chromosomes with the molecular inversion probes provided in Appendix A. In all of these examples, the number of molecular inversion probes that correspond to the target chosen depends on the amount of coverage one desires.

It is understood that one can modify the molecular inversion probes listed in Appendix A, while achieving a similar coverage and tile-capture layout as the probes listed in Appendix A. For example, the sequence of the central region of the molecular inversion probes may be different from the sequence of the central region provided in Appendix A without changing capture region of the probe. For molecular inversion probe sets, the sequence chosen for the central region is preferably the same across each molecular inversion probe in a set of probes. This allows the capture targets to be amplified with a single set of primers. It is also preferable that the central region is designed so that it is not complementary to the target sequences or any other sequence in the sample.

In addition, it should be appreciated that other molecular inversion probes than those listed in Appendix A may be used to tile-capture different regions of the genes listed in Table 5. Those molecular inversion probes may include a different first targeting arm, second targeting arm, and/or central region from the molecular inversion probes listed in Appendix A. In a non- limiting example, a modified molecular inversion probe may include the first targeting arm sequence of SEQ ID NO: 300, but have a different sequence for the central region and the second targeting arm. The specific sequences and length of the sequences chosen for the first targeting arm, second targeting arm, and/or central region depend on the desired capture region and coverage.

In certain embodiments, the molecular inversion probes for tile or staggered capture are selected to maximize performance with respect to both capture efficiency and robustness to common polymorphisms. In order to determine which probes maximize performance for a genomic target, methods of the invention, according to certain aspects, involve designing all possible probes capable of targeting a genomic interval and ranking the probes based on a number of score tuples or ranking factors. In certain embodiments, the possible probes are assigned score tuples including, but not limited to: 1) presence of quanine or cystosine as the 5'- most base of the ligation arm, 2) the number of dbSNP (version 130) entries intersecting targeting arm sites, 3) the root mean squared deviation of the targeting arms' predicted melting temperatures from optimal values derived from empirical studies of efficiencies. Using any combination of these score tuples, the possible probes for a certain genomic interval may be ranked, and the highest ranking probe for the genomic interval is preferably chosen for capture.

In certain aspects, method of the invention provide for shearing or fragmenting genomic nucleic acid prior to performing capture with a molecular inversion probe ( e.g. capture with one or more of the molecular inversion probes provided in Appendix A). Fragmenting the genomic nucleic acid prior to performing a capture reaction allows for greater exposure of a target site to a molecular inversion probe, which reduces failed capture and increases the percentage of molecular inversion probes that hybridize to targets within the genome. This advantageously yields a target abundance distribution that is significantly more uniform than if a native high molecular weight genomic nucleic acid is used. Molecular inversion techniques involving a fragmenting step are described in co-owned and co-assigned U.S. Serial Number 13/448,961, having U.S. Publication No. 2012/0252020, entitled "Capture Reactions."

Fragmenting the nucleic acid can be accomplished by any technique known in the art.

Exemplary techniques include mechanically fragmenting, chemically fragmenting, and/or enzymatically fragmenting. Mechanical nucleic acid fragmentation can be, for example, sonication, nebulization, and hydro- shearing (e.g., point-sink shearing). Enzymatic nucleic acid fragmenting includes, for example, use of nicking endonucleases or restriction endonucleases. The nucleic acid can also be chemically fragmented by performing acid hydrolysis on the nucleic acid or treating of the nucleic acid with alkali or other reagents. The fragment length can be adjusted based on the sizes of the nucleic acid targets to be captured. The nucleic acid fragments can be of uniform length or of a distribution of lengths. In certain embodiments, the nucleic acid is fragmented into nucleic acid fragments having a length of about 10 kb or 20 kb. In addition, the nucleic acid fragments can range from between 1 kb to 20 kb, with various distributions.

In certain embodiments, the nucleic acid is also denatured, which may occur prior to, during, or after the fragmenting step. The nucleic acid can be denatured using any means known in the art, such as pH-based denaturing, heat-based denaturing, formamide or urea, exonuclease degradation, or endonuclease nicking. In certain embodiments, the use of pH, such as in acid hydrolysis, alone or in combination with heat fragments and either partially or fully denatures the nucleic acid. This combined fragmenting and denaturing method can be used to fragment the nucleic acid for MIP capture or to fragment captured target nucleic acids or whole genomic DNA for shotgun library preparation.

In one aspect, a nucleic acid is fragmented by heating a nucleic acid immersed in a buffer system at a certain temperature for a certain period to time to initiate hydrolysis and thus fragment the nucleic acid. The pH of the buffer system, duration of heating, and temperature can be varied to achieve a desired fragmentation of the nucleic acid. In one embodiment, after a genomic nucleic acid is purified, it is resuspended in a Tris-based buffer at a pH between 7.5 and 8.0, such as Qiagen's DNA hydrating solution. The resuspended genomic nucleic acid is then heated to 65°C and incubated overnight (about 16-24 hours) at 65°C. Heating shifts the pH of the buffer into the low- to mid- 6 range, which leads to acid hydrolysis. Over time, the acid hydrolysis causes the genomic nucleic acid to fragment into single- stranded and/or double- stranded products. The above method of fragmenting can be modified by increasing the temperature and reducing the heating time. For example, a nucleic acid is fragmented by incubating the nucleic acid in the Tris-based buffer at a pH between 7.5 and 8.0 for 15 minutes at 92°C. In addition to adjusting the temperature and the duration of heating, the pH of the Tris- based buffer can be adjusted to achieve a desired nucleic acid fragmentation.

Once molecular inversion probes of the invention are hybridized to genomic or fragmented nucleic acid, the captured target may further be subjected to an enzymatic gap-filling and ligation step, such that a copy of the target sequence is incorporated into a circle. Capture efficiency of the MIP to the target sequence on the nucleic acid fragment can be improved by lengthening the hybridization and gap-filing incubation periods. (See, e.g., Turner EH, et al., Nat Methods. 2009 Apr 6: 1-2.).

The result of molecular inversion probe capture as described above is a library of circular target probes, which then can be processed in a variety of ways. In one aspect, adaptors for sequencing can be attached during common linker-mediated PCR, resulting in a library with non- random, fixed starting points for sequencing. In another aspect, for preparation of a shotgun library, a common linker-mediated PCR is performed on the circle target probes, and the post- capture amplicons are linearly concatenated, sheared, and attached to adaptors for sequencing. Methods for shearing the linear concatenated captured targets can include any of the methods disclosed for fragmenting nucleic acids discussed above. In certain aspects, performing a hydrolysis reaction on the captured amplicons in the presence of heat is the desired method of shearing for library production.

Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.

An example of a sequencing technology that can be used is Illumina sequencing. Illumina sequencing is based on the amplification of DNA on a solid surface using fold-back PCR and anchored primers. Genomic DNA is fragmented, and adapters are added to the 5' and 3' ends of the fragments. DNA fragments that are attached to the surface of flow cell channels are extended and bridge amplified. The fragments become double stranded, and the double stranded molecules are denatured. Multiple cycles of the solid-phase amplification followed by denaturation can create several million clusters of approximately 1,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore-labeled, reversibly terminating nucleotides are used to perform sequential sequencing. After nucleotide incorporation, a laser is used to excite the fluorophores, and an image is captured and the identity of the first base is recorded. The 3' terminators and fluorophores from each incorporated base are removed and the incorporation, detection and identification steps are repeated. Sequencing according to this technology is described in U.S. Pat. 7,960,120; U.S. Pat. 7,835,871; U.S. Pat. 7,232,656; U.S. Pat. 7,598,035; U.S. Pat.

6,911,345; U.S. Pat. 6,833,246; U.S. Pat. 6,828,100; U.S. Pat. 6,306,597; U.S. Pat. 6,210,891; U.S. Pub. 2011/0009278; U.S. Pub. 2007/0114362; U.S. Pub. 2006/0292611; and U.S. Pub. 2006/0024681, each of which are incorporated by reference in their entirety.

Sequencing generates a plurality of reads. Reads generally include sequences of nucleotide data less than about 150 bases in length, or less than about 90 bases in length. In certain embodiments, reads are between about 80 and about 90 bases, e.g., about 85 bases in length. In some embodiments, these are very short reads, i.e., less than about 50 or about 30 bases in length. A set of sequence reads can be analyzed by any suitable method known in the art. For example, in some embodiments, sequence reads are analyzed by hardware or software provided as part of a sequence instrument. In some embodiments, individual sequence reads are reviewed by sight (e.g., on a computer monitor). A computer program may be written that pulls an observed genotype from individual reads. In certain embodiments, analyzing the reads includes assembling the sequence reads and then genotyping the assembled reads.

In certain embodiments, the sequences obtained using the molecular inversion probe techniques of the invention are analyzed using the methods for evaluating of genetic test, which are described in co-pending and co-owned U.S. Provisional Serial Number 61/723,508, entitled "Validation of Genetic Test." The method involves obtaining a plurality of sequence reads, introducing a simulated mutation into at least one of the plurality of sequence reads, and analyzing the sequence reads to determine if the test identifies the simulated mutation. To mimic the expected genotype of a heterozygous carrier, the simulated mutation can be introduced into each of those sequence reads that span a location of the mutation with a probability of 0.5 (e.g., into about half of those sequence reads that should contain the location of the simulated mutation). The simulated mutation can be introduced by manipulating a data field in the sequence read such as, for example, a base sequence field or quality data field. The sequences can be manipulated by a computer program. For example, a program can be written using Java, Groovy, Python, Perl, or other languages, or a combination thereof, that can automatically insert simulated mutations into sequence reads. Computer-based methods can be used to automatically introduce a number of different simulated mutations into different ones of the plurality of sequence reads.

The sequence reads including the manipulated reads are analyzed to detect a genotype. Analysis can include any method known in the art, such as de novo assembly, alignment to a reference, or a combination thereof. In some embodiments, the sequence reads are assembled into a contig. The contig can be aligned to a reference genome. In certain embodiments, individual reads are then aligned back to the contig.

Sequence assembly can be done by methods known in the art including reference-based assemblies, de novo assemblies, assembly by alignment, or combination methods. Assembly can include methods described in U.S. Pat. 8,209,130 titled Sequence Assembly by Porecca and Kennedy, the contents of each of which are hereby incorporated by reference in their entirety for all purposes. In some embodiments, sequence assembly uses the low coverage sequence assembly software (LOCAS) tool described by Klein, et al., in LOCAS-A low coverage sequence assembly tool for re-sequencing projects, PLoS One 6(8) article 23455 (2011), the contents of which are hereby incorporated by reference in their entirety. Sequence assembly is described in U.S. Pat. 8,165,821; U.S. Pat. 7,809,509; U.S. Pat. 6,223,128; U.S. Pub.

2011/0257889; and U.S. Pub. 2009/0318310, the contents of each of which are hereby incorporated by reference in their entirety.

In certain embodiments, genetic test of the invention are validated using a genotyping by assembly-template alignment (GATA) technique, which is also described in co-pending and co- owned U.S. Provisional Serial Number 61/723,508, entitled "Validation of Genetic Test." FIG. 21 diagrams the validation of a genotyping by assembly-templated alignment (GATA) technique. Genetic analysis by GATA-based methods includes obtaining 401 sequence reads and

assembling 405 the reads into a contig, which is then aligned 409 to a reference. Differences are identified by comparison 413. The raw reads are aligned 417 to the contigs and positional and variant information is mapped to the reads from the reference via the contig, allowing

genotyping 421 to produce an observed genotyping. The GATA-based method is evaluated by introducing 403 at least one simulated mutation into the reads.

FIG. 22 illustrates obtaining sequence reads and inserting a simulated mutation. As shown in FIG. 22, if only wild type sample is sequenced, the raw sequence reads may only include wild type sequence. However, a mutation of interest may be known, for example, from the literature or it may be desirable to simply invent a difficult-to-detect mutation to use in methods of validating a genetic analysis. Here, a hypothetical 8 base pair deletion proximal to a C>A substitution is depicted. As shown in FIG. 22, the raw sequence reads are edited so that they include base sequence data, quality data, or both that would arise from sequencing the simulated mutation.

FIG. 23 shows an example in which a standard analytical method is performed for comparison to a GATA-based method. The standard analysis is demonstrated to not be able to detect a mutation. FIG. 23 depicts a workflow in which edited sequence reads (e.g., as depicted in FIG. 22) are aligned to a reference genome (here, using BWA and GATK). The alignment software properly aligns the wild type sequence reads to the reference genome, finding a perfect match and giving a result indicating that the sample is the wild type. However, the alignment software finds no valid alignment for the edited sequence reads and is unable to produce a result. Due to the fact that the expected genotype of the edited sequence reads is known a priori (and, in fact intentionally supplied by editing), an operator is able to identify that this analysis method— alignment of sequence reads to a reference genome— is incapable of detecting the mutation. For comparison, the sequence reads are also analyzed by a GATA-based method.

FIG. 24 shows analysis of sequence reads that include simulated mutations by GATA. In step 1, reads are assembled into contigs. Assembly can include any method including those discussed below. In step 2, each contig is aligned to a reference genome. Alignment can be by any method such as those discussed below, including, e.g., the bwa-sw algorithm implemented by BWA. As shown in FIG. 24, both align to the same reference position. Differences between the contig and the reference genome are identified and, as shown in FIG. 24, described by a CIGAR string.

In step 3, raw reads are aligned to contigs (using any method such as, for example, BWA with bwa- short and writing, for example, a CIGAR string). At step 4, raw read alignments are mapped from contig space to original reference space (e.g., via position and CIGAR

information). In step 5, genotyping is performed using the translated, aligned reads from step 4 (e.g., including raw quality scores for substitutions).

For step 1, reads may be assembled into contigs by any method known in the art.

Algorithms for the de novo assembly of a plurality of sequence reads are known in the art. One algorithm for assembling sequence reads is known as overlap consensus assembly. Assembly with overlap graphs is described, for example, in U.S. Pat. 6,714,874. In some embodiments, de novo assembly proceeds according to so-called greedy algorithms, as described in U.S. Pub. 2011/0257889, incorporated by reference in its entirety. In other embodiments, assembly proceeds by either exhaustive or heuristic pairwise alignment. Exhaustive pairwise alignment, sometimes called a "brute force" approach, calculates an alignment score for every possible alignment between every possible pair of sequences among a set. Assembly by heuristic multiple sequence alignment ignores certain mathematically unlikely combinations and can be computationally faster. One heuristic method of assembly by multiple sequence alignment is the so-called "divide-and-conquer" heuristic, which is described, for example, in U.S. Pub.

2003/0224384. Another heuristic method of assembly by multiple sequence alignment is progressive alignment, as implemented by the program ClustalW (see, e.g., Thompson, et al., Nucl. Acids. Res., 22:4673-80 (1994)).

With continuing reference to step 1 of FIG. 24, in some embodiments assembly into contigs involves making a de Bruijn graph. De Bruijn graphs reduce the computation effort by breaking reads into smaller sequences of DNA, called k-mers, where the parameter k denotes the length in bases of these sequences. In a de Bruijn graph, all reads are broken into k-mers (all subsequences of length k within the reads) and a path between the k-mers is calculated. In assembly according to this method, the reads are represented as a path through the k-mers. The de Bruijn graph captures overlaps of length k-1 between these k-mers and not between the actual reads. By reducing the entire data set down to k-mer overlaps, the de Bruijn graph reduces the high redundancy in short-read data sets. Assembly of reads using de Bruijn graphs is described in U.S. Pub. 2011/0004413, U.S. Pub. 2011/0015863, and U.S. Pub. 2010/0063742, incorporated by reference in their entirety. Assembly of reads into contigs is further discussed in U.S. Pat. 6,223,128, U.S. Pub. 2009/0298064, U.S. Pub. 2010/0069263, and U.S. Pub. 2011/0257889, each of which is incorporated by reference herein in its entirety.

2. Reducing Analytical Errors Associated with Bias in Nucleic Acid Preparations:

In some embodiments, aspects of the invention relate to preparative steps in DNA sequencing-related technologies that reduce bias and increase the reliability and accuracy of downstream quantitative applications. There are currently many genomics assays that utilize next-generation (polony-based) sequencing to generate data, including genome resequencing, RNA-seq for gene expression, bisulphite sequencing for methylation, and Immune-seq, among others. In order to make quantitative measurements (including genotype calling), these methods utilize the counts of sequencing reads of a given genomic locus as a proxy for the representation of that sequence in the original sample of nucleic acids. The majority of these techniques require a preparative step to construct a high-complexity library of DNA molecules that is representative of a sample of interest. This may include chemical or biochemical treatment of the DNA (e.g., bisulphite treatment), capture of a specific subset of the genome (e.g., padlock probe capture, solution hybridization), and a variety of amplification techniques (e.g., polymerase chain reaction, whole genome amplification, rolling circle amplification).

Systematic and random errors are common problems associated with genome

amplification and sequencing library construction techniques. For example, genomic sequencing library may contain an over- or under-representation of particular sequences from a source genome as a result of errors (bias) in the library construction process. Such bias can be particularly problematic when it results in target sequences from a genome being absent or undetectable in the sequencing libraries. For example, an under-representation of particular allelic sequences (e.g., heterozygotic alleles) from a genome in a sequencing library can result in an apparent homozygous representation in a sequencing library. As most downstream sequencing library quantification techniques depend on stochastic counting processes, these problems have typically been addressed by sampling enough (over- sampling) to obtain a minimum number of observations necessary to make statistically significant decisions. However, the strategy of oversampling is generally limited to elimination of low-count Poisson noise, and the approach wastes resources and increases the expense required to perform such experiments. Moreover, oversampling can result in a reduced statistical confidence in certain conclusions (e.g., diagnostic calls) based on the data. Accordingly, new approaches are needed for overcoming bias in sequencing library preparatory methods.

Aspects of the disclosure are based, in part, on the discovery of methods for overcoming problems associated with systematic and random errors (bias) in genome capture, amplification and sequencing methods, namely high variability in the capture and amplification of nucleic acids and disproportionate representation of heterozygous alleles in sequencing libraries. Accordingly, in some embodiments, the disclosure provides methods that reduce variability in the capture and amplification of nucleic acids. In other embodiments, the methods improve allelic representation in sequencing libraries and, thus, improve variant detection outcomes. In certain embodiments, the disclosure provides preparative methods for capturing target nucleic acids (e.g., genetic loci) that involve the use of differentiator tag sequences to uniquely tag individual nucleic acid molecules. In some embodiments, the differentiator tag sequence permits the detection of bias based on the frequency with which pairs of differentiator tag and target sequences are observed in a sequencing reaction. In other embodiments, the methods reduce errors caused by bias, or the risk of bias, associated with the capture, amplification and sequencing of genetic loci, e.g., for diagnostic purposes.

Aspects of the invention relate to associating unique sequence tags (referred to as differentiator tag sequences) with individual target molecules that are independently captured and/or analyzed (e.g., prior to amplification or other process that may introduce bias). These tags are useful to distinguish independent target molecules from each other thereby allowing an analysis to be based on a known number of individual target molecules. For example, if each of a plurality of target molecule sequences obtained in an assay is associated with a different differentiator tag, then the target sequences can be considered to be independent of each other and a genotype likelihood can be determined based on this information. In contrast, if each of the plurality of target molecule sequences obtained in the assay is associated with the same differentiator tag, then they probably all originated from the same target molecule due to over- representation (e.g., due to biased amplification) of this target molecule in the assay. This provides less information than the situation where each nucleic acid was associated with a different differentiator tag. In some embodiments, a threshold number of independently isolated molecules (e.g., unique combinations of differentiator tag and target sequences) is analyzed to determine the genotype of a subject.

In some embodiments, the invention relates to compositions comprising pools (libraries) of preparative nucleic acids that each comprise "differentiator tag sequences" for detecting and reducing the effects of bias, and for genotyping target nucleic acid sequences. As used herein, a "differentiator tag sequence" is a sequence of a nucleic acid (a preparative nucleic acid), which in the context of a plurality of different isolated nucleic acids, identifies a unique, independently isolated nucleic acid. Typically, differentiator tag sequences are used to identify the origin of a target nucleic acid at one or more stages of a nucleic acid preparative method. For example, in the context of a multiplex nucleic acid capture reaction, differentiator tag sequences provide a basis for differentiating between multiple independent, target nucleic acid capture events. Also, in the context of a multiplex nucleic acid amplification reaction, differentiator tag sequences provide a basis for differentiating between multiple independent, primary amplicons of a target nucleic acid, for example. Thus, combinations of target nucleic acid and differentiator tag sequence (target:differentiator tag sequences) of an isolated nucleic acid of a preparative method provide a basis for identifying unique, independently isolated target nucleic acids. FIG. 4A-C depict various non-limiting examples of methods for combining differentiator tag sequence and target sequences.

It will be apparent to the skilled artisan that differentiator tags may be synthesized using any one of a number of different methods known in the art. For example, differentiator tags may be synthesized by random nucleotide addition. Differentiator tag sequences are typically of a predefined length, which is selected to control the likelihood of producing unique

target:differentiator tag sequences in a preparative reaction (e.g., amplification-based reaction, a circularization selection-based reaction, e.g., a MIP reaction). Differentiator tag sequences may be, up to 5, up to 6, up to 7 up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 21, up to 22, up to 23, up to 24, up to 25, or more nucleotides in length. For purposes of genotyping, isolated nucleic acids are identified as independently isolated if they comprise unique combinations of target nucleic acid and differentiator tag sequences, and observance of threshold numbers of unique combinations of target nucleic acid and differentiator tag sequences provide a certain statistical confidence in the genotype.

During a library preparation process, each nucleic acid molecule may be tagged with a unique differentiator tag sequence in a configuration that permits the differentiator tag sequence to be sequenced along with the target nucleic acid sequence of interest (the nucleic acid sequence for which the library is being prepared, e.g., a polymorphic sequence). The incorporation of the nucleic acid comprising a differentiator tag sequence at a particular step allows the detection and correction of biases in subsequent steps of the protocol.

A large library of unique differentiator tag sequences may be created by using

degenerate, random-sequence polynucleotides of defined length. The differentiator tag sequences of the polynucleotides may be read at the final stage of the sequencing. The observations of the differentiator tag sequences may be used to detect and correct biases in the final sequencing read-out of the library. For example, the total possible number of differentiator tag sequences, which may be produced, e.g., randomly, is 4^N, where N is the length of the differentiator tag sequence. Thus, it is to be understood that the length of the differentiator tag sequence may be adjusted such that the size of the population of MIPs having unique differentiator tag sequences is sufficient to produce a library of MIP capture products in which identical independent combinations of target nucleic acid and differentiator tag sequence are rare. As used herein combinations of target nucleic acid and differentiator tag sequences, may also be referred to as "target:differentiator tag sequences".

In the final readout of a sequencing process, each read may have an additional unique differentiator tag sequence. In some embodiments, when differentiator tag sequences are distributed randomly in a library, all the unique differentiator tag sequences will be observed about an equal number of times. Accordingly, the number of occurrences of a differentiator tag sequence may follow a Poisson distribution.

In some embodiments, overrepresentation of target:differentiator tag sequences in a pool of preparative nucleic acids (e.g., amplified MIP capture products) is indicative of bias in the preparative process (e.g., bias in the amplification process). For example, target:differentiator tag sequence combinations that are statistically overrepresented are indicative of bias in the protocol at one or more steps between the incorporation of the differentiator tag sequences into MIPs and the actual sequencing of the MIP capture products.

The number of reads of a given target:differentiator tag sequence may be indicative (may serve as a proxy) of the amount of that target sequence present in the originating sample. In some embodiments, the numbers of occurrence of sequences in the originating sample is the quantity of interest. For example, using the methods disclosed herein, the occurrence of differentiator tag sequences in a pool of MIPs may be predetermined (e.g., may be the same for all differentiator tag sequences). Accordingly, changes in the occurrence of differentiator tag sequences after amplification and sequencing may be indicative of bias in the protocol. Bias may be corrected to provide an accurate representation of the composition of the original MIP pool, e.g., for diagnostic purposes. According to some aspects, a library of preparative nucleic acid molecules (e.g., MIPs, each nucleic acid in the library having a unique differentiator tag sequence, may be constructed such that the number of nucleic acid molecules in the library is significantly larger than the number prospective target nucleic acid molecules to be captured using the library. This ensures that products of the preparative methods include only unique target:differentiator tag sequence; e.g., in a MIP reaction the capture step would undersample the total population of unique differentiator tag sequences in the MIP library. For example, an experiment utilizing 1 μg of genomic DNA will contain about ^"150,000 copies of a diploid genome. For a MIP library, each MIP in the library comprising a randomly produced 12-mer differentiator tag sequence (^~1.6 million possible unique differentiator tag sequences), there would be more than 100 unique differentiator tag sequences per genomic copy. For a MIP library, each MIP in the library comprising a randomly produced 15-mer differentiator tag sequence (^~1 billion possible unique differentiator tag sequences), there would be more than 7000 unique differentiator tag sequences per genomic copy. Therefore, the probability of the same differentiator tag sequence being incorporated multiple times is incredibly small. Thus, it is to be appreciated that the length of the differentiator tag sequence is to be selected based on the amount of target sequence in a MIP capture reaction and the desired probability for having multiple, independent occurrences of target:differentiator tag sequence combinations.

FIG. 5 depicts a non-limiting method for genotyping based on target and differentiator tag sequences. Sequencing reads of target and differentiator tags sequences are collapsed to make diploid genotype calls. FIG. 6 depicts non-limiting results of a simulation of a MIP capture reaction in which MIP probes, each having a differentiator tag sequence of 15 nucleotides, are combined with 10000 target sequence copies (e.g., genome equivalents). In this simulated reaction, the probability of capturing one or more copies of a target sequence having the same differentiator tag sequence is 0.05. The Y axis reflects the number of observations. The X axis reflects the number of independent occurrences of target:differentiator tag combinations. FIG. 7 depicts a non-limiting graph of sequencing coverage, which can help ensure that alleles are sampled to sufficient depth (e.g., either lOx or 20x minimum sampling per allele, assuming 1000 targets). In this non-limiting example, the X axis is total per-target coverage required, and the Y axis is the probability that a given total coverage will result in at least lOx or 20x coverage for each allele. The skilled artisan will appreciate that as part of a MIP library preparation process, adapters may be ligated onto the ends of the molecules of interest. Adapters often contain PCR primer sites (for amplification or emulsion PCR) and/or sequencing primer sites. In addition, barcodes may be included, for example, to uniquely identify individual samples (e.g., patient samples) that may be mixed together. (See, e.g., USPTO Publication Number US 2007/0020640 Al (McCloskey et al.)

The actual incorporation of the random differentiator tag sequences can be performed through various methods known in the art. For example, nucleic acids comprising differentiator tag sequences may be incorporated by ligation. This is a flexible method, because molecules having differentiator tag sequence can be ligated to any blunt-ended nucleic acids. The sequencing primers must be incorporated subsequently such that they sequence both the differentiator tag sequence and the target sequence. Alternatively, the sequencing adaptors can be synthesized with the random differentiator tag sequences at their 3' end (as degenerate bases), so that only one ligation must be performed. Another method is to incorporate the differentiator tag sequence into a PCR primer, such that the primer structure is arranged with the common adaptor sequence followed by the random differentiator tag sequence followed by the PCR priming sequence (in 5' to 3' order). A differentiator tag sequence and adaptor sequence (which may contain the sequencing primer site) are incorporated as tags. Another method to incorporate the differentiator tag sequences is to synthesize them into a padlock probe prior to performing a gene capture reaction. The differentiator tag sequence is incorporated 3' to the targeting arm but 5' to the amplification primer that will be used downstream in the protocol. Another method to incorporate the differentiator tag sequences is as a tag on a gene- specific or poly-dT reverse- transcription primer. This allows the differentiator tag sequence to be incorporated directly at the cDNA level.

In some embodiments, at the incorporation step, the distribution of differentiator tag sequences can be assumed to be uniform. In this case, bias in any part of the protocol would change the uniformity of this distribution, which can be observed after sequencing. This allows the differentiator tag sequence to be used in any preparative process where the ultimate output is sequencing of many molecules in parallel.

Differentiator tag sequences may be incorporated into probes (e.g., MIPs) of a plurality when they are synthesized on-chip in parallel, such that degeneracy of the incorporated nucleotides is sufficient to ensure near-uniform distribution in the plurality of probes. It is to be appreciated that amplification of a pool of unique differentiator tag sequences may itself introduce bias in the initial pool. However, in most practical cases, the scale of synthesis (e.g., by column synthesis, chip based synthesis, etc.) is large enough that amplification of an initial pool of differentiator tag sequences is not necessary. By avoiding amplification or selection steps on the pool of unique differentiator tag sequences, potential bias may be minimized.

One example of the use of the differentiator tag sequences is in genome re-sequencing. Considering that the raw accuracy of most next-generation sequencing instruments is relatively low, it is crucial to oversample the genomic loci of interest. Furthermore, since there are two alleles at every locus, it is important to sample enough to ensure that both alleles have been observed a sufficient number of times to determine with a sufficient degree of statistical confidence whether the sample is homozygous or heterozygous. Indeed, the sequencing is performed to sample the composition of molecules in the originating sample. However, after multiple reads have been collected for a given locus, it is possible that due to bias (e.g., caused by PCR amplification steps), a large fraction of the reads are derived from a single originating molecule. This would skew the population of target sequences observed, and would affect the outcome of the genotype call. For example, it is possible that a locus that is heterozygous is called as homozygous, because there are only a few observations of the second allele out of many observations of that locus. However, if information is available on differentiator tag sequences, this situation could be averted, because the over-represented allele would be seen to also have an over-represented differentiator tag sequence (i.e., the sequences with the overrepresented differentiator tag sequence all originated from the same single molecule).

Therefore, the sequences and corresponding distribution of differentiator tag sequences can be used as an additional input to the genotype-calling algorithm to significantly improve the accuracy and confidence of the genotype calls.

In some aspects, the disclosure provides methods for analyzing a plurality of to target sequences which are genetic loci or portions of genetic loci (e.g., a genetic locus of Table 1). The genetic loci may be analyzed by sequencing to obtain a genotype at one or more polymorphisms (e.g., SNPs). Exemplary polymorphisms are disclosed in Table 2. The skilled artisan will appreciate that other polymorphisms are known in the art and may be identified, for example, by querying the Entrez Single Nucleotide Polymorphism database, for example, by searching with a

GenelD from Table 1.

transmembrane ABCC7;

group C

carboxylase, beta

(autosomal recessive, DFNB18; PDZ- pl4

The mutations listed in Table 2 are documented polymorphisms in several disease-associated genes (CFTR is mutated in cystic fibrosis, GBA is mutated in Gaucher disease, ASPA is mutated in Canavan disease, HEXA is mutated in Tay Sachs disease). The polymorphisms are of several types: insertion/deletion polymorphisms which will cause frameshifts (and thus generally interrupt protein function) unless the insertion/deletion length is a multiple of 3 bp, and substitutions which can alter the amino acid sequence of the protein and in some cases cause complete inactivation by introduction of a stop codon.

GCCTGGCCTGATGGGACATATTTT

CFTR 1080 rs62469433 CTAC A ATAT A AGT AT AGTATTGC A A A [ A/C] CC 12

ATCAGGAAGGGTGTTAACTATTT

CFTR 1080 rs61763210 GTTGTCTCCAAACTTTTTTTCAGGTG[-/AGA] 13

AGGTGGCCAACCGAGCTTCGGAAAG

CFTR 1080 rs61720488 TTTTTTC ATA A A AG ATTAT ATA A AGG [A/C] TA 14

TTGCTTTTGAATCACAAACACTA

CFTR 1080 rs61481156 ATCT AGTG AGC AGTC AGG A A AG AG A A [C/T] T 15

TCCAGATCCTGGAAATCAGGGTTA

CFTR 1080 rs61443875 TAGAGTATAAAAAAAAAAAAAAAAAA[-/A] 16

GTTTGAATGTTTTCTTGCATTCAGA

CFTR 1080 rs61312222 TGCAAATGCCAACTATCAAAGATATT[C/G]GA 17

GTATACTGTCAATAAACTTCATA

CFTR 1080 rs61159372 TCCTC A AC AGTTAG A A AC A AT ATTTT [C/G] AG 18

TGATTTCCCATGCCAACTTTACT

CFTR 1080 rs61094145 TTTTTGGTATTGTTGTTA A AT A AGTG [ A/G] G A 19

ATTCAATACAGTATAATGTCTGT

CFTR 1080 rs61086387 CTTGAAATCGGAT AT ATAT ATAT ATA[-/T 20

GT ATAT ATATATATATAT ATAT AT ATAT AT ACATATATATATATA]GTATTATCCCTGTTTTC ACAGTTTT

CFTR 1080 rs60996744 AG AGGGGCTGTG A AGG AC ACC A AGG A [A/G] G 21

AGACTAAGAGCCAGGAGGGAAAAC

CFTR 1080 rs60960860 TAG AGTTTATT AGCTTTT ACT ACTCT [A/G] CTT 22

AGTTACTTTGTGTTACAGAATA

CFTR 1080 rs60923902 ACTAGTGATGATGAGCTTCTTTTCAT[-/AT] 23

GTTTGTTGGCTGCATAAATGTCTTC

CFTR 1080 rs60912824 GC AG AG A A A AG AGGGGCTGTG A AGG A [C/G] A 24 CCAAGGAGGAGACTAAGAGCCAGG

CFTR 1080 rs60887846 TTC AG AGGTCTACC ACTGGTGC ATAC [G/T] CT 25

AATCACAGTGTCGAAAATTTTAC

CFTR 1080 rs60793174 AAGAAAGAGCAAAAGAGGGCAAACTT[C/T]T 26

CATACATTTTTGATGTCGAAACCA

CFTR 1080 rs60788575 CCTAAAGTTTAAAAAGAAAAAAAAAA[-/A] 27

GGAAGAAGGAATTAAAAATCCAAAG

CFTR 1080 rs60760741 GTGTGTGTGTGTATATATATATATAT[A/T]TA 28

TATATTTTTTTTTTCCTGAGCCA

CFTR 1080 rs60456599 AA ACTGTTG ATGTTTTC ATTT ATTTA [C/G] ATC 29

ATTGGAAAACTTTAGATTCTAG

CFTR 1080 rs60363249 TTT ATCC ATTCTT A ACC AG A AC AG AC [ A/G] TT 30

TTTTCAGAGCTGGTCCAGGAAAA

CFTR 1080 rs60355115 TTG A A ATCGG ATAT AT ATAT ATAT AT [A/G] T A 31

TATATATATATATATATATATAT

CFTR 1080 rs60308689 TAGTTTTTTATTTCCTCATATTATTT[-/T] 32

CAGTGGCTTTTTTCTTCCACATCTTT

CFTR 1080 rs60271242 AC ATAGTTCTC AGTGGTAC A ACT AC A [A/G] GT 33

GATTTCTCTTTTCTTATTTCTGG

CFTR 1080 rs60010318 AG AGC A ATGGC ATCCCTTGTCTTGTG [C/T] TA 34

TACAGGATGCAGCAATTTATAGG

CFTR 1080 rs59961323 TTCTGTCT AC AT A AG ATGTC ATACTA [A/G] AT 35

TATCTTTTCCAGCATGCATTCAG

CFTR 1080 rs59961270 C AGGGTGGC ATGTT AGGC AGTGCTT A [A/G] A 36

ATAAATGAGTTGGTTATACAAGTA

CFTR 1080 rs59837506 AGGACACACACACACACACACACACA[-/CA] 37

TGCACACACATTTAAATAGATGCAT CFTR 1080 rs59572090 TA A A A A ATTGGT AT A ATG A A ATTGC A [C/T] TT 38

GTAGTCTTTGGACATTTAAATCC

CFTR 1080 rs59548252 TTTC A AT ACTTA AG AGGTACGC AG AG [ A/G] A 39

AAGAGGGGCTGTGAAGGACACCAA

CFTR 1080 rs59519859 C AGC A ATG A ATATTTTG AGGCTG AGG [C/T] GC 40

TGAGGGGTAAAATTGCAGCCTGG

CFTR 1080 rs59509837 TTATGGTTTATATTTTGTGTCTTCT[-/C 41

TTT] A AC AC ATCTTTTCT AGC AG A ATTC A

CFTR 1080 rs59417037 GTATTTTAGTTTTTTTTTTTTGTTTG[-/T] 42

TTTGTTTTGTTTTGTTTTGTTTTTG

CFTR 1080 rs59159458 TGGGTGACTCCATTTTTACTTTTAGT[C/T]TGG 43

TCTGTTGAGGCCTCGTGAGAGA

CFTR 1080 rs59048119 TATTTTCATGTATTTTAGTTTTTTTTT[-/T 44

TTT] GTTTGTTTTGTTTTGTTTTGTTTTG

CFTR 1080 rs58970500 GTGTGTGTGTATATATATATATATAT[A/T]TA 45

TATTTTTTTTTTCCTGAGCCAAA

CFTR 1080 rs58942292 AACCTATTAGCATGTCTGGCAGAAAA[-/A] 46

TAGATACTTAATAAATTTCTTAAAT

CFTR 1080 rs58917054 GAGGCTT AG AC AGTTT A AGTA ACTC A [A/G] G 47

CATGGTTACACAACTAGCTAGGGC

CFTR 1080 rs58837484 GTGTG AGT ATTATG AG ACC ATATGTT [A/G] GG 48

AGATTTTATTTGGTATTGAGGAT

CFTR 1080 rs58829491 GA A ACCCC ACCCCTTCT ATAGTTTTC [C/T] CTT 49

TAATATTTACAATGGAACCATT

CFTR 1080 rs58805195 CATATATATATAGTGTGTGTGTGTGT[A/G]TA 50

TATATATATATATATATATTTTT

GBA 2629 rs60866785 CGAGCGAGAGAGAGAGAGAGAGAGAG[-/AG] 51 GAGCCGGCGCGAGAACTACGCATGC

GBA 2629 rs60239603 GGCAGGTAATATCTAGTACCTTACTT[A/T]TA 52

TTTCCTGAGCACATTCTACATTT

GBA 2629 rs56310840 GGCC AGG A ATGGG AGTGCTT AGGTGC [ A/G] G 53

AGGTGGCACTGTTCCCGCAGCTGC

GBA 2629 rs41264927 GA A A ACTCC ATCCCCTC AGGGTC AT [C/T] AG 54

ATGAAGAGAAGACCACAGGGGTT

GBA 2629 rs41264925 TGTAGGT A AGGGTC AC ATGTGGG AG A [C/G] G 55

CAGCTGTGGGTAGGTCAGCCCTGT

GBA 2629 rs36024691 CCAAGAAGGCGCCATTACACTCCAGC[-/C] 56

TGGGCGACAGGGCGAGACTCCCTCA

GBA 2629 rs36024092 TGCCACACCCAGCTAATTTGTGTGTG[-/G] 57

TATGTGTGTGTATGTATGTGTGTGT

GBA 2629 rs35682967 GTTCCTCCAGTAATTTTTTTTTTTTT[-/T] 58

GGTTTTGAGACAGAGTCTTGCCCTG

GBA 2629 rs35033592 ATCATGCCCAGATAATTTTTTTTTTT[-/T] 59

GTATTTTAGTAGACACAGGGTTTCA

GBA 2629 rs34732744 CGAGCGAGAGAGAGAGAGAGAGAGAG[-/AG] 60

GAGCCGGCGCGAGAACTACGCATGC

GBA 2629 rs34620635 CCTGTGAGGGGCACATTCCTTAGTAG[-/C] 61

TAAGGAGTTGGGGGTGTGAAGATCC

GBA 2629 rs34302637 ACAGGCTACTGGCTGGGCCCAGGCAA[-/A] 62

GGGGGCCTTGGCAGGAAAAGTTCCT

GBA 2629 rs33949225 GCGAGAGAGAGAGAGAGAGAGAGAGG[-/AG] 63

AGCCGGCGCGAGAACTACGCATGCG

GBA 2629 rs28678003 AAG A AG A A A A AT A A A A AG A A AGTGGG [C/T] C 64

AGACCGAGAGAACAGGAAGCCTGA GBA 2629 rs28559737 AAGG AC A A AGGC A A AG AG AC A A AGGC [G/T] C 65

AACACTGGGGGTCCCCAGAGAGTG

GBA 2629 rs28373017 TACCTAGTC ACTTCCTGCCTCC ATGG [C/T] GC 66

AAAAGGGGATGGGTGTGCCTCTT

GBA 2629 rsl2752133 CTCTTCCG AGGTTCC ACCCTG A AC AC [C/T] TT 67

CCTGCTCCCTCGTGGTGTAGAGT

GBA 2629 rs 12747811 TTCTGACTGGCAACCAGCCCCACTCT[C/T]TG 68

GGAGCCCTCAGGAATGAACTTGC

GBA 2629 rs 12743554 gctcagcctcccaggctggagtgcag[A/T]ggcgcgatc 69 tcggctcaccgcaacc

GBA 2629 rsl2041778 C ATG A ACC AC ATC A A ATG AG ATTTAG [C/T] GG 70

GAGTGGCACACACAGTCATGACC

GBA 2629 rs 12034326 AAGC AGCCCTGGGG AGTCGGGGCGGG [ A/G] C 71

CTGGATTGGAAAAGAGACGGTCAC

GBA 2629 rsl l558184 CTCC A AGTTCTGGG AGC AG AGTGTGC [A/G] G 72

CTAGGCTCCTGGGATCGAGGGATG

GBA 2629 rs 11430678 GTTCCTCCAGTAAttttttttttttt[-/G/T] 73 gttttgagacagagtcttgccctgt

GBA 2629 rs 11264345 CTAGT ACCTT ACTTCCCTC A AGTTC A [ A/T] TC 74

ATCTCACAGATATTTCCTGAGCA

GBA 2629 rs 10908459 aattagccgtgcgtggtggcgggtgc[C/T]tgtaatccc 75 acgtacttgggaggct

GBA 2629 rs 10796940 CC ATGGCC AGCCGGGG AGGGG ACGGG [ A/C] A 76

CACACAGACCCACACAGAGACTCA

GBA 2629 rs 10668496 agcgagagagagagagagagagagag[-/AG] 77 gagCCGGCGCGAGAACTACGCATGC

GBA 2629 rs7416991 CGTAGCAGTTAGCAGATGATAGGCGG[C/G/T] 78 GAAATCTTATTTCACAGGGCATTAA

GBA 2629 rs4024049 CTGGCCCTGGTG AC AGTGGGGCTGTG [C/T] GT 79

GGGGCCAGAGCCTTCTCAGAGGT

GBA 2629 rs4024048 C AG ATACTGGCCCTGGTG AC AGTGGG [ A/G] C 80

TGTGCGTGGGGCCAGAGCCTTCTC

GBA 2629 rs4024047 G AC AG ATACTGGCCCTGGTG AC AGTG [G/T] G 81

GCTGTGCGTGGGGCCAGAGCCTTC

GBA 2629 rs3841430 GGCTCctctctctctctctctctctc[-/TC] 82 gctc gctctctc gctctctcgctct

GBA 2629 rs3754485 GTTTC AG ACC AGCCTGGCC A AC AT AG [C/T] G A 83

AACCCCATCTCTACTAAAAATAA

GBA 2629 rs3205619 AGTGGGCG ATTGG ATGG AGCTG AGT A [C/T] G 84

GGGCCCATCCAGGCTAATCACACC

GBA 2629 rs2990227 CCGGGCTCCGTG A ATGTTTGTC AC AT [C/G] TC 85

TGAAGAACGTATGAATTACATAA

GBA 2629 rs2990226 GAATCCCAACCCCGACGCTCGTCGCC[C/G]G 86

GCTCCGTGAATGTTTGTCACATGT

GBA 2629 rs2990225 GCGAATCCCAACCCCGACGCTCGTCG[C/T]CG 87

GGCTCCGTGAATGTTTGTCACAT

GBA 2629 rs2990224 TGGGC AG A AGTC AGGGTCC A A AG A A A [G/T] G 88

GCAAAGAAAAGTGTcagtggctca

ASPA 443 rs63751297 TA AG A A AG ACGTTTTTG ATTTTTTTC [A/G] G A 89

CTTCTCTGGCTCCACTACCCTGC

ASPA 443 rs62071301 CTG ATTCCTGGCC AGG AGCGGTGGCT [C/T] AC 90

GCCTGTAATCCCAGCGCTTTGGG

ASPA 443 rs62071300 TA AAA ATGCTG ATTCCTGGCC AGG AG [C/T] GG 91

TGGCTCACGCCTGTAATCCCAGC

ASPA 443 rs55859596 CGGGGCTC AG A ACTTGT A AC AG A A A A [ A/T] T 105

AAAATATACTCCACTCAAGGGAAT

ASPA 443 rs55742972 TACTACACTTCACGGATACTGTACTT[-/G 106

TACTT] TTTTTCC A A ATTG A AGGTTTTTGGC

ASPA 443 rs55640436 TTGTTTTTGTTTTTGTTTTTGTTTTT[-/G 107

TTTTTGTTTTT] TG AG ATGG AGTCTCGCTCT

GTCGCC

ASPA 443 rs36225687 TTTGCCTTACTACACTTCACGGATAC [-/T 108

GT ACT] TGT ACTTTTTTTCC A A ATTG A AGGT

ASPA 443 rs36051310 GAGGTGGCTGAAAG AAGCACGTATCC [-/C] 109

TGATGGCATGGTTGCGGGTTATATG

ASPA 443 rs36034906 GAGAAA AGCAGTTCCTGGAAC ACCCC [-/C] 110

ACCCCTTAACCCCTTATCTCTGCTT

ASPA 443 rs36033666 TTACATATGTATACATGTGCCATGTT[-/T] 111

GGTGTGCCGCACCCATTAACTCGTC

ASPA 443 rs35730123 CTTTTTCCAGATTTTTTTTTTTTTTT[-/T] 112

GAGACAGAGTTTCACTCTTGTTGCC

ASPA 443 rs35629100 TTTGGAAATCTTAAGCTTTTATTTGG[-/G] 113

TGTCACAGAGAAACAGGATCTGTAT

ASPA 443 rs35614631 TACTTTAAGTTTTAGGGTACATGTGC[-/A] 114

CCATGTGCAGGTTTGTTACATATGT

ASPA 443 rs35225782 ATTC ATGACCAGCCAC ATAAATGCAC [-/A] 115

GTATTACTTCGCAAGCATGCCAATG

ASPA 443 rs35178659 GTGCACTAGAATTAGCTAAAGTGGGG[-/G] 116

AAAAAAAGATGCATTTGATGGTCTA

ASPA 443 rs35095578 AACCTCCACCTCCCAGGTTCAAGAGA[-/A] 117

TTCTCCTGCCTCAGCCTCCCAAGTA ASPA 443 rs35002210 CCTCCCTGTG ATCCGA AGT AGC AG AC [ A/G] TA 118

CTTAACTTCCATGGTGGATTGTT

ASPA 443 rs34744839 AAAACATTATTATATCTAGAAAAAAA[-/A] 119

TGTATCTTAACCATTGTGGGAAGTG

ASPA 443 rs34680506 TTGAAGGTAAAATCATAGGGAGTTGG[-/G] 120

AGCTGTCCTCTTGCGCTGAATCAGT

ASPA 443 rs34365618 ACTTGTGGCCTTTTTGGAGAGGTTAG[-/CA] 121

ACTCTGAAAACTCTGTCCCTGGACC

ASPA 443 rs34275920 GAAGGAGAAAAAGAGAGGAAATAAGT[-/T] 122

AAAATAATAAACACAATTAATAAAG

ASPA 443 rs34109510 TGTAT AC ATGTGCC ATGTTGGTGTGC [C/T] GC 123

ACCCATTAACTCGTCATTTAGCA

ASPA 443 rs34054576 TCACCTGTCACCTCCTATAGAACTTT[-/C] 124

CCCTGACCCTCCTCTATAGCATTAA

ASPA 443 rs34015272 ATAAATGATCATCATTCACAGTAGGG[-/G] 125

TTTTGTTTTGTTTTTTTTCTGGAAA

ASPA 443 rs34002091 ACAGACATATCTACAAACACACTTTT[-/T] 126

CACATATTTGTGTAAGTCATTTATG

ASPA 443 rs28940574 AA AG AC A ACT A A ACTA ACGCTC A ATG [ A/C] A 127

AAAAGTATTCGCTGCTGTTTACAT

ASPA 443 rs28940279 TACCGTGT ACCCCGTGTTTGTG A ATG [ A/C] GG 128

CCGCATATTACGAAAAGAAAGAA

ASPA 443 rs 17850703 C AGGGCTGG AGGT A A A ACC ATTT ATT [A/G] CT 129

AACCCCAGAGCAGTGAAGAAGTG

ASPA 443 rs 17222495 TTCTTC ATTGCCT ATTG A AG AG AG AG [C/T] GG 130

AATGCTTTGGTTGCCAGATATGG

ASPA 443 rs 17175228 C AC A AG ATCTC ATTACTC AGG AGCTG [C/T] CC 131 AAGTGTCTAATGTACTTAGTTAA

ASPA 443 rs 16953074 TTCTGTGTA AC ATTTC ATTT A AGC A A [ A/G] GG 132

ATTCGGCAAATCAAAAATTGTCA

ASPA 443 rs 16953070 TA A A ACGT ATTG A AGGTATTATTG AC [G/T] CT 133

GTTGAAGCAAAGAGAACAAAACA

HEXA 3073 rs62022858 ATCTGCTCTTCC AGTTGG ATG AC A AG [C/T] CT 134

TGCTGTCTAACACCTGCTGCAGA

HEXA 3073 rs62022857 CC ATTTTTTGTTGT ATTTTTTTTTTC [C/T] TG A A 135

TACTTTTTATCGCAGTTGGTT

HEXA 3073 rs62017872 CCCTGTCTCTAAAAGAAAAAAAAAAA[A/G]A 136

AAAAAAAAAGAAAACAAAACCCAA

HEXA 3073 rs62017871 AGTGGCTCC A A A A AGGTC ATGG A ACC [C/T] CT 137

TGAGGATGATGCAAATTGACTCT

HEXA 3073 rs61662730 TA A AGTT ACTTTTCTTTTATTG ACTT [C/T] CCC 138

TTATTTTTTAACCTTATGCTTT

HEXA 3073 rs61329913 CAGAGTTAAAAAAAAAAAAAAAAAAA[-/A] 139

GGAAGTAGCAGCAACAGCTTGGAAA

HEXA 3073 rs60920713 GTTGCCC AGGGTTG AGTGC AG AGGC A [C/T] AT 140

TTGGCTCACAGCAACCTCTGCC

HEXA 3073 rs60783213 AAGGCTTTTTTTTTTTTTTTTTTTTT[-/T 141

TTT] G AG AC AG AGTCTTGCTGTGTC ACCC

HEXA 3073 rs60644867 GCCT AC ATTCTGC A A AG AGG AGGG A A [C/G] A 142

TTCACAGCTCCATACTTGAACCCT

HEXA 3073 rs60288568 CC A A AGG AG A AT AGCTCT AGGGG AGG [C/G] A 143

GGTGGATGAGTATGCATGGGGGAG

HEXA 3073 rs59888548 GACTCCATCTCAAAAAAAAAAAAAAA[-/A] 144

TGCAGTCTAATGGCAGAATTAGACT HEXA 3073 rs59733856 TTATTT ATTTATTTATTTATTTTTG A [ A/G] AC A 145

GGGTCTCTGTTGTCCAGGCTGG

HEXA 3073 rs59427837 TTTTG AGGC AGGGTCTC ACTCTGTTG [C/T] CC 146

AGGGTTGAGTGCAGAGGCACATC

HEXA 3073 rs59171976 CGCCTTGCG A AGGCCCC AC AGCTTGC [C/T] TG 147

TGACAAACGTTCATAGGCAAATG

HEXA 3073 rs58706602 GG AGGTCTGT AC A A AGC ACC ACCT AC [C/T] TC 148

ATGGGTCAGTTTCCACAGCAGAA

HEXA 3073 rs58696963 GAATCTTATAATTC ACTGTGTACCTC [-/C 149

CTC] TGTTTCATATTTTCGC AATTGAACT

HEXA 3073 rs58610850 AAC ATAGT ATCTA AT ATAGCTTTAC A [C/T] CC 150

AAAGCCAAAATATGAATACACTG

HEXA 3073 rs58016062 TTGTTTTGTTTTGTTTGGGGGGGGGG[-/G] 151

TTGTTTTTCTGAGAGGGAGTCTTGC

HEXA 3073 rs57733983 C ATACC A A AGGGC AGCTGG AGGG ATAC [C/T] A 152

GACGGAAGTCATGTGGAGAGTGAA

HEXA 3073 rs57476645 C AGGTGTG AGCC ACC ACG ACC ACC A A [ A/T] T 153

TAGCTCTTTTTACTCCTTCCCTTC

HEXA 3073 rs56870003 AGTGGTAGCTGATTTTGCTTCTGGAT[A/C]CT 154

TTGCCACCTTCCCACTCTTTAAT

HEXA 3073 rs56338339 AAAGACCTGTTTCTTAAAAAAAAAAA[-/A 155

GA A A A A A A A A A A] G A A AG A A A AG A A A AG

AAAAAAACAG

HEXA 3073 rs55995352 TA A A A A ATCTTTC A ATG AGG AG ATGT [C/T] CC 156

CAGAGCAAGACAGCTGTAGGATG

HEXA 3073 rs55860138 AAAAGAAAAAAAAAAAAAAAAAAAAA[-/A] 157

GAAAACAAAACCCAAACCCATAAAG HEXA 3073 rs55743646 CCTGTCTCTAAAAGAAAAAAAAAAAA[A/G]A 158

AAAAAAAAGAAAACAAAACCCAAA

HEXA 3073 rs55665666 GTTATCATAGAAAAATATCACACTCT[-/GT] 159

CTGTATCCCCACTTCCAGAAACTGT

HEXA 3073 rs36106892 CAGGAGCTCATAGAATTACATACAAT[-/C] 160

TTTTTTTTTTTTTTTTGAGACAGCG

HEXA 3073 rs36091525 TTGAGAATCTTATAATTCACTGTGTA[-/C 161

CTC] CCTCTGTTTC ATATTTTCGCAATTG

HEXA 3073 rs35949555 CC ACTACC AC AGTGCCTAG AG A AC A A [C/T] A 162

TGTGTTTAATAATATTTAAATAAT

HEXA 3073 rs35827424 CCCTGTCTCTAAAAGAAAAAAAAAAA[-/A] 163

AAAAAAAAAAGAAAACAAAACCCAA

HEXA 3073 rs35729578 CCATTATATCATTCATTTCCCACTCA[-/T] 164

TTTCTTCATTCCAACCAAGATATAT

HEXA 3073 rs35649102 TCCGTCTCAAAAAAAAAAAAAAAAAG[-/A] 165

GAAAGGAATTATTCTCATGTATACA

HEXA 3073 rs35118677 CTGGGGCAGTTAAAAAGAAAAACAAA[-/C] 166

CCCTGGTCCCTGCCCTTGAGGAGAT

HEXA 3073 rs35005352 CTCCAGGGTCCCATTCCAGGACCACA[-/C] 167

GCCTGCTACCTCTGCAGCTCACTCA

HEXA 3073 rs34736306 GGATTGACATATACCAGTTAGACGGA[-/T] 168

TTTTTTTTTCCATAAACCAGGCTCA

HEXA 3073 rs34607939 AC A A ATA ATT ACT AC AT ATCTAC A AC [ A/G] TT 169

CCAGATACAGAAGAAATGGCCAA

HEXA 3073 rs34496117 TAAACACACTTGAAACATCATATAAA[-/A 170

TG] ATATTACTAC A AG ACTT A ACCGTA A

HEXA 3073 rs34300017 ACACAGGTAATCCATGTTTATTATAG[-/A] 171 AAAATGCCACATTACTCTTTATTGA

HEXA 3073 rs34206496 AGTTATCATAGAAAAATATCACACTC[-/TG] 172

TCTGTATCCCCACTTCCAGAAACTG

HEXA 3073 rs34110830 AATGAACTTACAGGAAGGTAATATAT[-/G] 173

GGAAATAAACATCTTATTGAATTTA

HEXA 3073 rs34093438 GGACCCCTGAAAGGCACAAGACACCC[-/T] 174

TTCAGGTTCACACTTCCTGAAAGCT

HEXA 3073 rs34085965 CC ACC A ATC ACC AG AGCCTTCTGCTC [ A/G] GG 175

GGTACCTGAGGGAAAACAAGCAA

HEXA 3073 rs34004907 AAAGACTGAAAAAACATTCATAACTA[-/T] 176

TTTTCTTGTTATCCTCGGAAATGTC

HEXA 3073 rs28942072 TATCTTCATCTTGGAGGAGATGAGGT[C/T]GA 177

TTTCACCTGCTGGAAGTCCAACC

HEXA 3073 rs28942071 TTGCCT ATG A ACGTTTGTC AC ACTTC [C/T] GCT 178

GTGAGTTGCTGAGGCGAGGTGT

HEXA 3073 rs28941771 GCTTGCTGTTGG ATAC ATCTCGCC AT [C/T] AC 179

CTGCCACTCTCTAGCATCCTGGA

HEXA 3073 rs28941770 CCGGGGCTTGCTGTTGG AT AC ATCTC [G/T] CC 180

ATTACCTGCCACTCTCTAGCATC

3. Nucleic Acid Target Length Evaluation:

In some embodiments, aspects of the invention relate to methods for detecting nucleic acid deletions or insertions in regions containing nucleic acid sequence repeats.

Genomic regions that contain nucleic acid sequence repeats are often the site of genetic instability due to the amplification or contraction of the number of sequence repeats (e.g., the insertion or deletion of one or more units of the repeated sequence). Instability in the length of genomic regions that contain high numbers of repeat sequences has been associated with a number of hereditary and non hereditary diseases and conditions. For example, "Fragile X syndrome, or Martin-Bell syndrome, is a genetic syndrome which results in a spectrum of characteristic physical, intellectual, emotional and behavioral features which range from severe to mild in manifestation. The syndrome is associated with the expansion of a single trinucleotide gene sequence (CGG) on the X chromosome, and results in a failure to express the FMR-1 protein which is required for normal neural development. There are four generally accepted forms of Fragile X syndrome which relate to the length of the repeated CGG sequence; Normal (29-31 CGG repeats) (SEQ ID NO: 6375), Premutation (55-200 CGG repeats) (SEQ ID NO: 6376), Full Mutation (more than 200 CGG repeats) (SEQ ID NO: 6377), and Intermediate or Gray Zone Alleles (40-60 repeats) (SEQ ID NO: 6378)."

Other examples include cancer, which has been associated with microsatellite instability

(MSI) involving an increase or decrease in the genomic copy number of nucleic acid repeats at one or more microsatellite loci (e.g., BAT-25 and/or BAT-26). There are currently many sequencing-based assays for determining the number of nucleic acid sequence repeats at a particular locus and identifying the presence of nucleic acid insertions or deletions. However, such techniques are not useful in a high throughput multiplex analysis where the entire length of a region may not be sequenced.

In contrast, in some embodiments, aspects of the invention relate to detecting the presence of an insertion or deletion at a genomic locus without requiring the locus to be sequenced (or without requiring the entire locus to be sequenced). Aspects of the invention are particularly useful for detecting an insertion or deletion in a nucleic acid region that contains high levels of sequence repeats. The presence of sequence repeats at a genetic locus is often associated with relatively high levels of polymorphism in a population due to insertions or deletions of one or more of the sequence repeats at the locus. The polymorphisms can be associated with diseases or predisposition to diseases (e.g., certain polymorphic alleles are recessive alleles associated with a disease or condition). However, the presence of sequence repeats often complicates the analysis of a genetic locus and increases the risk of errors when using sequencing techniques to determine the precise sequence and number of repeats at that locus.

In some embodiments, aspects of the invention relate to determining the size of a genetic locus by evaluating the capture frequency of a portion of that locus suspected of containing an insertion or deletion (e.g., due to the presence of sequence repeats) using a nucleic acid capture technique (e.g., a nucleic acid sequence capture technique based on molecular inversion probe technology). According to aspects of the invention, a statistically significant difference in capture efficiency for a genetic locus of interest in different biological samples (e.g., from different subjects) is indicative of different relative lengths in those samples. It should be appreciated that the length differences may be at one or both alleles of the genetic locus. Accordingly, aspects of the invention may be used to identify polymorphisms regardless of whether biological samples being interrogated at heterozygous or homozygous for the polymorphisms. According to aspects of the invention, subjects that contain one or more loci with an insertion or deletion can be identified by analyzing capture efficiencies for nucleic acids obtained from one or more biological samples using appropriate controls (e.g., capture efficiencies for known nucleic acid sizes, capture efficiencies for other regions that are not suspected of containing an insertion or deletion in the biological sample(s), or predetermined reference capture efficiencies, or any combination thereof. However, it should be appreciated that aspects of the invention are not limited by the nature or presence of the control. In some embodiments, if a statistically significant variation in capture efficiency is detected, a subject may be identified as being at risk for a disease or condition associated with insertions or deletions at that genetic locus. In some embodiments, the subject may be analyzed in greater detail in order to determine the precise nature of the insertion or deletion and whether the subject is heterozygous or homozygous for one or more insertions or deletions. For example, gel electrophoresis of an amplification (e.g., PCR) product of the locus, or Southern blotting, or any combination thereof can be used as an orthogonal approach to verify the length of the locus. In some embodiments, a more exhaustive and detailed sequence analysis of the locus can be performed to identify the number and types of insertions and deletions. However, other techniques may be used to further analyze a locus identified as having an abnormal length according to aspects of the invention.

Accordingly, aspects of the invention relate to detecting abnormal nucleic acid lengths in genomic regions of interest. In some embodiments, the invention aims to estimate the size of genomic regions that are hard to be accessed, such as repetitive elements. However, it should be appreciated that methods of the invention do not require that the precise length be estimated. In some embodiments, it is sufficient to determine that one or more alleles with abnormal lengths are present at a locus of interest (e.g., based on the detection of abnormal capture efficiencies). In a non-limiting example, fragile X can be used to illustrate aspects of the invention where the size of trinucleotide repeats (genotype) is linked to a symptom (phenotype). However, it should be appreciated that fragile X is a non-limiting example and similar analyses may be performed for other genetic loci (e.g., independently or simultaneously in multiplex analyses).

Use of molecular inversion probes (MIPs) has been demonstrated for detection of single nucleotide polymorphisms (Hardenbol et al. 2005 Genome Res 15:269-75) and for preparative amplification of large sets of exons (Porreca et al. 2007 Nat Methods 4:931-6, Krishnakumar et al. 2008 Proc Natl Acad Sci USA 105:9296-301). In both cases, oligonucleotide probes are designed which have ends ('targeting arms') that hybridize up-stream and down-stream of the locus that is to be amplified. In some embodiments, aspects of the invention are based on the recognition that the effect of length on probe capturing efficiency can be used in the context of an assay (e.g., a high throughput and/or multiplex assay) to allow the length of sequences to be determined without requiring sequencing of the entire region being evaluated. This is particularly useful for repeat regions that are prone to changes in size. As illustrated in FIG. 8, which is reproduced from Deng et al., Nature Biotech. 27:353-60, (see Supplemental FIG. 1G of Deng et al.,) illustrates that shorter sequences are captured with higher efficiency that longer sequences using MIPs. The statistical package R and its effects module were used for this analysis. A linear model was used, and each individual factor was assumed to be independent. The dashed lines represent a 95% confidence interval. Shorter target sequences were captured with higher efficiency than long target sequences (p<2xl0^~16). However, the use of this differential capture efficiency for systematic sequence length analysis was not previously recognized.

In some embodiments, following probe hybridization, polymerase fill-in and ligation reactions are performed to convert the hybridized probe to a covalently-closed, circular molecule containing the desired target. PCR or rolling circle amplification plus exonuclease digestion of non-circularized material is performed to isolate and amplify the circular targets from the starting nucleic acid pool. Since one of the main benefits of the method is the potential for a high degree of multiplexing, generally thousands of targets are captured in a single reaction containing thousands of probes.

According to aspects of the invention, repetitive regions are surrounded by non-repetitive unique sequences, which can be used to amplify the repeat-containing regions using, for example, PCR or padlock (MlP)-based method. In addition to the repetitive regions, a probe (e.g., a MIP or padlock probe) can be designed to include at least a sequence that is sufficient to be uniquely identified in the genome (or target pool). After the probe is circularized and amplified, the amplicon can be end- sequenced so that the unique sequence can be identified and served as the "representative" of the repetitive region as illustrated in FIG. 9. FIG. 9 illustrates a non-limiting scheme of padlock

(MIP) capture of a region that includes both repetitive regions (thick wavy line) and the adjacent unique sequence (thick strait line). The regions of the probe are indicated with the targeting arms shown as regions "1" and "3." An intervening region that may be, or include, a sequencing primer binding site is shown as "2." After the padlock is circularized and amplified, it can be end-sequenced to obtain the sequence of the unique sequence, which represents the repetitive region of interest. Although capturing efficiency is overall negatively correlated with target length, different probe sequences may have unique features. Therefore, multiple probes could be designed and tested so that an optimal one is chosen to be sensitive enough to differentiate repetitive sizes of roughly 0-150 bp, 150-600 bp, and beyond, which represent normal, premutation and full mutation of fragile X syndrome, respectively. However, it should be appreciated that other probe sizes and sequences can be designed, and optionally optimized, to distinguish a range of repeat region size differences (e.g., length differences of about 3-30 bases, about 30-60 bases, about 60-90 bases, about 90-120 bases, about 120-150 bases, about 150-300 bases, about 300-600 bases, about 600-900 bases, or any intermediate or longer length difference). It should be appreciated that a length difference may be an increase in size or a decrease in size.

In some embodiments, an initial determination of an unexpected capture frequency is indicative of the presence of size difference. In some embodiments, an increase in capture frequency is indicative of a deletion. In some embodiments, a decrease in capture frequency is indicative of an insertion. However, it should be appreciated that depending on specific sequence parameters and the relative sizes of the capture probes, the target region, and the deletions or insertions, a change in capture frequency can be associated with either an increase or decrease in target region length. In some embodiments, the precise nature of the change can be determined using one or more additional techniques as described herein.

Accordingly, in some aspects a MIP probe includes a linear nucleic acid strand that contains two hybridization sequences or targeting arms, one at each end of the linear probe, wherein each of the hybridization sequences is complementary to a separate sequence on a the same strand of a target nucleic acid, and wherein these sequences on the target nucleic acid flank the two ends of the target nucleic acid sequence of interest. It should be appreciated that upon hybridization, the two ends of the probe are inverted with respect to each other in the sense that both 5' and 3' ends of the probe hybridize to the same strand to separate regions flanking the target region (as illustrated in FIG. 9 for example).

In some embodiments, the hybridization sequences are between about 10-100 nucleotides long, for example between about 10-30, about 30-60, about 60-90, or about 20, about 30, about 40, or about 50 nucleotides long. However, other lengths may be used depending on the application. In some embodiments, the hybridization Tms of both targeting arms of a probe are designed or selected to be similar. In some embodiments, the hybridization Tms of the targeting arms of a plurality of probes designed to capture different target regions are selected or designed to be similar so that they can be used together in a multiplex reaction. Accordingly, a typical size of a MIP probe prior to fill-in is about 60-80 nucleotides long. However, other sizes can be used depending on the sizes of the targeting arms and any other sequences (e.g., primer binding or tag sequences) that are present in the MIP probe. In some embodiments, MIP probes are designed to avoid sequence-dependent secondary structures. In some embodiments, MIP probes are designed such that the targeting arms do not overlap with known polymorphic regions. In some embodiments, targeting arms that can be used for capturing the repeat region of the Fragile X locus can have the following sequences or complementary to these sequences depending on the strand that is captured.

left: CTCCGTTTCGGTTTCACTTC (SEQ ID NO: 181)

right: ATCTTCTCTTCAGCCCTGCT (SEQ ID NO: 182)

The typical captured size using these targeting arms is about 100 nucleotides in length (e.g., about 30 repeats of a tri-nucleotide repeat).

In some embodiments, the number of reads obtained for the "representative" of the repetitive region is not informative to estimate the target length because it is dependent on the total number of reads obtained. To overcome this, it is useful to include one or more probes that target other "control" regions where no or minimal polymorphism exists among populations. Because of the systematic consistency of capturing efficiency (see, e.g., FIG. 9), the ratio of reads obtained for the repetitive "representative" to reads obtained for the control region(s) will be tuned using DNA with defined numbers of repeats. Ultimately, the ratio can serve as a measure of the repeat length as illustrated in FIG. 10. FIG. 10 illustrates a non-limiting hypothetical relationship between target gap size and the relative number of reads of the repetitive region, which is measured by the ratio of the repeat "representative" reads vs. the "control" region reads. The unit of y-axis is arbitrary.

In some embodiments, to better tell targets with similar size range apart, the whole repetitive region can be sequenced by making a shotgun library (e.g., by making a shotgun library from a captured sequence, for example a sequence captured using a MIP probe). The longer the repeat is, the more short reads of repeats will be obtained. Therefore, the target length will contribute twice to the relative number of "repetitive" reads, which will gain better resolution of differentiating targets. In some embodiments, the expectation is that the number of reads from any given repeat will be a direct function of the number of repeats present. However, in some embodiments, a Poisson sampling-induced spread may need to be considered and in some embodiments may be sufficiently large to limit the resolution.

When a precise measurement of the length of both alleles from a diploid sample is desired, further manipulations may be required. This is because the capture efficiency measured will actually be the average efficiency of the two alleles. To effectively achieve separate

measurements for each allele, barcodes (e.g., sequence tags) can be used that allow the efficiency of individual capture events (from individual genomic loci) to be followed. FIG. 11A-C shows the approach. For a given locus, MIPs are synthesized to contain one of a large number differentiator tags in their backbone such that the probability of any two MIPs in a reaction having the same differentiator tag sequence is low. MIP capture is performed on the sample; the reaction will be biased for shorter target lengths, and therefore the reaction product will be comprised of more 'short' circles than 'long' circles. Each circle should bear a unique differentiator tag sequence. Then, linear RCA (IRCA) is performed on the circles. In the IRCA reaction, circles are converted into long, linear concatemers of themselves. The IRCA reaction for a given circle stops when the concatemer has reached a 'fixed' length (based on the processivity/error rate of the polymerase). Concatemers derived from smaller circles will therefore contain more copies of the differentiator tag, and concatemers derived from larger circles will contain fewer copies of the differentiator tag. The number of each differentiator tag sequence is counted, for example, by next- generation sequencing. When number of occurrences is plotted against differentiator tag ID, the data will naturally cluster into two groups reflecting the lengths of the two alleles in the diploid sample. The allele lengths can therefore be read directly off this graph, after absolute length calibration using known standards. In some embodiments, a sequencing technique (e.g., a next-generation sequencing technique) is used to sequence part of one or more captured targets (e.g., or amplicons thereof) and the sequences are used to count the number of different barcodes that are present. Accordingly, in some embodiments, aspects of the invention relate to a highly-multiplexed qPCR reaction.

Other non-limiting examples of loci at which insertions or deletions or repeat sequences may be associated with a disease or condition are provided in Tables 3 and 4. It should be appreciated that the presence of an abnormal length at any one or more of these loci may be evaluated according to aspects of the invention. In some embodiments, two or more of these loci or other loci may be evaluated in a single multiplex reaction using different probes designed to hybridize under the same reaction conditions to different target nucleic acid in a biological sample.

TABLE 4

Non-Polyglutamine Diseases

Normal/

Type Gene Codon wildtype Pathogenic

FRAXA FMR1, on the X- CGG 6-53 230+ (Fragile X chromosome

syndrome)

FXTAS (Fragile FMR1, on the X- CGG 6-53 55-200 X- associated chromosome

tremor/ataxia

syndrome)

FRAXE AFF2 or FMR2, GCC 6-35 200+ (Fragile XE on the X- mental chromosome retardation)

FRDA FXN or X25, GAA 7-34 100+

(Friedreich's (frataxin)

ataxia)

DM (Myotonic DMPK CTG 5-37 50+

dystrophy)

SCA8 OSCA or SCA8 CTG 16-37 110-250

(Spinocerebellar

ataxia Type 8)

SCA12 PPP2R2B or CAG 7-28 66-78

(Spinocerebellar SCA12 On

ataxia Type 12) 5' end

The following examples illustrate aspects and embodiments of the invention and are not intended to be limiting or restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

4. Increasing Detection Sensitivity:

In some embodiments, aspects of the invention relate to methods for increasing the sensitivity of nucleic acid detection assays.

There are currently many genomic assays that utilize next-generation (e.g., polony-based) sequencing to generate data, including genome resequencing, RNA-seq for gene expression, bisulphite sequencing for methylation, and Immune- seq, among others. In order to make quantitative measurements (including genotype calling), these methods utilize the counts of sequencing reads of a given genomic locus as a proxy for the representation of that sequence in the original sample of nucleic acids. The majority of these techniques require a preparative step to construct a high-complexity library of DNA molecules that is representative of a sample of interest. Current assays use one of several alternative nucleic acid preparative techniques (e.g., amplification, for example PCR-based amplification; sequence- specific capture, for example, using immobilized capture probes; or target capture into a circularized probe followed by a sequence analysis step. In order to reduce errors associated with the unpredictability (stochastic nature) of nucleic acid isolation and sequence analysis techniques, current methods to involve oversampling a target nucleic acid preparation in order to increase the likelihood that all sequences that are present in the original nucleic acid sample will be represented in the final sequence data. For example, a genomic sequencing library may contain an over- or under- representation of particular sequences from a source nucleic acid sample (e.g., genome preparation) as a result of stochastic variations in the library construction process. Such variations can be particularly problematic when they result in target sequences from a genome being absent or undetectable in a sequencing library. For example, an under-representation of particular allelic sequences (e.g., heterozygotic alleles) from a genome in a sequencing library can result in an apparent homozygous representation in a sequencing library.

In contrast, aspects of the invention relate to basing a nucleic acid sequence analysis on results from two or more different nucleic acid preparatory techniques that have different systematic biases in the types of nucleic acids that they sample rather than simply oversampling the target nucleic acid. According to some embodiments, different techniques have different sequence biases that are systematic and not simply due to stochastic effects during nucleic acid capture or amplification. Accordingly, in some embodiments, the degree of oversampling required to overcome variations in nucleic acid preparation needs to be sufficient to overcome the biases. In some embodiments, the invention provides methods that reduce the need for oversampling by combining nucleic acid and/or sequence results obtained from two or more different nucleic acid preparative techniques that have different biases.

According to the invention, different techniques have different characteristic or systematic biases. For example, one technique may bias a sample analysis towards one particular allele at a genetic locus of interest, whereas a different technique would bias the sample analysis towards a different allele at the same locus. Accordingly, the same sample may be identified as being different depending on the type of technique that is used to prepare nucleic acid for sequence analysis. This effectively represents a sensitivity issue, because each technique has a different relative sensitivities for polymorphic sequences of interest.

Currently, the ability of DNA sequencing to detect mutations is limited by the ability of the upstream sample isolation (e.g., by amplification, immobilization enrichment, circularization capture, etc.) methods to reliably isolate the locus of interest. If one wishes to make

heterozygote base-calls for a diploid genome (e.g. a human sample presented for molecular diagnostic sequencing), it is important in some embodiments that the isolation method produces near- or perfectly-uniform amounts of the two alleles to be sequenced (at least sufficiently uniform to be "called" unambiguously as a heterozygote or a homozygote for a locus of interest).

Sample preparative methods may fall into three classes: 1) single- or several-target amplification (e.g., uniplex PCR, 'multiplex' PCR), 2) multi-target hybridization enrichment (e.g., Agilent SureSelect 'hybrid capture' [Gnirke et al 2009, Nature methods 27: 182-9], Roche/Nimblegen 'sequence capture' [Hodges et al 2007, Nature genetics 39: 1522-7], and 3) multi-target circularization selection (e.g. molecular inversion probes or padlock probes, [Porreca et al 2007, Nature methods 4:931-6, Turner et al 2009, Nature methods 6:315-6], 'selectors' [Dahl et al 2005, Nucleic acids research 33:e71]). Each of these methods can result in a pool of isolated product that does not adequately represent the input abundance distribution. For example, the two alleles at a heterozygous position can become skewed far from their input 50:50 ratio to something that results in a missed basecall during downstream sequencing. For example, if the ratio was skewed from 50:50 to 10:90, and the sample was sequenced to lOx average coverage, there is a high probability that one of the two alleles would not be observed once in the ten sequencing reads. This would reduce the sensitivity of the sequencing method by converting a heterozygous position to homozygous (where potentially the 'mutant' allele was the one not observed). In some embodiments, a skewed ratio is a particular issue that decreases the sensitivity of detecting mutations present in a heterogeneous tumor tissue. For example, if only 10% of the cells analyzed in a heterogeneous sample harbored a heterozygous mutation, the mutation would be expected to be present in 5% of sequence reads, not 50%. In this scenario, the need for robust, sensitive detection may be even more acute.

The methods disclosed herein are based, in part, on the discovery that certain classes of isolation methods have different modes of bias. The disclosure provide methods for increasing the sensitivity of the downstream sequencing by using a combination of multiple isolation methods (e.g., one or more from at least two of the classes disclosed herein) for a sample. This is particularly important in molecular diagnostics where high sensitivity is required to minimize the chances of 'missing' a disease-associated mutation. For example, given a nominal false- negative error rate of 1x10 for sequencing following circularization selection, and a false- negative error rate of 1x10 for sequencing following hybridization enrichment, one can achieve a final false-negative rate of lxl0^~6 by performing both techniques on the sample (assuming failures in each method are fully independent). For a recessive disease with carrier frequency of 0.1, caused by a single fully-penetrant mutant allele, the number of missed carrier diagnoses would decrease from 1000 per million patients tested to 1 per million patients tested. Furthermore, if the testing was used in the context of prenatal carrier screening, the number of affected children born as a result of missing the carrier call in one parent would decrease from 25 per million to 25 per billion born.

Additionally, the disclosure provides combinations of preparative methods to effectively increase sequencing coverage in regions containing disease-associated alleles. Since

heterozygote error rate is largely tied to both deviations from 50:50 allele representation, and in the case of next-generation DNA sequencing deviations from average abundance (such that less abundant isolated targets are more likely to be undersampled at one or both alleles), selectively increasing coverage in these regions will also selectively increase sensitivity. Furthermore, MIPs that detect presence or absence of specific known disease-associated mutations can be used to increase sensitivity selectively. In some embodiments, these MIPs would have a targeting arm whose 3 '-most region is complementary to the expected mutation, and has a fill-in length of 0 or more bp. Thus, the MIP will form only if the mutation is present, and its presence will be detected by sequencing.

Additionally, algorithms disclosed herein may be used to determine base identity with varying levels of stringency depending on whether the given position has any known disease-associated alleles. Stringency can be reduced in such positions by decreasing the minimum number of observed mutant reads necessary to make a consensus base-call. This will effectively increase sensitivity for mutant allele detection at the cost of decreased specificity.

An embodiment of the invention combines MIPs plus hybridization enrichment, plus optionally extra MIPs targeted to specific known, common disease-associated loci, e.g., to detect the presence of a polymorphism in a target nucleic acid. A non-limiting example is illustrated in FIG. 12 that illustrates a schematic using MIPs plus hybridization enrichment, plus optionally extra MIPs targeted to specific known, common disease-associated loci, e.g., to detect the presence of a polymorphism in a target nucleic acid.

FIGS. 13 and 14 illustrate different capture efficiencies for MlP-based captures. FIG. 13 shows a graph of per-target abundance with MIP capture. In this graph, bias largely drives the heterozygote error rate, since targets which are less abundant here are less likely to be covered in sufficient depth during sequencing to adequately sample both alleles. This is from Turner et al 2009, Nature methods 6:315-6. Hybridization enrichment results in a qualitatively similar abundance distribution, but the abundance of a given target is likely not correlated between the two methods. FIG. 14 shows a graph of correlation between two MIP capture reactions from Ball et al 2009, Nature biotechnology 27:361-8. Each point represents the target abundance in replicate 1 and replicate 2. Pearson correlation r=0.956. This indicates that MIP capture reproducibly biases targets to specific abundances. Hybridization enrichment is similarly correlated from one capture to the next.

According to aspects of the invention, such biases can be detected or overcome by

systematically combining different capture and/or analytical techniques in an assay that interrogates a plurality of loci in a plurality of subject samples. Accordingly, it should be appreciated that in any of the embodiments described herein (e.g., tiling/staggering, tagging, size-detection, sensitivity enhancing algorithms, or any combination thereof), aspects of the invention involve preparing genomic nucleic acid and/or contacting them with one or more different probes (e.g., capture probes, hybridization probes, MIPs, others etc.). In some embodiments, the amount of genomic nucleic acid used per subject ranges from 1 ng to 10 micrograms (e.g., 500 ng to 5 micrograms). However, higher or lower amounts (e.g., less than 1 ng, more than 10 micrograms, 10-50 micrograms, 50-100 micrograms or more) may be used. In some embodiments, for each locus of interest, the amount of probe used per assay may be optimized for a particular application. In some embodiments, the ratio (molar ratio, for example measured as a concentration ratio) of probe to genome equivalent (e.g., haploid or diploid genome equivalent, for example for each allele or for both alleles of a nucleic acid target or locus of interest) ranges from 1/100, 1/10, 1/1, 10/1, 100/1, 1000/1. However, lower, higher, or intermediate ratios may be used.

In some embodiments, the amount of target nucleic acid and probe used for each reaction is normalized to avoid any observed differences being caused by differences in concentrations or ratios. In some embodiments, in order to normalize genomic DNA and probe, the genomic DNA concentration is read using a standard spectrophotometer or by fluorescence (e.g., using a fluorescent intercalating dye). The probe concentration may be determined experimentally or using information specified by the probe manufacturer.

Similarly, once a locus has been captured (e.g., on a MIP or other probe or in another form), it may be amplified and/or sequenced in a reaction involving one or more primers. The amount of primer added for each reaction can range from 0.1 pmol to 1 nmol, 0.15 pmol to 1.5 nmol (for example around 1.5 pmol). However, other amounts (e.g., lower, higher, or intermediate amounts) may be used.

In some embodiments, it should be appreciated that one or more intervening sequences (e.g., sequence between the first and second targeting arms on a MIP capture probe), identifier or tag sequences, or other probe sequences that are not designed to hybridize to a target sequence (e.g., a genomic target sequence) should be designed to avoid excessive complementarity (to avoid cross -hybridization) to target sequences or other sequences (e.g., other genomic sequences) that may be in a biological sample. For example, these sequences may be designed have a sufficient number of mismatches with any genomic sequence (e.g., at least 5, 10, 15, or more mismatches out of 30 bases) or as having a Tm (e.g., a mismatch Tm) that is lower (e.g., at least 5, 10, 15, 20, or more degrees C. lower) than the hybridization reaction temperature.

It should be appreciated that a targeting arm as used herein may be designed to hybridize (e.g., be complementary) to either strand of a genetic locus of interest if the nucleic acid being analyzed is DNA (e.g., genomic DNA). However, in the context of MIP probes, whichever strand is selected for one targeting arm will be used for the other one. However, in the context of RNA analysis, it should be appreciated that a targeting arm should be designed to hybridize to the transcribed RNA. It also should be appreciated that MIP probes referred to herein as "capturing" a target sequence are actually capturing it by template-based synthesis rather than by capturing the actual target molecule (other than for example in the initial stage when the arms hybridize to it or in the sense that the target molecule can remain bound to the extended MIP product until it is denatured or otherwise removed).

It should be appreciated that in some embodiments a targeting arm may include a sequence that is complementary to one allele or mutation (e.g., a SNP or other polymorphism, a mutation, etc.) so that the probe will preferentially hybridize (and capture) target nucleic acids having that allele or mutation. However, in many embodiments, each targeting arm is designed to hybridize (e.g., be complementary) to a sequence that is not polymorphic in the subjects of a population that is being evaluated. This allows target sequences to be captured and/or sequenced for all alleles and then the differences between subjects (e.g., calls of heterozygous or homozygous for one or more loci) can be based on the sequence information and/or the frequency as described herein.

It should be appreciated that sequence tags (also referred to as barcodes) may be designed to be unique in that they do not appear at other positions within a probe or a family of probes and they also do not appear within the sequences being targeted. Thus they can be used to uniquely identify (e.g., by sequencing or hybridization properties) particular probes having other characteristics (e.g., for particular subjects and/or for particular loci).

It also should be appreciated that in some embodiments probes or regions of probes or other nucleic acids are described herein as comprising or including certain sequences or sequence characteristics (e.g., length, other properties, etc.). However, it should be appreciated that in some embodiments, any of the probes or regions of probes or other nucleic acids consist of those regions (e.g., arms, central regions, tags, primer sites, etc., or any combination thereof) of consist of those sequences or have sequences with characteristics that consist of one or more characteristics (e.g., length, or other properties, etc.) as described herein in the context of any of the embodiments (e.g., for tiled or staggered probes, tagged probes, length detection, sensitivity enhancing algorithms or any combination thereof).

It should be appreciated that probes, primers, and other nucleic acids designed or used herein may be synthetic, natural, or a combination thereof. Accordingly, as used herein, the term "nucleic acid" refers to multiple linked nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to an exchangeable organic base, which is either a pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a purine (e.g., adenine (A) or guanine (G)). "Nucleic acid" and "nucleic acid molecule" may be used interchangeably and refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms shall also include polynucleosides (i.e., a polynucleotide minus a phosphate) and any other organic base containing nucleic acid. The organic bases include adenine, uracil, guanine, thymine, cytosine and inosine. Unless otherwise stated, nucleic acids may be single or double stranded. The nucleic acid may be naturally or non-naturally occurring. Nucleic acids can be obtained from natural sources, or can be synthesized using a nucleic acid synthesizer (i.e., synthetic). Harvest and isolation of nucleic acids are routinely performed in the art and suitable methods can be found in standard molecular biology textbooks. (See, for example, Maniatis' Handbook of Molecular Biology.) The nucleic acid may be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA, cDNA, rRNA, miRNA, or a combination thereof. Non-naturally occurring nucleic acids such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) can also be used. The invention also contemplates the use of nucleic acid derivatives. As will be described herein, the use of certain nucleic acid derivatives may increase the stability of the nucleic acids of the invention by preventing their digestion, particularly when they are exposed to biological samples that may contain nucleases. As used herein, a nucleic acid derivative is a non-naturally occurring nucleic acid or a unit thereof. Nucleic acid derivatives may contain non-naturally occurring elements such as non-naturally occurring nucleotides and non-naturally occurring backbone linkages.

Nucleic acid derivatives may contain backbone modifications such as but not limited to phosphorothioate linkages, phosphodiester modified nucleic acids, phosphorothiolate modifications, combinations of phosphodiester and phosphorothioate nucleic acid,

methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates,

phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof. The backbone composition of the nucleic acids may be homogeneous or heterogeneous.

Nucleic acid derivatives may contain substitutions or modifications in the sugars and/or bases. For example, they include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position (e.g., an 2'-0-alkylated ribose group). Nucleic acid derivatives may include non-ribose sugars such as arabinose. Nucleic acid derivatives may contain substituted purines and pyrimidines such as C-5 propyne modified bases, 5- methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2- thiouracil and pseudoisocytosine. In some embodiments, substitution(s) may include one or more substitutions/modifications in the sugars/bases, groups attached to the base, including biotin, fluorescent groups (fluorescein, cyanine, rhodamine, etc), chemically-reactive groups including carboxyl, NHS, thiol, etc., or any combination thereof.

A nucleic acid may be a peptide nucleic acid (PNA), locked nucleic acid (LNA), DNA, RNA, or co-nucleic acids of the same such as DNA-LNA co-nucleic acids. PNA are DNA analogs having their phosphate backbone replaced with 2-aminoethyl glycine residues linked to nucleotide bases through glycine amino nitrogen and methylenecarbonyl linkers. PNA can bind to both DNA and RNA targets by Watson-Crick base pairing, and in so doing form stronger hybrids than would be possible with DNA or RNA based oligonucleotides in some cases.

PNA are synthesized from monomers connected by a peptide bond (Nielsen, P. E. et al. Peptide Nucleic Acids, Protocols and Applications, Norfolk: Horizon Scientific Press, p. 1-19 (1999)). They can be built with standard solid phase peptide synthesis technology. PNA chemistry and synthesis allows for inclusion of amino acids and polypeptide sequences in the PNA design. For example, lysine residues can be used to introduce positive charges in the PNA backbone. All chemical approaches available for the modifications of amino acid side chains are directly applicable to PNA. Several types of PNA designs exist, and these include single strand PNA (ssPNA), bisPNA and pseudocomplementary PNA (pcPNA).

The structure of PNA/DNA complex depends on the particular PNA and its sequence. ssPNA binds to single stranded DNA (ssDNA) preferably in antiparallel orientation (i.e., with the N- terminus of the ssPNA aligned with the 3' terminus of the ssDNA) and with a Watson-Crick pairing. PNA also can bind to DNA with a Hoogsteen base pairing, and thereby forms triplexes with double stranded DNA (dsDNA) (Wittung, P. et al., Biochemistry 36:7973 (1997)).

A locked nucleic acid (LNA) is a modified RNA nucleotide. An LNA form hybrids with DNA, which are at least as stable as PNA/DNA hybrids (Braasch, D. A. et al., Chem &Biol. 8(1): 1-7 (2001)). Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some embodiments by adding positive charges to it. LNAs have been reported to have increased binding affinity inherently.

Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs. Therefore, production of mixed LNA/DNA sequences is as simple as that of mixed PNA/peptide sequences. The stabilization effect of LNA monomers is not an additive effect. The monomer influences conformation of sugar rings of neighboring deoxynucleotides shifting them to more stable configurations (Nielsen, P. E. et al. Peptide Nucleic Acids, Protocols and Applications, Norfolk: Horizon Scientific Press, p. 1-19 (1999)). Also, lesser number of LNA residues in the sequence dramatically improves accuracy of the synthesis. Most of biochemical approaches for nucleic acid conjugations are applicable to LNA/DNA constructs.

These and other aspects of the invention are illustrated by the following non-limiting examples.

EXAMPLES

The following examples illustrate non-limiting embodiments of the invention.

Example 1

Design a Set of Capture Probes for a Human Target Exon

All targets are captured as a set of partially-overlapping subtargets. For example, in the tiling approach, a 200 bp target exon might be captured as a set of 12 subtargets, each 60 bp in length (FIG. 1). Each subtarget is chosen such that it partially overlaps two or three other targets.

In some embodiments, all probes are composed of three regions: 1) a 20 bp 'targeting arm' comprised of sequence which hybridizes immediately upstream from the sub-target, 2) a 30 bp 'constant region' comprised of sequence used as a pair of amplification priming sites, and 3) a second 20 bp 'targeting arm' comprised of sequence which hybridizes immediately downstream from the sub-target. Targeting arm sequences will be different for each capture probe in a set, while constant region sequence will be the same for all probes in the set, allowing all captured targets to be amplified with a single set of primers. Targeting arm sequences should be designed such that any given pair of 20 bp sequences is unique in the target genome (to prevent spurious capture of undesired sites). Additionally, melting temperatures should be matched for all probes in the set such that hybridization efficiency is uniform for all probes at a constant temperature (e.g., 60 C). Targeting arm sequences should be computationally screened to ensure they do not form strong secondary structure that would impair their ability to basepair with the genomic target.

Hybridize Capture Probes to Human Genomic Sample

Assemble hybridization reaction: ul capture probe mix (^"2.5 pmol) ul lOx Ampligase buffer (Epicentre)

6.0 ul 500 ng/ul human genomic DNA (^"16.7 fmol)

l l ul dH20

In a thermal cycler, heat reaction to 95 C for 5 min to denature genomic DNA, then cool to 60 C. Allow to incubate at 60 C for 40 hours.

Convert Hybridized Probes into Covalently-Closed Circular Products Containing Subtargets Prepare fill-in/ligation reaction mixture:

0.25 ul 2 mM dNTP mix (Invitrogen)

2.5 ul lOx Ampligase buffer (Epicentre)

5.0 ul 5 U/ul Taq Stoffel fragment (Applied Biosystems)

12.5 ul 5 U/ul Ampligase (Epicentre)

4.75 ul dH20

Add 1.0 ul of this mix to the hybridized probe reaction, and incubate at 60 C for 10 hours.

Purify Circularized Probe/Subtarget Products from Un-Reacted Probes and Genomic DNA

Prepare exonuclease reaction mixture:

21 ul fill-in/ligation reaction product

ul lOx exonuclease I buffer (New England Biolabs)

2.0 ul 20 U/ul exonuclease I (New England Biolabs)

2.0 ul 100 U/ul exonuclease III (New England Biolabs)

Incubte at 37 C for 60 min, then heat- inactivate by incubating at 80 C for 15 min. Immediately cool to 4 C for storage.

Amplify Circular Material by PCR Using Primers Specific to the 'Constant Region' of the Probes

Prepare PCR mixture:

5.0 ul lOx Accuprime reaction buffer (Invitrogen)

(SEQ ID NO: 183)

1.5 ul 10 uM CP-2-FA (5 '-GCACGATCCGACGGTAGTGT-3 ')

(SEQ ID NO: 184)

1.5 ul 10 uM CP-2-RA (5 '-CCGTAATCGGGAAGCTGAAG-3 ')

0.4 ul 25 mM dNTP mix (Invitrogen)

2.0 ul heat-inactivated exonuclease reaction mix

1.5 ul lOx SybrGreen (Invitrogen)

0.4 ul 2.5 U/ul Accuprime Pfx polymerase (Invitrogen)

37.7 ul dH20

Thermal cycle in real-time thermal cycler according to the following protocol, but stop cycling before amplification yield plateaus (generally 8-12 cycles):

95C for 5 min

95C for 30 sec

58C for 60 sec

72C for 60 sec

goto 2, N more times

Prepare a Shotgun Next-Generation Sequencing Library for Analysis

Purify desired amplicon population from non-specific amplification products by gel extraction.

Concatemerize amplicons into high-molecular weight products suitable for shearing Mechanically shear, using either a nebulizer, BioRuptor, Hydroshear, Covaris, or similar instrument. DNA should be sheared into fragments several hundred basepairs in length. Ligate adapters required for amplification by the sequencing platform used. If necessary, purify ligated product from unligated product and adapters.

Example 2

Use of Differentiator Tag Sequences to Detect and Correct Bias in a MIP-Capture Reaction of a Set of Exon Targets

The first step in performing the detection/correction is to determine how many differentiator tag sequences are necessary for the given sample. In this example, 1000 genomic targets corresponding to 1000 exons were captured. Since the differentiator tag sequence is part of the probe, it will measure/report biases that occur from the earliest protocol steps. Also, being located in the backbone, the differentiator tag sequence can easily be sequenced from a separate priming site, and therefore not impact the total achievable read-length for the target sequence. MIP probes are synthesized using standard column-based oligonucleotide synthesis by any number of vendors (e.g. IDT), and differentiator tag sequences are introduced as 'degenerate' positions in the backbone. Each degenerate position increases the total number of differentiator tag sequences synthesized by a factor of 4, so a 10 nt degenerate region implies a differentiator tag sequence complexity of ^~le6 species.

Hybridize Capture Probes to Human Genomic Sample

Assemble hybridization reaction:

1.0 ul capture probe mix (^"2.5 pmol)

2.0 ul lOx Ampligase buffer (Epicentre)

6.0 ul 500 ng/ul human genomic DNA (^"16.7 fmol)

11 ul dH20

0.25 ul 2 mM dNTP mix (Invitrogen) 2.5 ul lOx Ampligase buffer (Epicentre)

5.0 ul 5 U/ul Taq Stoffel fragment (Applied Biosystems)

12.5 ul 5 U/ul Ampligase (Epicentre)

4.75 ul dH20

Prepare exonuclease reaction mixture:

21 ul fill-in/ligation reaction product

2.0 ul lOx exonuclease I buffer (New England Biolabs)

2.0 ul 20 U/ul exonuclease I (New England Biolabs)

2.0 ul 100 U/ul exonuclease III (New England Biolabs)

Incubate at 37 C for 60 min, then heat-inactivate by incubating at 80 C for 15 min. Immediately cool to 4 C for storage.

Prepare PCR mixture:

5.0 ul lOx Accuprime reaction buffer (Invitrogen)

(SEQ ID NO: 183)

1.5 ul 10 uM CP-2-FA (5 '-GCACGATCCGACGGTAGTGT-3 ')

(SEQ ID NO: 184)

1.5 ul 10 uM CP-2-RA (5 '-CCGTAATCGGGAAGCTGAAG-3 ')

0.4 ul 25 mM dNTP mix (Invitrogen)

2.0 ul heat-inactivated exonuclease reaction mix

1.5 ul lOx SybrGreen (Invitrogen)

0.4 ul 2.5 U/ul Accuprime Pfx polymerase (Invitrogen) 37.7 ul dH20

95C for 5 min

95C for 30 sec

58C for 60 sec

72C for 60 sec

goto 2, N more times

Prepare a shotgun next- generation sequencing library for analysis

Concatemerize amplicons into high-molecular weight products suitable for shearing Mechanically shear, using either a nebulizer, BioRuptor, Hydroshear, Covaris, or similar instrument. DNA should be sheared into fragments several hundred basepairs in lenth.

Ligate adapters required for amplification by the sequencing platform used. If necessary, purify ligated product from unligated product and adapters.

Perform Sequencing of Library According to Manufacturer's Directions (e.g. Illumina, ABI, etc), Reading Both the Target Sequence and the Differentiator Tag Sequence.

Analyze Data by Correcting for any Biases Detected by Quantitation of Differentiator Tag Sequence Abundance.

Construct a table of target:differentiator tag abundances from the read data, e.g.:

Target Differentiator

ID tag sequence ID Count

1 3547 1

2 4762 1

1 9637 1 3 4762 1

1 2984 1

All 'count' entries should be T, since any particular target:differentiator tag mapping will not occur more than once by chance, and therefore will only be observed if bias was present somewhere in the sample preparation process. For any target:differentiator tag combination observed more than once, all such reads are 'collapsed' into a single read before consensus basecalls are determined. This will cancel the effect of bias on consensus basecall accuracy. FIG. 5 depicts a method for making diploid genotype calls in which repeat

target:differentiator tag combination are collapsed.

Example 3

Differentiator Tag Sequence Design for MIP Capture Reactions

For a set of targets, the number of differentiator tag sequences necessary to be confident (within some statistical bounds) that a certain differentiator tag sequence will not be observed more than once by chance in combination with a certain target sequence was determined. The total number of unique differentiator tag sequences for a certain differentiator tag sequence length is determined as

For a molecular inversion probe capture reaction that uses MIP probes having differentiator tag sequences, the probability of performing the capture reaction and capturing one or more copies of a target sequence having the same differentiator tag sequence is calculated as: p=l-[N!/(N-M)!]/[NM], wherein N is the total number of possible unique differentiator tag sequences and M is the number of target sequence copies in the capture reaction. Thus, by varying the differentiator tag sequence length it is possible to perform a MIP capture reaction in which the probability of capturing one or more copies of a target sequence having the same differentiator tag sequence is set at a predetermined probability value. For example, for a differentiator tag sequence of 15 nucleotides in length, there are

1,073,741,824 possible differentiator tag sequences. A MIP capture reaction in which MIP probes, each having a differentiator tag sequence of 15 nucleotides, are combined with 10000 target sequence copies (e.g., genome equivalents), the probability of capturing one or more copies of a target sequence having the same differentiator tag sequence is 0.05. In this example, the MIP reaction will produce very few (usually 0, but occasionally 1 or more) targets where multiple copies are tagged with the same differentiator tag sequence. FIG. 6 depicts results of a simulation for 100000 capture reactions having 15 nucleotide differentiator tag sequences and 10000 target sequences.

Example 4

Assessment of the Probability for Obtaining Enough Sequencing Reads to Make Accurate Base- Calls at Multiple Independent Loci, as a Function of Sequencing Coverage.

Monte Carlo simulations were performed to determine sequencing coverage requirements. The simulations assume 10000 genomic copies of a given locus (target) half mom alleles and half dad alleles. The simulations further assume 1% efficiency of capture for the MIP reaction. The simulation samples from a capture mix 100 times without replacement to create a set of 100 capture products. The simulation then samples from the set of 100 capture products with replacement (assuming unbiased amplification) to generate 'reads' from either mom or dad. The number of reads sampled depends on the coverage. The number of independent reads from both mom and dad necessary to make a high-quality base-call (assumed to be 10 or 20 reads) were then determined. The process was repeated 1000 times for each coverage level, and the fraction of times that enough reads from both parents were successfully obtained was determined. This fraction was raised to the power 1000, assuming we have 1000 independent loci that must obtain successful base-calls, plotted (See FIG. 7). Result show that roughly 50x coverage is required to capture each allele >=10x with >0.95 probability.

Example 5

MIP Capture of 'Target' Locus and 'Control' Loci

In some embodiments, to accurately quantify the efficiency of target locus capture, at least three sets of control loci are captured in parallel that have a priori been shown to serve as proxies for various lengths of target locus. For example, if the target locus is expected to have a length between 50 and 1000 bp, then sets of control loci having lengths of 50, 250, and 1000 bp could be captured (e.g. 20 loci per set should provide adequate protection from outliers), and their abundance digitally measured by sequencing. These loci should be chosen such that minimal variation in efficiency between samples and on multiple runs of the same sample is observed (and are therefore 'efficiency invariant'). These will serve as 'reference' points that define the shape of the curve of abundance-vs-length. Determining the length of the target is then simply a matter of 'reading' the length from the appropriate point on the calibration curve.

In some embodiments, the statistical confidence one has in the estimate of target length from this method is driven largely by three factors: 1) reproducibility/variation of the abundance data used to generate the calibration curve; 2) goodness of fit of the regression to the 'control' datapoints; 3) reproducibility of abundance data for the target locus being measured. Statistical bounds on 1) and 2) will be known in advance, having been measured during development of the assay. Additionally, statistical bounds on 3) will be known in general in advance, since assay development should include adequate population sampling and measure of technical reproducibility. Standard statistical methods should be used to combine these three measures into a single P value for any given experimental measure of target abundance.

In some embodiments, given the set of calibration observations, and a linear regression fit to that data, the regression can be used to predict the length value for n observations of the target locus whose length is unknown. First, choose an acceptable range for the confidence interval of the length estimate. For example, in the case of distinguishing "normal" (87-93 bp) from "premutation" (165-600 bp) potential cases of Fragile X, the goal is to measure length to sufficient precision to distinguish 93 bp from 165 bp. The predicted response value, computed when n observations is substituted into the equation for the regressed line, will have arbitrary precision. However, if for example a 95% confidence level is desired, that 95% confidence interval must be sufficiently short that it does not overlap both the "normal" and "premutation" length ranges. Continuing the example, if one calculates a length of 190 from n=400 MIP observations, and based on the regression from calibration data, the 95% confidence interval is 190 +/-20 bp, one can conclude the sample represents a "premutation" length with 95% certainty. Conversely, if the calibration data were less robust, error estimates of the regression would be higher, leading to larger confidence intervals on the predicted response value. In some embodiments, if the 95% CI were calculated as 190 +/-100 bp from n=400, one could not determine whether the predicted response value corresponds to a "normal" or "premutation" length.

In some embodiments, the confidence interval for a predicted response is calculated as:

The estimate for the response γ is identical to the estimate for the mean of the response: {tilde over (y)}=b₀+b₁x*. The confidence interval for the predicted value is given by y+t*sy, where γ is the fitted value corresponding to x*. The value t* is the upper (l-C)/2 critical value for the t(n-2) distribution.

In some embodiments, a technique for analyzing a locus of interest can involve the following steps.

Convert Hybridized Probes into Covalently-Closed Circular Products Containing

Subtargets

Prepare fill-in/ligation reaction mixture:

0.25 ul 2 mM dNTP mix (Invitrogen)

2.5 ul lOx Ampligase buffer (Epicentre)

5.0 ul 5 U/ul Taq Stoffel fragment (Applied Biosystems)

12.5 ul 5 U/ul Ampligase (Epicentre)

4.75 ul dH20

Prepare exonuclease reaction mixture:

21 ul fill-in/ligation reaction product

2.0 ul lOx exonuclease I buffer (New England Biolabs) 2.0 ul 20 U/ul exonuclease I (New England Biolabs)

2.0 ul 100 U/ul exonuclease III (New England Biolabs)

Prepare PCR mixture:

5.0 ul lOx Accuprime reaction buffer (Invitrogen)

1.5 ul 10 uM CP-2-FA-Ilmn (platform- specific amplification sequence plus 'circle constant region' -specific sequence)

1.5 ul 10 uM CP-2-RA-Ilmn (platform- specific amplification sequence plus

'circle constant region' -specific sequence)

0.4 ul 25 mM dNTP mix (Invitrogen)

2.0 ul heat-inactivated exonuclease reaction mix

1.5 ul lOx SybrGreen (Invitrogen)

0.4 ul 2.5 U/ul Accuprime Pfx polymerase (Invitrogen)

37.7 uldH20

95C for 5 min

95C for 30 sec

58C for 60 sec

72C for 60 sec

goto 2, N more times

Perform Sequencing (e.g., Next-Generation Sequencing) on Sample for Digital Quantitation According to Manufacturer's Instructions (e.g., Illumina, Abi)

Example 6 MIP- Capture Reaction of a Set of Exon Target Nucleic Acids

MIP probes are synthesized using standard column-based oligonucleotide synthesis by any number of vendors (e.g. IDT).

Hybridize Capture Probes to Human Genomic Sample

Assemble hybridization reaction:

1.0 ul capture probe mix (^"2.5 pmol)

2.0 ul lOx Ampligase buffer (Epicentre)

6.0 ul 500 ng/ul human genomic DNA (^"16.7 fmol)

11 ul dH20

Convert Hybridized Probes into Covalently-Closed Circular Products Containing Target Nucleic Acids

Prepare fill-in/ligation reaction mixture:

0.25 ul 2 mM dNTP mix (Invitrogen)

2.5 ul lOx Ampligase buffer (Epicentre)

5.0 ul 5 U/ul Taq Stoffel fragment (Applied Biosystems)

12.5 ul 5 U/ul Ampligase (Epicentre)

4.75 ul dH20

Purify circularized probe/target nucleic acid products from un-reacted probes and genomic DNA

Prepare exonuclease reaction mixture:

21 ul fill-in/ligation reaction product

2.0 ul lOx exonuclease I buffer (New England Biolabs)

2.0 ul 20 U/ul exonuclease I (New England Biolabs)

2.0 ul 100 U/ul exonuclease III (New England Biolabs) Incubate at 37 C for 60 min, then heat-inactivate by incubating at 80 C for 15 min.

Immediately cool to 4 C for storage.

Prepare PCR mixture:

5.0 ul lOx Accuprime reaction buffer (Invitrogen)

(SEQ ID NO: 183)

1.5 ul 10 uM CP-2-FA (5 '-GCACGATCCGACGGTAGTGT-3 ')

(SEQ ID NO: 184)

1.5 ul 10 uM CP-2-RA (5 '-CCGTAATCGGGAAGCTGAAG-3 ')

0.4 ul 25 mM dNTP mix (Invitrogen)

2.0 ul heat-inactivated exonuclease reaction mix

1.5 ul lOx SybrGreen (Invitrogen)

0.4 ul 2.5 U/ul Accuprime Pfx polymerase (Invitrogen)

37.7 ul dH20

95 C for 5 min

95 C for 30 sec

58 C for 60 sec

72 C for 60 sec

goto 2, N more times

Prepare a Shotgun Next-Generation Sequencing Library for Analysis

Concatemerize amplicons into high-molecular weight products suitable for shearing Mechanically shear, using either a nebulizer, BioRuptor, Hydroshear, Covaris, or similar instrument. DNA should be sheared into fragments several hundred basepairs in length.

Perform Sequencing of Library According to Manufacturer's Directions (e.g. Illumina, ABI, etc), Reading the Target Sequence to Determine Abundance of the Target Nucleic Acid.

Example 7

Use of MIPs, Hybridization, and Mutation-Detection Mips to Genotype a Set of 1000 Targets

MIPs, hybridization, and mutation-detection MIPs are used to genotype a set of 1000 targets. The protocol permits detection of any of 50 specific known point mutations First, separate MIP, hybridization, and mutation-detection MIP reactions are performed on a biological sample. A MIP capture reaction is performed essentially as described in Turner et al 2009, Nature methods 6:315-6. A set of MIPs is designed such to that each probe in the set flanks one of the 1000 targets. Separately, a hybridization enrichment reaction is performed using the Agilent SureSelect procedure. Prior to selection, the genomic DNA to be enriched is converted into a shotgun sequencing library using Illumina's 'Fragment Library' kit and protocol. Agilent's web interface is used to design a set of probes which will hybridize to the target nucleic acids. Separately, a set of probes are designed

(mutation-detection MIPs) which will form MIPs only if mutations (e.g., specific polymorphisms) are present. Each mutation-detection MIP has a 3 '-most base identity that is specific for a single known mutation. A reaction with this set of mutation-detection MIPs is performed to selectively detect the presence of any mutant alleles.

Once all three reactions have been performed, the two MIP reactions are combined (e.g., at potentially non-equimolar ratios to further increase sensitivity of mutation detection) into a single tube, and run as one sample on the next-generation DNA sequencing instrument. The hybridization-enriched reaction is run as a separate sample on the next-generation DNA sequencing instrument. Reads from each 'sample' are combined by a software algorithm which forms a consensus diploid genotype at each position in the target set by evaluating the total coverage at each position, the origin of each read in that total coverage, the quality score of each individual read, and the presence (or absence) of any reads derived from mutation- specific MIPs overlapping the region.

Example 8

Carrier screening is performed either pre-conception or during pregnancy to determine a couple's risk of having a child with a recessive genetic disorder. The number of individuals who could benefit from such screening is substantial, as roughly 2 million women give birth to their first child each year in the US. The disorders for which testing is recommended vary based on a number of different patient-specific factors. For instance, the American Congress of

Obstetricians and Gynecologists recommends that screening for cystic fibrosis be offered to all women of reproductive age, and that testing be performed for additional disorders if indicated by family history, partner's carrier status, or ethnicity.

Today, carrier screening is typically performed using focused genotyping technologies that are designed to interrogate specific mutations within a gene of interest. However, because of cost and complexity, these tests often do not include all known disease causing mutations. In contrast, next- generation DNA sequencing (NGS) can comprehensively genotype a set of genes in a cost-efficient manner, and is therefore poised to supplant current technologies for routine, high-volume carrier screening.

For NGS to be used for carrier screening in a clinical setting, it must satisfy at least three requirements. First, analytical accuracy must be both high and well characterized within the clinically relevant genes or regions. Previous reports have demonstrated a broad

range of accuracy values, and in some cases it is unclear whether these values hold within the relevant regions of the genome. In addition, accuracy for insertions and deletions

is generally either substantially lower or uncharacterized, and measured to lower precision. Second, the NGS workflow employed should yield data sufficient to cover the vast majority of targeted bases at a depth sufficient to make high-quality genotype calls.

It has been noted, however, that the percentage of bases callable at a given depth varies widely with both the sample preparation workflow and the total amount of sequencing

8,10. Finally, the workflow must be highly robust and reproducible, which can often be achieved through automation. However, typical NGS sample preparation workflows are not amenable to high-throughput automation because of rate-limiting mechanical

shearing, reaction purifications, size selections, and kitted reagent costs (typically $50- $200 per sample).

The following is an integrated NGS workflow that meets these requirements for carrier screening. The workflow combines automated, optimized molecular inversion probe target capture with molecular barcoding to maximize the sample throughput of a next-generation DNA sequencing machine, and employs a novel read assembly-based alignment method that enables accurate identification of both substitution and insertion/deletion lesions. The workflow is applied to sequence the protein-coding regions of fifteen genes in which loss-of-function mutations cause recessive Mendelian disorders often included as part of routine carrier screening, and demonstrate through realistic simulation and comparison to Sanger sequencing data that our approach achieves high accuracies. METHODS AND MATERIALS

Molecular Inversion Probe Design

Molecular inversion probes were designed to capture the coding regions and certain well- characterized non-coding regions of 15 genes (See Table 5 below). The 5' targeting arm (ligation arm) and 3' targeting arm (extension arm) comprised a total of 40 nucleotides, and were designed to flank 130 bp target regions. Probes were selected to maximize performance with respect to both capture efficiency and robustness to common polymorphisms. All possible probes targeting a genomic interval were designed and assigned score tuples consisting of: 1) presence of guanine or cytosine as the 5 '-most base of the ligation arm, 2) the number of dbSNP (version 130) entries intersecting targeting arm sites, and 3) the root mean squared deviation of the arms' predicted melting temperatures from optimal values derived from empirical studies of capture efficiency. Using these tuples, probes were ranked sequentially by 1, 2, and 3, and the probe with the highest rank was chosen. Probes were designed to 'tile' across targets with a period of 25 bp such that multiple probes with orthogonal targeting arm sequences captured every genomic position. The molecular inversion probes are provided in Appendix A. Appendix A also includes the upstream and downstream regions corresponding to each molecular inversion probe, which is shown by the start position and end position coordinates of each targeting arm relative to the target sub-region's coordinates on the Human Genome 18 (HG 18). Appendix B lists the genomic sub-regions targeted by the molecular inversion probes of Appendix A.

Table 5 shows diseases and genes the workflow is designed to interrogate, and the corresponding genes and nucleotides targeted.

Usher syndrome type IF 602083 PCDH15 6,508

Niemann-Pick disease type A/B 257200 / 607616 SMPD1 2,056

TOTAL 42,858

Target Capture, Barcoding, and NGS

Genomic DNA was purchased from the Coriell Cell Repositories (Camden, NJ) or isolated from whole blood by the Gentra Puregene method (Qiagen) modified to conclude with an overnight incubation at 65°C. Overnight incubation at an elevated temperature led to DNA shearing and an increased fraction of callable bases. All samples were considered "IRB Exempt" by Liberty IRB, our independent Institutional Review Board. On Tecan automation, 1.5 ug of genomic DNA was annealed with 1 ul of molecular inversion probe mix in IX Ampligase buffer (Epicentre Biotechnologies) for 5 min at 95°C followed by 24 hr at 54°C. 17 ul of fill-in mix (4 U Taq Stoffel fragment [Life Technologies], 10 U Ampligase [Epicentre Biotechnologies], 23.1 uM dNTP mix) was added by Tecan automation and incubated for 1 hr at 54°C. 50 U

Exonuclease I and 50 U Exonuclease III (Enzymatics Inc.) were then added by Tecan automation and incubated for 1 hr at 37 °C followed by 10 min at 98°C. The capture reaction product was amplified in two separate PCR reactions designed to attach a molecular barcode and Illumina cluster amplification sequences to the ends of each molecule so as to enable sequencing from each end of the captured region. Tecan automation was used to set up the PCR, which was carried out with 3.75 ul of capture product, 15 pmol of each primer, 10 nmol dNTPs, and 1 U VeraSeq polymerase (Enzymatics, Inc) in lx Veraseq buffer. Cycling conditions were: 98°C 30 sec, 17-22X (98°C 10 sec, 54°C 30 sec, 72°C 15 sec), 4°C forever.

Following PCR, equal volumes of product from multiple samples were pooled using Tecan automation, then purified using a Qiaquick column (Qiagen). The library pool

concentration was quantified on a Bioanalyzer 2100 (Agilent Technologies) and diluted to 10 nM. Single-read sequencing (85 bp for genomic tag and 15 bp for barcode/index) was performed on the Hiseq 2000 (Illumina, Inc) according to the manufacturer's instructions. Each pool of libraries was sequenced in 7 lanes, with the 8th lane used for the manufacturer- supplied PhiX control library. NGS Data Analysis with Alignment Only Algorithm

Raw .bcl files were converted to qseq files using bclConverter (Illumina). Fastq files were generated by 'de-barcoding' genomic reads using the associated barcode reads; reads for which barcodes yielded no exact match to an expected barcode, or contained one or more low-quality basecalls, were discarded. The remaining reads were aligned to hgl8 on a per-sample basis using BWA version 0.5.7 for short alignments and genotype calls were made using GATK version 1.0.4168 after base quality score re-calibration, realignment (with GATK version 1.0.5083) and targeting arm removal. High- confidence genotype calls were defined as having depth >= 50 and strand bias score <= 0. Clinical significance of variant calls was determined by matching against a VCF- formatted database of disease-causing mutations curated from the literature, with equivalent insertion/deletion regions calculated as previously described.

NGS Data Analysis with Genotyping by Assembly- Templated Alignment Algorithm De-barcoded fastq files were obtained as described above and partitioned by capture region (exon) using the target arm sequence as a unique key. Reads were assembled in parallel by exon using SSAKE version 3.7 with parameters "-m 30 -o 15". The resulting contigs were aligned to hgl8 using BWA version 0.5.7 for long alignments with parameter "-r 1". Short read alignment was performed as described above except that sample contigs (rather than hgl8) were used as the input reference sequence. Software was developed in Java to accurately transfer coordinate and variant data (gaps) from local sample space to global reference space for every BAM-formatted alignment. Genotyping and base quality recalibration were performed on the coordinate- translated BAM files using GATK version 1.6.5. Sanger Sequencing

PCR was carried out with the genomic DNA described in Target capture, barcoding, and NGS using a modified version of the protocol from Zimmerman et al., using PCR primers from Jones et al., except Ml 3 tails were removed. See Zimmerman RS, Cox S, Lakdawala NK, et al. A novel custom resequencing array for dilated cardiomyopathy. Genet Med. May 2010;12(5):268- 278; Jones S, Zhang X, Parsons DW, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. Sep 2008;321(5897): 1801-1806. Briefly, 15 ul reactions were performed with 25 ng of genomic DNA, 1U of AmpliTaq Gold (Applied Biosystems), and 10 fmol of each PCR primer in a PCR mix containing 4.8% DMSO (v/v), 1M betaine, 2.5mM magnesium chloride, luM dNTPs (total), and IX GeneAmp PCR Gold Buffer (Applied Biosystems). Cycling conditions were: 95°C 10 min, 30X (95°C 30 sec, 60°C 30 sec, 72°C 30 sec), 72°C 10 min, 8°C forever. PCR products were sent to either

Beckman Coulter Genomics or Genewiz where cleanup and chain termination bi-directional Sanger sequencing was performed on an ABI 3730x1 according to standard protocols. Data was retrieved in electropherogram (abl) format. Sanger Data Analysis and Cross- Validation to NGS

Mutation Surveyor software ("MS", Softgenetics) version 4.0.5 was used in batch-mode with default parameters to align abl files to target reference sequence and make genotype calls. Positions where MS base calls did not match in the forward and reverse directions were removed from consideration. All high-quality NGS genotype calls within 10 bp (inclusive) of target exons were subjected to cross-validation against VCF-converted MS variant calls. This process is described in more detail below.

Calls were compared by (i) lesion type (substitution, insertion, deletion, or combination thereof), (ii) lesion pattern (sequence difference compared to the reference), and (iii) genomic position (or equivalent position for insertions and deletions). NGS calls were classified true positive (TP), discordant (non-reference) variant genotype (DVG), or false positive (FP) if they matched MS calls by (i-iii), (iii) only, or none of the above criteria, respectively. MS variant calls with no corresponding NGS variant call were classified false negative (FN). Indel calls classified as DVG were re-classified as TP because GATK 1.0.4168 does not report zygosity for such calls. All concordant reference calls were considered true negative (TN). Each discordant call (DVG, FP, and FN), along with a subset of concordant calls, was subject to expert manual review and discarded or reclassified as appropriate. False positive rate was calculated as FP / (FP +TN). False negative rate was calculated as FN / (FN + TP). Compound heterozygous NGS calls (two different non-reference alleles) were cross-validated against Sanger data manually by aligning traces to a reference manipulated to contain one of the two variant alleles. In these cases TP genotype calls were reported as simple heterozygous by MS. Assessment of Detectability of Clinical Mutations by Simulation

145 Coriell samples were sequenced and analyzed by Genotyping by Assembly-Templated Alignment (GATA, described above). Applications were developed in Java and Groovy to input aligned reads (BAM records) from each sample and manipulate specific data fields (base sequence and qualities) to resemble the appropriate DNA lesion pattern of a given clinically relevant mutation. To simulate heterozygous carriers input reads covering the mutation were chosen at random for sequence manipulation with an average probability of 0.5. All reads, whether manipulated or not, were output in fastq format for subsequent GATA analysis as described. This process was repeated for each of 81 mutations of clinical significance whereupon genotyped (observed) alleles were cross- referenced back to the original simulated (expected) allele. Samples for which the allele was already present were excluded from simulation (e.g. many Coriell samples in the set contained the common CFTR F508del mutation). Mutations with detection rates <100 between the expected and observed alleles were classified as undetectable by NGS.

Determining Clinical Significance of Variant Allele Calls

Each NGS -detected variant allele is annotated for functional (clinical) significance by determining its relative position within the corresponding consensus coding sequence (CCDS). For the genes under consideration here these are: PCDH15 (CCDS7248.1), SMPD1

(CCDS44531.1), ABCC8 (CCDS31437.1), HEXA (CCDS 10243.1), BLM (CCDS 10363.1), ASPA (CCDS 11028.1), G6PC (CCDS 11446.1), MCOLN1 (CCDS 12180.1), BCKDHA

(CCDS 12581.1), CLRN1 (CCDS3153.1), BCKDHB (CCDS4994.1), DLD (CCDS5749.1), CFTR (CCDS5773.1), FANCC (CCDS35071.1), and IKBKAP (CCDS6773.1). Clinically significant (reportable) mutations include alterations to the conserved 2 basepairs flanking each exon (splice site), the native start codon, or the last codon (readthrough), as well as truncating (nonsense and frameshift) mutations. Additionally, GATK occasionally reports alternate insertion patterns with non-native bases (e.g. 'N') chosen from a minority of reads. These were classified 'indeterminate' and reportable to prompt follow-up confirmation.

RESULTS

i. Completeness and Reproducibility Automated target capture and molecular barcoding were performed followed by NGS on a set of 194 samples derived from immortalized cell lines (55 containing specific disease- causing mutations, and 139 chosen to represent ethnic diversity) and 59 samples derived from whole blood (as shown in Table 6 below). All exons were targeted including 10 nt of flanking intronic sequence, plus additional intronic regions known to contain disease- causing mutations in 15 genes causative of 14 recessive Mendelian diseases (Table 5) using tiling molecular inversion probes (see Methods). A total of 25,907,612,945 basepairs of de-multiplexed sequence were generated, corresponding to an average per- base coverage per sample of 2,399X (min 89 IX, max 4,000X). Out of the 42,858 bases targeted for capture in each sample, we made high- confidence genotype calls at an average of 97.3% (min 92.2%, max 99.8%) for cell line-derived DNA and 99.9% (min 99.8%, max 99.9%) for blood-derived DNA (See Table 5 above).

Table 6 shows the set of 94 samples derived from immortalized cell lines and 59 samples derived from whole blood.

1 line 3

GM0426 Cell Disease 113,557,31 2,660 98.6 Yes Yes 8 line 0

GM0433 Cell Disease 115,608,79 2,708 94.3 Yes Yes 0 line 0

GM0504 Cell Disease 113,811,44 2,666 96.0 Yes Yes 2 line 9

GM0696 Cell Disease 51,321,434 1,202 99.0 Yes Yes 6 line

GM0738 Cell Disease 87,834,174 2,057 99.3 Yes Yes 1 line

GM0744 Cell Disease 108,471,71 2,541 99.4 Yes Yes 1 line 7

GM0755 Cell Disease 106,594,63 2,497 99.3 Yes Yes 2 line 0

GM0773 Cell Disease 137,685,13 3,225 94.7 Yes Yes 2 line 1

GM0785 Cell Disease 98,376,083 2,304 96.6 Yes Yes 7 line

GM0833 Cell Disease 131,459,59 3,079 96.9 Yes Yes 8 line 1

GM1127 Cell Disease 119,881,29 2,808 99.3 Yes Yes 5 line 9

GM1127 Cell Disease 85,993,084 2,014 99.3 Yes Yes 7 line

GM1127 Cell Disease 125,921,30 2,949 92.9 Yes Yes 8 line 3

GM1128 Cell Disease 121,485,71 2,845 99.2 Yes Yes 0 line 2

GM1128 Cell Disease 107,022,43 2,507 99.7 No Yes 1 line 3

GM1128 Cell Disease 105,909,02 2,481 99.5 Yes Yes 2 line 9 GM1128 Cell Disease 128,624,24 3,013 96.5 Yes Yes 3 line 1

GM1128 Cell Disease 125,265,00 2,934 99.8 Yes Yes 4 line 8

GM1128 Cell Disease 105,205,58 2,464 97.3 Yes Yes 5 line 0

GM1128 Cell Disease 121,267,78 2,840 96.4 Yes Yes 7 line 7

GM1128 Cell Disease 116,071,39 2,719 98.0 Yes Yes 8 line 7

GM1137 Cell Disease 106,105,64 2,485 95.9 Yes Yes 0 line 7

GM1146 Cell Disease 120,852,66 2,831 97.9 Yes Yes 8 line 9

GM1147 Cell Disease 146,901,68 3,441 97.0 Yes Yes 2 line 2

GM1149 Cell Disease 119,104,14 2,790 99.3 Yes Yes 6 line 9

GM1149 Cell Disease 103,338,59 2,420 99.0 Yes Yes 7 line 4

GM1172 Cell Disease 111,446,56 2,610 99.1 Yes Yes 3 line 5

GM1185 Cell Disease 132,558,32 3,105 99.7 Yes Yes 9 line 1

GM1186 Cell Disease 133,241,17 3,121 99.5 Yes Yes 0 line 0

GM1244 Cell Disease 112,979,13 2,646 99.3 Yes Yes 4 line 0

GM1258 Cell Disease 142,275,40 3,332 98.4 Yes Yes 5 line 0

GM1278 Cell Disease 77,781,835 1,822 99.0 Yes Yes 5 line

GM1296 Cell Disease 105,134,32 2,462 99.2 Yes Yes 0 line 3

GM1320 Cell Disease 116,426,91 2,727 97.0 Yes Yes 5 line 2

GM1342 Cell Disease 142,163,14 3,330 96.5 Yes Yes 3 line 1

GM1359 Cell Disease 127,607,78 2,989 98.6 Yes Yes 1 line 3

GM1619 Cell Disease 80,190,257 1,878 92.7 Yes Yes 3 line

GM1702 Cell HuVar 114,118,59 2,673 98.9 No Yes 3 line 8

GM1707 Cell TGP 125,212,95 2,933 98.5 No Yes 4 line 6

GM1707 Cell TGP 114,067,84 2,672 95.1 No Yes 5 line 1

GM1707 Cell TGP 135,956,92 3,184 97.3 No Yes 8 line 5

GM1707 Cell TGP 109,131,65 2,556 93.8 No Yes 9 line 0

GM1708 Cell TGP 104,079,00 2,438 98.2 No Yes 0 line 0

GM1720 Cell HuVar 100,286,17 2,349 95.8 No Yes 3 line 0

GM1720 Cell HuVar 133,095,16 3,117 99.7 No Yes 7 line 5

GM1722 Cell HuVar 75,792,351 1,775 98.7 No Yes 8 line

GM1723 Cell HuVar 138,157,41 3,236 97.1 No Yes 1 line 8

GM1723 Cell HuVar 115,522,25 2,706 97.1 No Yes 3 line 6

GM1724 Cell HuVar 114,147,39 2,673 99.3 No Yes 2 line 2 GM1724 Cell HuVar 88,905,331 2,082 99.3 No Yes 7 line

GM1725 Cell HuVar 134,029,72 3,139 97.8 No Yes 1 line 8

GM1728 Cell HuVar 104,284,77 2,443 95.5 Yes Yes 2 line 7

GM1728 Cell HuVar 124,885,88 2,925 98.0 No Yes 6 line 6

GM1730 Cell HuVar 115,253,37 2,699 95.5 Yes Yes 1 line 5

GM1730 Cell HuVar 126,663,09 2,967 95.6 Yes Yes 2 line 1

GM1730 Cell HuVar 148,723,81 3,483 96.8 No Yes 3 line 5

GM1730 Cell HuVar 140,507,36 3,291 95.3 No Yes 4 line 0

GM1731 Cell HuVar 112,930,12 2,645 96.5 No Yes 0 line 3

GM1731 Cell HuVar 146,713,29 3,436 96.0 Yes Yes 5 line 5

GM1731 Cell HuVar 120,214,96 2,816 96.1 No Yes 7 line 4

GM1731 Cell HuVar 131,177,75 3,072 98.4 Yes Yes 8 line 3

GM1731 Cell HuVar 74,599,530 1,747 96.3 Yes Yes 9 line

GM1732 Cell HuVar 143,908,02 3,371 98.9 Yes Yes 0 line 6

GM1736 Cell HuVar 72,217,715 1,691 99.3 Yes Yes 0 line

GM1736 Cell HuVar 138,241,78 3,238 97.5 No Yes 1 line 9

GM1736 Cell HuVar 109,391,82 2,562 95.4 Yes Yes 2 line 7

GM1736 Cell HuVar 136,216,56 3,190 97.4 No Yes 3 line 3

GM1736 Cell HuVar 124,580,79 2,918 98.8 Yes Yes 4 line 4

GM1736 Cell HuVar 145,974,76 3,419 96.8 Yes Yes 5 line 3

GM1736 Cell HuVar 121,059,29 2,835 95.3 Yes Yes 6 line 1

GM1736 Cell HuVar 124,286,28 2,911 97.7 Yes Yes 7 line 0

GM1736 Cell HuVar 122,309,22 2,865 97.4 Yes Yes 8 line 8

GM1736 Cell HuVar 151,606,78 3,551 97.2 No Yes 9 line 8

GM1739 Cell HuVar 120,466,85 2,822 96.9 Yes Yes 2 line 2

GM1739 Cell HuVar 129,362,19 3,030 96.1 Yes Yes 3 line 9

GM1739 Cell HuVar 133,049,78 3,116 96.4 No Yes 4 line 0

GM1739 Cell HuVar 145,469,08 3,407 97.0 No Yes 5 line 9

GM1739 Cell HuVar 131,796,12 3,087 96.1 No Yes 6 line 4

GM1796 Cell HapMa 110,772,39 2,594 92.3 Yes Yes 2 line P 6

GM1796 Cell HapMa 131,430,39 3,078 95.4 No Yes 5 line P 1

GM1796 Cell HapMa 108,405,81 2,539 92.6 No Yes 6 line P 5

GM1796 Cell HapMa 133,849,48 3,135 95.2 Yes Yes 7 line P 2 GM1796 Cell HapMa 104,839,65 2,455 96.0 Yes Yes 8 line P 9

GM1796 Cell HapMa 170,762,90 4,000 98.7 Yes Yes 9 line P 0

GM1797 Cell HapMa 129,700,47 3,038 96.5 Yes Yes 0 line P 2

GM1797 Cell HapMa 146,346,72 3,428 97.2 Yes Yes 1 line P 2

GM1797 Cell HapMa 139,495,48 3,267 96.2 Yes Yes 2 line P 6

GM1797 Cell HapMa 116,085,42 2,719 93.2 No Yes 3 line P 1

GM1801 Cell HuVar 120,955,00 2,833 95.2 No Yes 5 line 0

GM1801 Cell HuVar 128,904,00 3,019 96.0 Yes Yes 7 line 6

GM1803 Cell HuVar 104,807,68 2,455 97.4 Yes Yes 4 line 2

GM1804 Cell HuVar 117,441,95 2,751 95.4 No Yes 3 line 3

GM1804 Cell HuVar 149,341,51 3,498 96.8 No Yes 4 line 8

GM1806 Cell HuVar 90,615,125 2,122 94.3 Yes Yes 7 line

GM1807 Cell HuVar 120,359,15 2,819 94.0 No Yes 3 line 4

GM1807 Cell HuVar 130,655,29 3,060 96.1 No Yes 5 line 2

GM1808 Cell HuVar 127,693,61 2,991 97.0 No Yes 4 line 2

GM1808 Cell HuVar 116,883,42 2,738 95.3 Yes Yes 7 line 5

GM1808 Cell HuVar 113,522,77 2,659 93.8 No Yes 9 line 5

GM1809 Cell HuVar 139,175,35 3,260 95.6 Yes Yes 0 line 1

GM1809 Cell HuVar 140,749,31 3,297 96.1 No Yes 1 line 1

GM1850 Cell HapMa 116,001,92 2,717 99.2 Yes Yes 7 line P 7

GM1852 Cell HapMa 123,974,59 2,904 99.4 Yes Yes 4 line P 3

GM1852 Cell HapMa 68,506,615 1,605 98.2 Yes Yes 6 line P

GM1852 Cell HapMa 103,011,72 2,413 99.2 No Yes 9 line P 9

GM1853 Cell HapMa 93,010,560 2,178 98.9 Yes Yes 2 line P

GM1853 Cell HapMa 88,541,054 2,074 98.2 Yes Yes 7 line P

GM1854 Cell HapMa 107,018,41 2,507 99.3 Yes Yes 0 line P 9

GM1855 Cell HapMa 110,404,28 2,586 99.3 No Yes 8 line P 0

GM1856 Cell HapMa 94,941,108 2,224 98.7 No Yes 1 line P

GM1856 Cell HapMa 109,707,90 2,570 99.4 Yes Yes 2 line P 7

GM1856 Cell HapMa 132,909,80 3,113 95.9 No Yes 3 line P 7

GM1866 Cell Disease 142,395,24 3,335 99.4 Yes Yes 8 line 5

GM1879 Cell Disease 117,599,23 2,754 99.2 Yes Yes 9 line 0

GM1880 Cell Disease 109,551,22 2,566 99.2 Yes Yes 0 line 4 GM1880 Cell Disease 87,204,605 2,042 98.8 Yes Yes 2 line

GM1888 Cell Disease 138,604,38 3,246 95.1 Yes Yes 6 line 6

GM1899 Cell HapMa 108,306,94 2,537 94.4 Yes Yes 2 line P 2

GM1899 Cell HapMa 96,468,405 2,259 99.5 No Yes 5 line P

GM1899 Cell HapMa 111,633,42 2,615 98.8 No Yes 7 line P 5

GM1899 Cell HapMa 99,785,735 2,337 99.4 Yes Yes 8 line P

GM1899 Cell HapMa 127,162,92 2,978 97.7 No Yes 9 line P 0

GM1900 Cell HapMa 66,999,861 1,569 98.0 Yes Yes 0 line P

GM1900 Cell HapMa 126,196,39 2,956 94.9 No Yes 3 line P 3

GM1900 Cell HapMa 143,461,74 3,360 96.3 No Yes 5 line P 9

GM1900 Cell HapMa 116,823,48 2,736 99.1 No Yes 7 line P 2

GM1901 Cell HapMa 121,510,89 2,846 99.8 Yes Yes 2 line P 3

GM1909 Cell HapMa 104,709,69 2,452 95.0 Yes Yes 3 line P 3

GM1909 Cell HapMa 108,885,87 2,550 98.2 Yes Yes 9 line P 3

GM1910 Cell HapMa 120,459,30 2,821 99.4 Yes Yes 1 line P 3

GM1911 Cell HapMa 71,500,299 1,675 99.2 No Yes

6 line P

GM1912 Cell HapMa 119,050,42 2,788 99.2 Yes Yes 7 line P 1

GM1913 Cell HapMa 66,366,273 1,554 96.9 No Yes 0 line P

GM1913 Cell HapMa 97,725,686 2,289 98.9 Yes Yes 7 line P

GM1914 Cell HapMa 110,866,36 2,597 99.1 Yes Yes 1 line P 3

GM1914 Cell HapMa 117,906,14 2,762 99.2 No Yes 4 line P 3

GM1915 Cell HapMa 84,729,187 1,984 99.2 Yes Yes 2 line P

GM1915 Cell HapMa 90,111,210 2,111 99.1 Yes Yes 9 line P

GM1917 Cell HapMa 74,654,792 1,749 96.6 No Yes 2 line P

GM1919 Cell HapMa 127,763,78 2,992 99.8 Yes Yes 2 line P 0

GM1920 Cell HapMa 114,675,88 2,686 99.7 Yes Yes 0 line P 6

GM1920 Cell HapMa 117,546,44 2,753 98.8 Yes Yes 3 line P 6

GM1920 Cell HapMa 84,803,031 1,986 98.7 Yes Yes 7 line P

GM1920 Cell HapMa 59,249,941 1,388 97.0 Yes Yes 9 line P

GM1922 Cell HapMa 70,582,882 1,653 98.0 Yes Yes 3 line P

GM1924 Cell HapMa 74,942,748 1,755 99.0 Yes Yes 0 line P

GM1977 Cell HapMa 91,506,428 2,143 93.3 No Yes 6 line P

GM1978 Cell HapMa 93,214,221 2,183 94.4 No Yes 0 line P GM1978 Cell HapMa 147,554,99 3,456 97.0 No Yes 2 line P 7

GM1978 Cell HapMa 99,895,304 2,340 95.9 Yes Yes 9 line P

GM1979 Cell HapMa 112,615,95 2,638 95.6 Yes Yes 4 line P 9

GM2028 Cell HapMa 118,388,59 2,773 94.9 Yes Yes 1 line P 0

GM2033 Cell HapMa 134,954,11 3,161 94.4 Yes Yes 2 line P 6

GM2033 Cell HapMa 109,399,56 2,562 92.8 Yes Yes 5 line P 1

GM2034 Cell HapMa 96,681,315 2,264 92.5 Yes Yes 1 line P

GM2034 Cell HapMa 149,562,21 3,503 98.3 Yes Yes 2 line P 0

GM2034 Cell HapMa 112,547,10 2,636 97.6 Yes Yes 4 line P 7

GM2034 Cell HapMa 96,682,870 2,264 92.7 No Yes 9 line P

GM2035 Cell HapMa 128,110,98 3,001 95.9 No Yes 7 line P 8

GM2036 Cell HapMa 105,976,91 2,482 92.2 No Yes 0 line P 1

GM2036 Cell HapMa 114,582,01 2,684 94.9 No Yes 3 line P 2

GM2073 Cell Disease 86,947,571 2,036 98.4 Yes Yes 7 line

GM2074 Cell Disease 131,676,64 3,084 99.5 Yes Yes 1 line 2

GM2074 Cell Disease 120,425,67 2,821 99.4 Yes Yes 5 line 8

GM2084 Cell HapMa 88,592,183 2,075 98.5 No Yes 5 line P

GM2084 Cell HapMa 94,474,722 2,213 98.9 No Yes 6 line P

GM2084 Cell HapMa 132,183,92 3,096 94.6 No Yes 7 line P 0

GM2084 Cell HapMa 94,859,450 2,222 99.2 Yes Yes 9 line P

GM2085 Cell HapMa 89,746,969 2,102 98.9 Yes Yes 0 line P

GM2085 Cell HapMa 105,058,24 2,461 98.9 No Yes 1 line P 8

GM2085 Cell HapMa 103,469,22 2,423 99.2 Yes Yes 2 line P 3

GM2085 Cell HapMa 67,451,488 1,580 99.0 Yes Yes 3 line P

GM2085 Cell HapMa 125,360,57 2,936 95.9 No Yes 4 line P 5

GM2085 Cell HapMa 125,206,71 2,933 95.1 Yes Yes 6 line P 1

GM2085 Cell HapMa 102,707,14 2,406 99.3 Yes Yes 8 line P 3

GM2085 Cell HapMa 107,012,00 2,506 98.9 No Yes 9 line P 9

GM2086 Cell HapMa 146,690,57 3,436 96.3 No Yes 1 line P 3

GM2086 Cell HapMa 121,310,10 2,841 99.4 Yes Yes 2 line P 7

GM2086 Cell HapMa 106,527,16 2,495 99.4 Yes Yes 6 line P 4

GM2086 Cell HapMa 88,099,219 2,063 99.4 Yes Yes 9 line P

GM2087 Cell HapMa 84,570,991 1,981 99.0 Yes Yes 0 line P GM2087 Cell HapMa 104,048,64 2,437 98.9 No Yes 1 line P 5

GM2087 Cell HapMa 90,867,460 2,128 99.0 No Yes 2 line P

GM2087 Cell HapMa 108,700,92 2,546 99.3 No Yes 3 line P 5

GM2092 Cell Disease 120,376,41 2,819 99.2 Yes Yes 4 line 4

GM2108 Cell Disease 66,554,012 1,559 97.3 Yes Yes 0 line

bloodOl Blood N/A 67,892,054 1,594 99.9 No No blood02 Blood N/A 75,235,946 1,766 99.9 No No blood03 Blood N/A 71,324,606 1,674 99.9 No No blood04 Blood N/A 58,883,762 1,382 99.9 No No blood05 Blood N/A 74,862,133 1,757 99.9 No No blood06 Blood N/A 77,267,380 1,814 99.9 No No blood07 Blood N/A 55,719,056 1,308 99.9 No No blood08 Blood N/A 64,495,882 1,514 99.9 No No blood09 Blood N/A 67,663,353 1,588 99.9 No No bloodlO Blood N/A 57,362,443 1,347 99.9 No No bloodl l Blood N/A 53,823,416 1,264 99.9 No No bloodl2 Blood N/A 73,097,398 1,716 99.9 No No bloodl3 Blood N/A 73,858,165 1,734 99.9 No No bloodl4 Blood N/A 87,675,439 2,058 99.9 No No bloodl5 Blood N/A 74,484,474 1,749 99.8 No No bloodl6 Blood N/A 59,096,764 1,387 99.8 No No bloodl7 Blood N/A 65,114,672 1,529 99.9 No No bloodl8 Blood N/A 41,759,247 980 99.9 No No bloodl9 Blood N/A 71,949,103 1,689 99.9 No No blood20 Blood N/A 81,225,381 1,907 99.9 No No blood21 Blood N/A 70,214,097 1,648 99.9 No No blood22 Blood N/A 72,674,504 1,706 99.9 No No blood23 Blood N/A 74,340,749 1,745 99.9 No No blood24 Blood N/A 64,015,737 1,503 99.9 No No blood25 Blood N/A 73,147,784 1,717 99.8 No No blood26 Blood N/A 41,950,444 985 99.8 No No blood27 Blood N/A 62,771,860 1,474 99.8 No No blood28 Blood N/A 47,085,570 1,105 99.8 No No blood29 Blood N/A 74,840,986 1,757 99.9 No No blood30 Blood N/A 73,612,767 1,728 99.9 No No blood31 Blood N/A 70,446,967 1,654 99.9 No No blood32 Blood N/A 86,513,773 2,031 99.9 No No blood33 Blood N/A 78,330,087 1,839 99.9 No No blood34 Blood N/A 76,890,117 1,805 99.9 No No blood35 Blood N/A 63,472,751 1,490 99.9 No No blood36 Blood N/A 77,259,799 1,814 99.9 No No blood37 Blood N/A 74,384,590 1,746 99.9 No No blood38 Blood N/A 87,075,653 2,044 99.9 No No blood39 Blood N/A 61,490,312 1,444 99.9 No No blood40 Blood N/A 83,490,415 1,960 99.9 No No blood41 Blood N/A 94,474,694 2,218 99.9 No No blood42 Blood N/A 79,180,999 1,859 99.9 No No blood43 Blood N/A 70,106,334 1,646 99.9 No No blood44 Blood N/A 66,239,225 1,555 99.9 No No blood45 Blood N/A 76,565,215 1,797 99.8 No No blood46 Blood N/A 66,932,062 1,571 99.9 No No blood47 Blood N/A 37,972,652 891 99.8 No No blood48 Blood N/A 66,880,850 1,570 99.9 No No blood49 Blood N/A 65,267,319 1,532 99.9 No No blood50 Blood N/A 63,720,579 1,496 99.9 No No blood51 Blood N/A 64,485,398 1,514 99.9 No No blood52 Blood N/A 90,657,228 2,128 99.9 No No blood53 Blood N/A 83,058,297 1,950 99.9 No No blood54 Blood N/A 86,145,665 2,022 99.9 No No blood55 Blood N/A 77,159,945 1,811 99.9 No No blood56 Blood N/A 88,169,014 2,070 99.9 No No blood57 Blood N/A 60,859,847 1,429 99.9 No No blood58 Blood N/A 72,504,883 1,702 99.9 No No blood59 Blood N/A 83,924,327 1,970 99.9 No No

The DNA extraction protocol used for our blood samples concluded with an overnight incubation at 65°C in a Tris-based buffer. Subsequent experiments showed that this step reduced the mean size of the purified DNA; shearing was likely caused by acid hydrolysis during a temperature-induced pH shift of the buffer. Lower molecular mass genomic DNA is more readily denatured, and therefore more accessible to molecular inversion probes, which improves capture reaction performance. Consistent with this hypothesis, overnight incubation temperature lowered to 25°C significantly reduces the percentage of target bases that yield high confidence genotype calls.

To assess reproducibility, a subset of 126 samples derived from cell line DNA (Appendix

A) was processed twice, each time by a different operator on different liquid handling equipment. At least 92% of bases were called at >= 50x coverage in all samples, with high agreement between replicates (Pearson correlation coefficient 0.868). Out of 5,177,206 total genotype calls compared, 17 were discordant, for a concordance rate of 0.999997. These occurred at only 5 unique genomic positions, consistent with systematic sequencing error as the primary cause.

Sanger Concordance

To assess the overall accuracy of our NGS genotype calls, the genotype calls from the NGS pipeline were compared to those generated by automated analysis (Mutation Surveyor, MS) of bi-directional Sanger sequence of PCR amplicons in a subset of 194 samples. Within a total of 6,997,906 bp of sequence called by both methods, 3,973 concordant and 1,220 discordant single nucleotide variant (SNV) genotype calls were observed. Through manual inspection of the Sanger trace(s) corresponding to discordant genotype calls, it was determined that 1,139 were MS errors, generally caused by low quality traces or misalignment of traces to reference.

Supporting the conclusion that the majority of discordant calls corresponded to incorrect Sanger calls, the Ti/Tv ratio of concordant genotype calls was observed as 3.19, and 0.61 for discordant Sanger calls eliminated as MS errors. The remaining 81 discordant genotype calls that could not be resolved because the corresponding traces were ambiguous, were re-amplified and re- sequenced. For 71 of these calls, this process yielded new Sanger data that led to the conclusion that the original automated Sanger calls were incorrect. An additional discordant call was resolved by another approach as a NGS true negative (Figures 15A-B), leaving 9 high- confidence discordant SNV calls (Table 7), corresponding to 8 NGS false positives and 1 NGS false negative. Table 7 shows a comparison of NGS genotype calls (alignment-only algorithm) to S anger-derived Mutation Surveyor genotype calls. Sanger genotype calls were considered truth. TP, true positive calls (non-reference NGS, non-reference Sanger); FP, false positive calls (non- reference NGS, reference Sanger); FN, false negative calls (reference NGS, non-reference Sanger); TN, true negative calls (reference NGS, reference Sanger). dbSNP membership determined relative to version 129. Indel calls were considered unique if they differed by sequence pattern or equivalence region. Known indels are disease-causing mutations present in previously- annotated samples.

TABLE 7 TP FP ¥U in

dbSNP 2,495 0 1

Heterozygous

not dbSNP 247 8 0

SNV dbSNP 1,245 0 0 6,992,746

Homozygous

not dbSNP 13 0 0

Unique 231 3 1

Total 61 396 3

Indd Unique 17 27 7 6,992,358

Known 31 - 0

The NGS SNV false positive rate was 1.14x10-6 (95% Wilson binomial confidence interval [5.80x10-7 2.26x10-6]). The false positive calls occurred at 5 unique genomic loci, 3 of which were at adjacent positions in a single exon of gene MCOLN1 due to realignment within GATK.

FIG. 16A-D shows GM18540 is an aneuploid cell line and hence yields skewed allelic fractions. FIG. 16A gives an IGV view of NGS data from GM18540 for the genotype call of interest (shown between vertical lines). FIG. 16B shows bi-directional Sanger data for the variant-containing region. FIG. 16C provides a histogram of allele ratios for all non-reference genotype calls in chromosome 11 derived from whole-genome shotgun sequencing (WGSS) of GM 18540 and control sample GM 18537. FIG. 16D shows genome- wide relative coverage for GM18540. WGSS coverage data for each of the autosomes was binned into 50 Kb intervals and the log-ratio of the per-sample mean normalized values was plotted versus chromosome position. Dashed vertical lines denote chromosome boundaries; within a chromosome the ratios are arranged according to genomic position.

The NGS SNV false negative rate was 2.50x10-4 (95% Wilson binomial confidence interval [1.28x10-5 1.41x10-3]). The false negative call observed occurred in chromosome 11 of a sample previously characterized as aneuploid. Out of 473 NGS reads covering the false negative locus, 9.5% supported the correct heterozygous A/C genotype call (FIG. 16A), with Sanger sequencing showing low peak height for the alternate A allele (FIG. 16B). Shotgun full- genome sequencing of this sample demonstrated a bimodal distribution of allele ratios for heterozygous calls in chromosome 11 (FIG. 16C), and illustrated variable chromosome copy numbers (FIG. 16D), supporting the conclusion that this sample was aneuploid. For indels, a total of 61 true positives, 394 false positives (27 unique alleles) and 3 false negatives (2 unique alleles, both in exon 1 of SMPDl) were observed. Of 31 clinically- relevant disease mutations, all 31 were detected.

iii. Detection of Pathogenic Mutations

The ability to detect variants that cause the Mendelian diseases targeted by the panel

(Table 5) in the set of 194 cell line-derived samples was assessed. 55 of these samples were derived from individuals who were either carriers of or affected by one of the diseases being assayed and collectively contained a total of 95 previously- characterized disease mutations. During the design of our NGS workflow, we determined that three of these lesions would be inaccessible by our approach— two were large deletions spanning multiple exons, and one was contained within a region of paralogous sequence in the tenth exon of CFTR (Table 8). Of the 92 mutations we could expect to detect by NGS, we detected all 92 (Table 8). We also identified truncating (and likely disease-causing) mutations in two affected samples where previously only one mutation was known (Figures 17A-D, Table 8), as well as 9 carriers in the set of 139 previously-uncharacterized HapMap, Thousand Genomes Project, and Human Diversity Panel samples (Table 8).

Table 8 shows pathogenic mutations detected in cell line-derived samples. Mutations highlighted in red and underlined were determined a priori to be inaccessible by NGS and therefore not evaluated here. Mutations listed in italicized font represent mutations in affected individuals that were previously unknown. Mutations listed in bolded font were present in Hapmap samples previously unannotated with respect to carrier status.

GM04330 CFTR 1812-lOA Yes 444delA Yes

GM06966 CFTR E92X Yes PHE508DEL Yes

GM07381 CFTR IVS19DS, +10 KB, OT Yes PHE508DEL Yes

(3849+lOkbOT)

GM07441 CFTR 621+lOT Yes IVS16,G>A,+1 Yes

(3120+1G>A)

GM07552 CFTR ARG553TER Yes PHE508DEL Yes

GM07732 CFTR E60X Yes PHE508DEL Yes

GM07857 CFTR M1101K Yes M1101K Yes

GM08338 CFTR GLY551ASP Yes - -

GM 11275 CFTR 1-BP DEL, 3659C Yes PHE508DEL Yes

GM 11277 CFTR ILE507DEL Yes ILE507DEL Yes

GM11278 CFTR Q493X Yes PHE508DEL Yes

GM 11280 CFTR 621+lOT Yes 711+lOT Yes

GM11281 CFTR 621+lOT Yes PHE508DEL Yes

GM 11282 CFTR 621+lOT Yes GLY85GLU Yes

GM11283 CFTR ALA455GLU N/A PHE508DEL Yes

GM 11284 CFTR ARG560THR Yes PHE508DEL Yes

GM11285 CFTR Y1092X Yes PHE508DEL Yes

GM 11287 CFTR P574H Yes PHE508DEL Yes

GM11288 CFTR G178R Yes PHE508DEL Yes

GM 11370 CFTR 444delA Yes IVSl l-lOA Yes

GM 11472 CFTR ASN1303LYS Yes GLY 1349 ASP Yes

GM 11496 CFTR GLY542TER Yes GLY542TER Yes

GM 11497 CFTR GLY542TER Yes - -

GM 11723 CFTR TRP1282TER Yes - - GM11859 CFTR 2789+5G>A Yes 2789+5G>A Yes

GM11860 CFTR IVS19DS, +10 KB, OT Yes IVS19DS, +10 KB, OT Yes

(3849+lOkbOT) (3849+lOkbOT)

GM 12444 CFTR IVSIOAS, G>A, -1 Yes

(1717-lOA)

GM12585 CFTR ARG1162TER Yes - -

GM12785 CFTR ARG347PRO Yes GLY551ASP Yes

GM 12960 CFTR ARG334TRP Yes c.3368-2A>T Yes

GM 13423 CFTR G85E Yes D1152H Yes

GM13591 CFTR ARG117HIS Yes PHE508DEL Yes

GM18668 CFTR CFTRdele2,3 N/A PHE508DEL Yes

GM 18799 CFTR 2184delA Yes PHE508DEL Yes

GM 18800 CFTR 1898+1 G>A Yes PHE508DEL Yes

GM 18802 CFTR Y122X Yes R1158X Yes

GM18886 CFTR 2143delT Yes PHE508DEL Yes

GM20737 CFTR R347H Yes - -

GM20741 CFTR 3876delA Yes - -

GM20745 CFTR S549N Yes - -

GM20924 CFTR R75X Yes - -

GM21080 CFTR 394delTT Yes - -

GM11468 G6PC R83C Yes Q347X Yes

GM00502 HEXA 1278insTATC Yes 1421+lOC Yes

GM03461 HEXA 1421+lOC Yes G269S Yes

GM05042 IKBKAP 2507+6T>C Yes 2507+6T>C Yes

GM02533 MCOLN1 IVS3-2A>G Yes del exl-ex7 N/A

GM03252 SMPD1 L302P Yes - - GM 13205 SMPD1 fsP330 Yes - -

GM16193 SMPD1 R496L Yes Arg608DEL Yes

GM19116 CFTR ARG334TRP Yes - -

GM17363 IKBKAP 2507+6T>C Yes - -

GM17366 IKBKAP 2507+6T>C Yes - -

GM17365 IKBKAP 2507+6T>C Yes - -

GM17364 IKBKAP 2507+6T>C Yes - -

GM17360 MCOLN1 IVS3-2A>G Yes - -

GM17362 HEXA 1278insTATC Yes - -

GM18015 HEXA c.739C>T Yes - -

GM17362 HEXA G269S Yes - -

Genotyping by Assembly - Templated Alignment

Although substitutions comprise the majority of coding variation in the human genome, insertions and deletions (indels) are often clinically relevant. Indels, especially when large or present in cis with substitutions, are notoriously difficult to detect with short NGS reads.

Assembly of short reads can improve indel detection sensitivity, but this is often at the cost of decreased SNV and indel specificity due to the presence of spurious contiguous sequence (contigs). An algorithm was devised termed Genotyping by Assembly-Templated Alignment (GATA), that first forms an assembly from reads partitioned into subsets by targeting arm sequence, then performs base quality- and coverage- informed genotyping by alignment of raw reads back to the assembled contigs (Figures 18A-18E).

Figures 18A-E depicts the next- generation DNA sequencing workflow. Genomic DNA samples are input to a molecular inversion probe capture reaction. Each target (depicted bgrey and black regions) is captured by multiple probes that anneal to non-overlapping genomic intervals. PCR is performed using primers containing patient-specific barcodes, yielding barcode libraries. Equal volumes of the libraries are pooled and enter Illumina's Hiseq high- throughput sequencing workflow as shown in FIG. 18B. Following sequencing, reads enter either the alignment only (AO, left) as depicted in FIG. 18C or Genotyping by Assembly- Templated Alignment (GATA, right) analysis pipeline as depicted in FIG. 18D. As shown in FIG. 18C, AO first partitions reads by sample molecular barcode, then in parallel for all samples performs short read alignment, base quality recalibration, realignment around putative indels, and genotyping. As shown in FIGS. 18D-E, GATA partitions reads first by sample molecular barcode, then by target. Reads are assembled into contigs that are then aligned to the reference genome. Raw reads are then aligned to the contigs, and raw read mapping and variant information relative to the reference is determined using reference-contig and read-contig alignments. Finally, base quality score recalibration and genotyping are performed on the mapped, raw reads.

The performance of GATA for indel genotyping was compared to the more conventional genotyping-by-alignment only (AO) algorithm used in the Sanger concordance studies. Across a set of 147 samples analyzed, both indel sensitivity and specificity were increased with GATA relative to AO (Table 9). GATA detected 23 unique insertions and deletions, which were confirmed by manual review of Sanger traces. Of these, 9 (39%) were not detected by AO in one or more samples, including BLM c.2207_2212delinsTAGATTC— the most common disease- causing mutation for Bloom syndrome in people of Ashkenazi Jewish descent— as well as several alleles in SMPD1 (Table 10), the gene associated with Niemann-Pick disease (FigureS 19A-D). Performance for substitutions was identical for both detection methods (AO and GATA).

Table 9 shows genotyping by assembly-templated alignment (GATA) improves detection of insertions and deletions. Raw variant alleles (positive calls) from 147 samples were filtered by depth and strand bias and categorized according to NGS data analysis method, alignment only (AO) or GATA. Calls were classified with GATA considered truth as true positive (TP), false positive (FP), and false negative (FN). Discordant calls, in all cases, were confirmed by manual review of corresponding Sanger traces and found to be GATA TP or TN, rather than FP or FN. Variant calls flagged as low-confidence are considered uncalled. Polymorphisms in the first ex on of SMPD1 accounted for the majority of uncalled and discordant alleles, which were not considered in accuracy calculations. Table 9

AO GATA

TP 104 211

FP 28 0

FN 47 0

Uncalled* 70 10

Sensitivity 0.696 1.0

Precision 0.786 1.0

Table 10 shows the frequency distribution of variant genotypes for the STR at SMPDl exon 1 representing various combinations of (i) the minor reference allele (0), (ii) a substitution (snp),(iii) insertions (+6 and +12bp in length), and (iv) deletions (-6, -12, and -18bp in length) as determined by GATA and confirmed by manual inspection of Sanger traces.

+6/snp 2

+6/0 1

-12/-12 1

As seen in FIGS. 19A-D, GATA correctly genotypes insertions and deletions that are undetectable by the Alignment Only method. Read from top to bottom, each figure provides tracks for cumulative depth of coverage (vertical grey bars); representative MIP alignments (horizontal grey bars) with mismatches (letters), and gaps (dashed lines); chromatogram;

reference DNA and amino acid sequence for FIG. 19A heterozygous BLM

c.2207_2212delinsTAGATTC in sample GM04408 as well as several alleles in the first exon of SMPD1 including FIG. 19B a heterozygous 18 bp deletion in sample GM20342 (minus strand), FIG. 19C a heterozygous 12 bp insertion and homozygous substitution in sample GM17282 (plus strand), and FIG. 19D compound heterozygous 6 and 12 bp deletions in sample GM00502

(minus strand). Chromatogram trace offsets corresponding to specific heterozygous insertion and deletion patterns are indicated with slanted lines color coded by reference base. For clarity offsets are shown for FIG. 19C and FIG. 19D only. Simulation to Assess Detectability of Rare Pathogenic Mutations

While detectability for all disease-causing mutations present in the sample set was empirically demostrated, there exist a number of disease-causing mutations for which samples cannot be readily obtained. To assess whether the NGS workflow can detect these additional mutations, the stimulations were performed in silico. Since detectability can be affected by any element of the workflow, a simulator was implemented that employed read sets from actual samples rather than model reads derived from the reference genome at uniform coverage. This allowed for realistic representation of target abundance distribution, neighboring in cis variants, as well as cycle- and context-dependent sequencing errors. Disease-causing variants were introduced into raw reads by a Bernoulli process, with an average 0.5 probability of introducing the lesion, to simulate the heterozygous genotypes carrier screening aims to detect.

A total of 81 heterozygous variants were simulated in a read set of at least 144 samples with the exception of c.l521_1523delCTT (F508del), the most common disease-causing mutation for cystic fibrosis in Caucasian populations, as shown in Table 11. This mutation was present in several samples, which were removed from simulation analysis (Materials and Methods). Of the simulated variants 67 (83%) were correctly genotyped in all (generally

145/145) samples and only four relatively large (>7 bp) deletions were undetected in one or more samples. High-confidence genotype calls were not made for the remaining 10 variants. No variants were found to be undetectable in all samples. Table 11 gives the performance results of GATA for detecting clinically-relevant mutations by simulation.

CFTR_c.l56_163dupATTGGAAA 145 145 0 0

CFTR_c.522_526delAATAA 145 145 0 0

ABCC8_c.259_268dell0 145 141 3 1

CFTR_c.l616_1617dupTA 145 145 0 0

CFTR_c.3068_3072delTAGTG 145 145 0 0

FANCC_c.356_360del5 145 145 0 0

CFTR_c.861_865delCTTAA 145 145 0 0

ABCC8_c.2835_2838delGAGA 145 145 0 0

CFTR_c.319_326delGCTTCCTA 145 145 0 0

CFTR_c.2249_2256del8 145 145 0 0

CFTR_c.l792_1798delAAAACTA 145 145 0 0

CFTR_c.2241_2248delGATACTGC 145 145 0 0

G6PC_c.462_466delTTTGT 145 145 0 0

CFTR_c.35_36insTATCA 145 145 0 0

HEXA_c.l471_1475delTCTGA 145 145 0 0

PCDH15_c.996_999delGGAT 145 145 0 0

ASPA_c.568_574del7 145 144 0 1

CFTR_c.3184_3188dupCTATG 145 145 0 0

SMPDl_c.l657_1663delACCGCCT 145 145 0 0

CFTR_c.ll62_1168delACGACTA 145 145 0 0

BCKDHB_c.l63_166dupACTT 145 145 0 0

BCKDHA_c.861_868delAGGCCCCG 145 145 0 0

CFTR_c.3773dupT 145 145 0 0

CFTR_c.ll55_1156dupTA 145 145 0 0 CFTR_c.3889dupT 145 145 0 0

HEXA_c.l274_1277dupTATC 145 145 0 0

CFTR_c.262_263delTT 144 144 0 0

CFTR_c.326_327delAT 145 145 0 0

CFTR_c.3691delT 145 145 0 0

CFTR_c.3528delC 144 144 0 0

BLM_c.2407dupT 145 145 0 0

CFTR_c.l521_1523delCTT 131 131 0 0

HEXA_c.915_917delCTT 145 145 0 0

G6PC_c.379_380dupTA 145 145 0 0

CFTR_c.2012delT 144 144 0 0

SMPDl_c.l829_1831delGCC 144 144 0 0

CFTR_c.l029delC 145 127 18 0

CFTR_c.2737_2738insG 145 145 0 0

CFTR_c.2947_2948delTT 145 142 3 0

CFTR_c.l911delG 145 145 0 0

CFTR_c.803delA 145 145 0 0

CFTR_c.l519_1521delATC 145 145 0 0

CFTR_c.805_806delAT 145 18 127 0

CFTR_c.2215delG 145 137 8 0

FANCC_c.67delG 145 145 0 0

CFTR_c.935_937delTCT 145 145 0 0

CFTR_c.2175dupA 145 145 0 0

CFTR_c.3530delA 145 145 0 0 CFT _c.531delT 145 145 0 0

CFTR_c.l021_1022dupTC 145 127 18 0

CFTR_c.3659delC 145 145 0 0

DLD_c.l04dupA 144 144 0 0

CFTR_c.2052dupA 144 144 0 0

CFTR_c.313delA 145 145 0 0

G6PC_c.79delC 145 145 0 0

CFTR_c.442delA 145 145 0 0

CFTR_c.l477_1478delCA 145 145 0 0

CFTR_c.l545_1546delTA 145 145 0 0

BCKDHA_c.ll7delC 145 145 0 0

CFTR_c.l418delG 145 145 0 0

CFTR_c.l976delA 145 145 0 0

CFTR_c.3536_3539delCCAA 145 145 0 0

CFTR_c.948delT 145 145 0 0

CFTR_c.2052delA 145 145 0 0

BCKDHB_c.595_596delAG 145 145 0 0

G6PC_c.980_982delTCT 145 145 0 0

CFTR_c.3039delC 145 145 0 0

Discussion

Robustness, completeness, and accuracy are three of the main factors that define the utility of a genetic carrier testing workflow in a clinical laboratory. By utilizing a target enrichment methodology that is performed in a single tube and requires no mechanical shearing or purifications of individual samples, methods of the invention provide an automated NGS workflow that yields highly-reproducible results across samples and operators. This reproducibility ensures that samples will not have to be rerun frequently, minimizing both turnaround time and per-sample cost.

Because each clinically meaningful basepair must be sequenced before an actionable medical report can be generated, a high level of completeness minimizes the amount of costly re- work necessary for a sample. Methods of the invention demonstrate completeness that is consistent with low to no re- work for the samples studied, and substantially better than other previously-reported methods using multiplex target capture or PCR with NGS. This

improvement is likely the result of a number of optimizations we have made relative to previous reports including the use of a tiling MIP design that ensures multiple probes capture each base and the use of a DNA extraction protocol that effortlessly shears the DNA to a lower molecular mass.

Regarding accuracy, the only observed SNV false negative was in a sample that exhibited skewed allele ratios along the chromosome, which should not commonly occur when testing for germline mutations in clinical specimens derived from whole

blood. Additionally, the SNV false positive rate of approximately 1.1 per million

basepairs corresponds to a low confirmation burden for clinical testing and surpasses values previously reported. Given the small target set and the rare nature of indels, it is difficult to give a precise measurement of our accuracy for indels in genera. However, this data suggests that the use of GATA substantially improves our ability to detect small

lesions. Additionally, a sensitivity of 100% by both AO and GATA was observed across the set of disease-causing insertions and deletions in carrier and affected samples.

It is worth noting that measuring accuracy to a sufficient level of precision and generality can be challenging within conserved coding regions because selective pressure limits the spectrum of variation present. While a large number of samples were sequenced, the

relatively small size of our target limited the number of unique alleles observable and meant that approximately 90% of such variants were common (i.e. present in

dbSNP). Nonetheless, there is no a priori reason to believe that the measured accuracy will not generalize to other rare and private mutations present in the targeted

loci. Supporting this point, these simulations using real data and controlled for sample-to-sample variability indicate that one can detect a number of very rare disease causing alleles of different types and sequence contexts, including insertions (up to 12bp), deletions (up to 22bp) and complex combinations thereof.

The reference standard one considers ground truth can impose a ceiling on measurable accuracy. Automated analysis of what is widely deemed the 'gold standard' for DNA sequencing was employed: bi-directional Sanger traces derived from PCR amplicons. FIGS. 20A-20B shows NGS detects allele dropout in Sanger sequencing reactions. FIG. 20A-1, FIG.20A-2, and FIG. 20A-3 show dropout of reference allele leads to homozygous non-reference call by Sanger sequencing, but heterozygous non-reference call by NGS, in BLM exon 12 of GM18034. Shown from top to bottom, (FIG. 20A-1) original PCR primer pair: expected reference sequence trace, sample forward trace, sample reverse trace; (FIG. 20A-2) re-designed PCR primer pair: expected reference sequence trace, sample forward trace, sample reverse trace; IGV of NGS data.

FIG. 20B-1, FIG.20B-2, and FIG. 20B-3 shows dropout of non-reference allele leads to homozygous reference call by Sanger sequencing, but heterozygous non-reference call by NGS, in DLD exon 9 of sample GM11370. Shown from top to bottom, expected reference sequence trace, sample forward trace, sample reverse trace. Shown from top to bottom, (FIG. 20B-1) original PCR primer pair: expected reference sequence trace, sample forward trace, sample reverse trace; (FIG. 20B-2) re-designed PCR primer pair: expected reference sequence trace, sample forward trace, sample reverse trace; IGV of NGS data. Project genotyping data was employed, 12 NGS false negatives and 7 false positives would have been observed in the subset of samples characterized by this approach. Because these were all shown by Sanger analysis to be HapMap Project genotyping errors, this would have underestimated both sensitivity and specificity.

The NGS workflow detected allele dropout in the Sanger data, a known limitation of that technology (FIGS. 20 A- 1 through 20B-3) and not surprising since each base sequenced by NGS was captured by multiple probes with independent targeting arms. Had the less laborious and more commonly-used reference of Hapmap Project genotyping data been employed, 12 NGS false negatives and 7 false positives would have been observed in the subset of samples characterized by this approach (Table 12). This would have underestimated both sensitivity and specificity. Table 12 shows concordance of NGS genotypes with HapMap data. All NGS positions called with high confidence (minimum 50x coverage and strand bias <= 0) that intersected Hapmap release 27 phase II+III genotyping data were evaluated, for a total of 5,337 genotypes across 83 samples. True negative: reference called by both NGS and HapMap; true positive: non- reference (heterozygous or homozygous) called by both NGS and HapMap; false positive: non- reference called by NGS, reference called by HapMap; false negative: reference called by NGS, non-reference called by HapMap. Specificity: TN / (TN + FP); sensitivity: TP / (TP + FN).

Indel detection methods that only employ gapped alignment of short reads to reference are often limited by false positives introduced by systematic, context-dependent

sequencing error, and false negatives introduced by failure of the aligner to open or

extend gaps. An assembly-based paradigm would address these limitations but raw

contigs do not always carry base quality and coverage information. The GATA

algorithm combines these approaches to deliver sensitive and specific indel detection

with SNV performance on par with a traditional alignment- only pipeline.

Many alleles detected exclusively by GATA were from a short tandem repeat (STR) region encoding the N-terminal signal peptide in SMPD1 (Table 10). Consistent with

previous reports, GATA detected non-reference alleles in 96% of samples, a rate that is strikingly high because hgl8 contains a minor allele that is frequently substituted

(V36A). While common hexanucleotide indels at this locus are clinically benign, any pathogenic mutation present in cis would likely be missed using a conventional approach for variant detection. Indeed, when reads were aligned independently, several genomic positions in this region consistently fell below our specified coverage threshold. GATA therefore should yield higher sensitivity for rare mutations linked to polymorphisms in the first exon of SPMD1 and potentially other STR loci as well.

The simulation methodology applied here attempts to assess detectability of rare pathogenic mutations in a highly realistic manner. Simply deriving reads from a

reference genome modified to include the mutation of interest can overestimate the

detection probability because of real-world factors that would otherwise render the

mutation undetectable. Additionally, we are able to determine whether a mutation is

sometimes, rather than always or never, detectable because it is simulated in the read sets of hundreds of samples; e.g., this could occur in a particular genetic background with a low-frequency in cis variant that interferes with alignment of reads containing the

mutation. Nonetheless, certain mutation types, in particular large deletions are still not amenable to this paradigm because they could fundamentally alter the distribution of reads generated across the relevant region. In these cases, either human samples or

synthetic templates remain the only way to assess detectability.

In conclusion, an automated, integrated workflow that converts human genomic

DNA isolated from blood or cell lines into clinically-relevant variant calls was presented by this example. High genotype concordance was achieved with conventional electrophoretic sequencing across a set of 15 genes. In addition, this example demonstrates the ability to detect a range of important disease-causing mutations. The pipeline analysis presented allows for sensitive and specific detection of indels, while simultaneously incorporating raw base quality and coverage into SNV genotype calls. Realistic simulation on actual run data indicates that a number of pathogenic mutations undetectable by a traditional alignment-based genotyping approach are accessible by GATA. Collectively, the data shows that this workflow has met three of the major requirements of a clinical carrier screening assay, supporting the notion that NGS is ready for clinical use.

It should be appreciated that the preceding examples are non-limiting and aspects of the invention may be implemented as described herein using alternative techniques and/or protocols that are available to one or ordinary skill in the art. It will be clear that the methods may be practiced other than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present disclosure are possible in light of the above teachings and, therefore, are within the scope of the claims. Preferred features of each aspect of the disclosure are as for each of the other aspects mutatis mutandis. The documents including patents, patent applications, journal articles, or other disclosures mentioned herein are hereby incorporated by reference in their entirety. In the event of conflict, the disclosure of present application controls, other than in the event of clear error.

Claims

What is claimed is:

1. A method of analyzing a target nucleic acid for carrier screening, the method comprising: introducing a plurality of molecular inversion probes to a sample derived from a human and suspected of containing a target nucleic acid, wherein each probe is complementary to nucleotides flanking a sub-region of the target nucleic acid and comprises a first targeting arm and a second targeting arm, and wherein each sub-region is different and overlapping with at least one other sub-region;

capturing a plurality of the sub-regions of the target nucleic acid with at least a portion of the molecular inversion probes, wherein the plurality of sub-regions of the target nucleic acid are from a combination of genes comprising ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (ABCC8), aspartoacylase (ASPA), branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA), branched chain keto acid dehydrogenase El, beta polypeptide

(BCKDHB), Bloom Syndrome, RecQ Helicase-Like (BLM), cystic fibrosis transmembrane conductance regulator (CFTR), clarin 1 (CLRN1), dihydrolipoamide dehydrogenase (DLD), Fanconi anemia, complementation group C (FANCC), glucose-6-phosphatase, catalytic subunit (G6PC), hexosaminidase A alpha polypeptide (HEXA), inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein (IKBKAP), mucolipin 1 (MCOLN1), protocadherin-related 15 (PCDH15), and sphingomyelin phosphodiesterase 1, acid lysosomal (SMPD1); and

analyzing the captured sub-regions for one or more genetic disorders, thereby analyzing the target nucleic acid.

2. The method of claim 1, wherein the plurality of molecular inversion probes are a combination of nucleic acid fragments comprising SEQ ID NO: 185 through SEQ ID NO: 3,254.

3. The method of claim 1, wherein each of the plurality of the molecular inversion probes includes a central region flanked by the first targeting arm and the second targeting arm.

4. The method of claim 3, wherein the central region is different for at least two of the plurality of the molecular inversion probes.

5. The method of claim 3, wherein the central region is the same for each of the plurality of the molecular inversion probes.

6. The method of claim 1, wherein the capturing step comprises:

hybridizing the at least a portion of the molecular inversion probes to the plurality of sub- regions;

converting the hybridized probes into circularized probes containing a copy of the sub- regions; and

amplifying the circularized probe/ sub-region products.

7. The method of claim 6, wherein the amplification step requires a single set of primers.

8. The method of claim 1, wherein the one or more genetic disorders comprise the combination of Familial hyperinsulinism, Canavan disease, Maple Syrup Urine disease, Bloom syndrome, Cystic fibrosis, Dihydrolipoamide dehydrogenase deficiency, Fanconi anemia, Glycogen Storage disease, Tay-Sachs diseases, Familial dysautonomia, Mucolipidosis, Usher syndrome, and Neimann-Pick disease.

9. The method of claim 1, wherein the plurality of sub-regions comprises coding and non-coding regions of the target nucleic acid.