AU2015202048B2 - Ligation-based detection of genetic variants - Google Patents

Abstract

The present invention provides assays systems and methods for detection of genetic variants in a sample, including copy number variation and single nucleotide polymorphisms. The invention preferably employs the technique of tandem ligation, i.e. the ligation of two or more fixed sequence oligonucleotides and one or more bridging oligonucleotides complementary to a region between the fixed sequence oligonucleotides.

Description

LIGATION-BASED DETECTION OF GENETIC VARIANTS CROSS-REFERENCE TO RELATED APPLICATIONS 2015202048 22 Apr 2015 [0001] The present application claims priority to U.S. Ser. No. 13/013,732, filed January 25, 2011 and U.S. Ser. No. 61/371,605, filed August 6, 2010, both of which are herein incorporated by reference in their entirety.

[0001Α] This application is a divisional of Australian Patent Application No. 2011285512, the entire contents of which are herein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to multiplexed selection, amplification, and detection of targeted regions from a genetic sample.

BACKGROUND OF THE INVENTION

[0003] In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that he articles and methods referenced herein do not constiUlte prior art under the applicable statutory provisions.

[0004] Genetic abnormalities account for a wide number of pathologies, including pathologies caused by chromosomal aneuploidy (e.g., Down's syndrome), germline mutations in specific genes (e.g., sickle cell anemia), and pathologies caused by somatic mutations (e.g., cancer). Diagnostic methods for determining such genetic anomalies have become standard techniques for identifying specific diseases and disorders, as well as providing valuable information on disease source and treatment options.

[0005] Copy-number variations are alterations of genomic DNA hat correspond to relatively large regions of the genome that have been deleted or amplified on certain chromosomes. CNVs can be caused by genomic rearrangements such as 1 2015202048 22 Apr 2015 deletions, duplications, inversions, and translocations. Copy nuntber variation has been associated with various forms of cancer (Cappuzzo F, Hirsch, et al. (2005) 97 (9): 643-655) neurological disorders, including autism (Sebat, j., et al. (2007) Science 316 (5823) 445-9), and schizophrenia St Clair D (2008). Schizophr Bull 35 (1): 9-12. Detection of copy number variants of a chromosome of interest or a portion thereof in a specific cell population can be a powerful tool to identify genetic diagnostic or prognostic indicators of a disease or disorder.

[0006] Detection of copy number variation is also useful in detecting chromosomal aneuploidies in fetal DNA. Conventional methods of prenatal diagnostic testing cunently requires removal of a sample of fetal cells directly from fee utenis for genetic analysis, using either chorionic villus sampling (CVS) between 11 and 14 weeks gestation or amniocentesis after 15 weeks. However, these invasive procedures carry a risk of miscarriage of around 1% (Mujezinovic and Alfirevic, Obstet Gynecol (2007) 110:687-694). A reliable and convenient method for non-invasive prenatal diagnosis has long been sought to reduce this risk of miscarriage and allow earlier testing.

[0007] Single nucleotide polymorphisms (SNPs) are single nucleotide differences at specific regions of the genome. The average human genome typically has more than three million SNPs when compared to a reference genome. SNPs have been associated with various diseases, including cancer, cardiovascular disease, cystic fibrosis, and diabetes. Detection of SNPs can be a powerflil tool to identify genetic diagnostic or prognostic indicators of a disease or disorder. It is often desirable to detect many different SNPs in the same sample.

[0008] Re-sequencing is the use of DNA sequence detection, often in a portion of the genome. Re-sequencing can be applied towards the analysis of a genetic sample 2 2015202048 22 Apr 2015 from any source including mammals, other animal species, plants, bacteria, virnses, and the like. Re-sequencing can be used for many applications including but not limited to clinical applications and environmental applications. One use of re-sequencing for clinical applications is the detemination of the DNA sequence in a disease-causing gene. Examples of gene re-sequencing for medical diagnostic or prognostic indications include the re-sequencing of BRCAl and BRCA2 for breast cancer risk. An example of an environmental application would be the detection of a specific pathogen in a water source.

[0009] There is thus a need for methods of screening for copy number variations, SNPs and re-sequencing that employs an efficient, reproducible multiplexed assay. The present invention addresses this need. 3 2015202048 22 Apr 2015

SUMMARY OF THE INVENTION

[WIOIO] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential featares of he claimed subject matter, nor is it intended to be used to limit he scope of the claimed subject matter. Other features, details, utilities, and advantages of he claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[00011] The present invention provides assays systems and mehods for detection of copy number variation, polymorphisms, mutations and re-sequencing. The invention employs the technique of selecting genomic regions using fixed sequence oligonucleotides and joining them via ligation and/or extension. In a prefened aspect this is accomplished by tandem ligation, i.e. the ligation of two or more non-adjacent, fixed sequence oligonucleotides and a bridging oligonucleotide that is complementary to a region between and directly adjacent to the portion of the nucleic acid region of interest complementary to the fixed sequence oligonucleotides.

[00012] In one general aspect, the invention provides an assay system for detecting a nucleic acid region of interest in a genetic sample, contprising the steps of providing a genetic sample; inrioducing a first and second fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complement^ regions in the nucleic acid of interest; introducing one or more bridging oligonucleotides under conditions that allow the fixed sequence oligonucleotides to specifically 4 2015202048 22 Apr 2015 hybridize to complementary regions in the nucleic acid of interest, wherein the one or more bridging oligonucleotides are complementary to a region of the nucleic acid between and immediately adjacent to the region complementary to he first and second fixed sequence oligonucleotides; ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acid region of interest; amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region; and detecting and quantifying he amplification products, wherein detection of he amplification product provides detection of the nucleic acid region in the genetic sample. The amplification products are optionally isolated and quantified to determine the relative fiequency of the nucleic acid region in the genetic sample.

[00013] In another general aspect, the invention provides an assay system for detecting a nucleic acid region of interest in a genetic sample, comprising the steps of providing a genetic sample; infioducing a first and second fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in he nucleic acid of interest; inrioducing one or more bridging oligonucleotides complementary to a region of the nucleic acid of interest between the regions complementary to the first and second fixed sequence oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to the nucleic acid of interest, wherein at least one or more bases on either or both ends of the bridging oligonucleotide are not immediately adjacent to the fixed sequence oligonucleotides; extending the one or more bridging oligonucleotides so that the bridging oligonucleotides are immediately adjacent to the fixed sequence oligonucleotides; ligating the hybridized and extended oligonucleotides to create a 5 2015202048 22 Apr 2015 contiguous ligation product; amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region of interest; and detecting and quantifying the amplification products, wherein detection of the amplification product provides detection of the nucleic acid region in the genetic sample. The amplification products are optionally isolated and quantifie detemine the relative frequency of the nucleic acid region in the genetic sample.

[00014] The relative frequency of the nucleic acid in the sample can be used to detemine not only copy number variation for that particular nucleic acid region, but also in conjunction with and/or in comparison to other nucleic acids, it may be used to determine the copy number variation of larger genomic regions, including chromosomes.

[00015] The fixed sequence oligonucleotides used in the assay system preferably comprise universal primer regions that are used in amplification of the contiguous ligation product. Alternatively, he universal primer sequences can be added to the contiguous ligation products following the ligation of he hybridized fixed sequence and bridging oligonucleotides, e.g.y through he introduction of adapters comprising such universal primer sequences to the ends of the contiguous ligation product.

[116] The bridging oligonucleotides are preferably shorter oligonucleotides, preferably between 1-10 nucleotides and more preferably between 3-7 nucleotides, and can be designed to provide degeneracy within the sequence of the bridging oligonucleotides, e.g., the bridging oligonucleotides are provided as full or partial randomers with various sequence variations to ensure detection of the selected nucleic region even if the region contains a polymorphic reside. The degeneracy of the bridging oligonucleotide can be determined based on the 6 2015202048 22 Apr 2015 predicted polymorphisms that may be present in the selected nucleic acid region. Alternatively, he pool of bridging oligonucleotides used in a reaction can provide degeneracy for one or more position of he bridging oligonucleotide. In one aspect, the pool of bridging oligonucleotides used in a reaction can provide degeneracy for each position of the bridging oligonucleotide. In yet another aspect, the pool of bridging molecules used in a reaction can provide degeneracy for each internal position of the bridging oligonucleotide, with the nucleotides adjacent to the ligation sites remaining constant in the pool of bridging oligonucleotides used within the set. In another aspect, the bridging oligo is longer than 10 nucleotides and preferably 18-30 nucleotides. In a preferred aspect, a single bridging oligonucleotide complementary to a region of the nucleic acid of interest is hybridized between the region ز to the first and second fixed sequence oligonucleotides. In another aspect, two or more bridging oligonucleotides are hybridized within the region between the fixed sequence oligonucleotides, and preferably the bridging oligonucleotides hybridize to adjacent regions on he nucleic acid of interest. In this situation, ligation occurs between the fixed sequence oligonucleotides and the adjacent bridging oligonucleotides as well as between adjacent bridging oligonucleotides. In another aspect, there are one or more base gaps between the serial bridging oligonucleotides and/or one or more base gaps between the bridging oligonucleotides and fixed sequence oligonucleotides. These gaps can be extended, e.g., by use of polymerase and dNTPs prior to ligation.

[00017] It is an advantage that using degenerate bridging oligonucleotides obviates the need to predetermine the maternal and fetal polymorphic content for a 7 2015202048 22 Apr 2015 selected nucleic acid region prior to employing the detection methods of he assay system.

[00018] In one aspect of the invention, the first and second fixed sequence oligonucleotides are introduced to he genetic sample and specifically hybridized to the complement^ portions of the nucleic acids of interest prior to inttoduction of the bridging oligonucleotides. The hybridized regions are optionally isolated following he specific hybridization of the fixed sequence oligonucleotides to remove any excess unbound oligonucleotides in the reaction.

[00019] In another aspect, the bridging oligonucleotides are inttoduced to the genetic sample at the same time the fixed sequence oligonucleotides are introduced, and all are allowed to hybridize to a contiguous portion of he nucleic acid region of interest.

[00020] In certain aspects, the fixed sequence oligonucleotides of the invention comprise one or more indices. These indices may serve as sunogate sequences for the identification of he nucleic acid region of interest, a locus, or a particular allele of a locus. In particular, these indices may serve as sunogate detection sequences for the detection of hybridization of the nucleic acid region of interest to an array. Other indices may be used to conespond an amplification product to a particular sample, or to identify experimental error within the assay methods. In particular assays, the amplification product from the contiguous ligation product is identified and quantified using one or more indices as a sunogate to the actual sequence of the amplification product.

[00021] In specific assay systems, the first or second fixed sequence oligonucleotide comprise an allele index that associates a specific allele with that complementary fixed sequence oligonucleotide. 2015202048 22 Apr 2015 [00022] In another genera] aspect of the invention, an assay system is provided for detecting a nucJeic acid region of interest in a maternal sample comprising both maternal and fetal cell free DNA. This assay system comprises the steps of providing a maternal sample comprising cell free DNA from both maternal and fetal sources; introducing a first and second non-adjacent, fixed sequence oligonucleotide to the genetic sample under conditions that allow the fixed sequence oligonucleotides to specifically hybridize to complementary regions in the nucleic acid of interest; introducing one or more bridging oligonucleotides under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in he nucleic acid of interest, wherein one or more bridging oligonucleotides are complementary to a region of the nucleic acid between and immediately adjacent to he region complementary to he first and second fixed sequence oligonucleotides; ligating the hybridized oligonucleotides to create a contiguous ligation product complementary to the nucleic acid region of interest; amplifying the contiguous ligation product to create amplification products having the sequence of the nucleic acid region; and detecting and quantifying the amplification products; wherein quantification of the amplification product provides a relative frequency of the nucleic acid region in the maternal sample.

[00023] The relative frequency of the nucleic acid in the sample can be used to detemine not only copy number variation for that particular nucleic acid region, but also in conjunction with and/or in comparison to other nucleic acids, it may be used to determine the copy number variation of larger genomic regions, including chromosomal imbalance between maternal and fetal nucleic acid regions due to aneuploidy in the fetus. 2015202048 22 Apr 2015 [00024] invention also provides compositions hat ae useful in ligation-based nucleic acid detection assays such as those of he present invention. Accordingly, he invention provides sets of oligonucleotides for ligation-based detection of a nucleic acid region of interest, comprising a first oligonucleotide that comprises sequences complementary to he sequences of a first portion of a nucleic acid region, a universal primer sequence, and optionally one or more indices; a second oligonucleotide that comprises sequences complementary to the sequence of a second portion of a nucleic acid region and a universal primer sequence; and one or more bridging oligonucleotides hat are complementary to he region immediately adjacent and between he nucleic acid region complementary to he first and second oligonucleotides. In certain aspects, the set of oligonucleotides comprises two or more bridging oligonucleotides wih the ability to identify different polymorphisms within the nucleic acid of interest. In oher aspects, the bridging molecules provide degeneracy for each position of the bridging oligonucleotide. In yet other aspects, the bridging molecules provide degeneracy for each internal position of the bridging oligonucleotide, wih he nucleotides adjacent to he ligation sites remaining constant in the pool of bridging oligonucleotides used wihin the set.

[00025] These aspects and oher features and advantages of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE FIGURES

[00026] FIG. 1 illusriates a first general schematic for a ligation-based assay system of the invention. 10 2015202048 22 Apr 2015 [127] FIG. 2 illustrates a second general schematic for a ligation-based assay system of the invention.

[00028] FIG. 3 illustrates a multiplexed assay system for detection of two or more regions of interest.

[00029] FIG. 4 illustrates a first multiplexed assay system for detection of two or more alleles within a region of interest.

[130] FIG. 5 illustrates a second multiplexed assay system for detection of two or more alleles within a region of interest.

[131] FIG. 6 illustrates a third multiplexed assay system for detection of two or more alleles within a region of interest.

[132] FIG. 7 illustrates a fourth multiplexed assay system for detection of two or more alleles within a region of interest.

[00033] FIG. 8 illustrates a fifth multiplexed assay system for detection of two or more alleles within a region of interest.

[00034] FIG. 9 illustrates a first genera] schematic for assay system utilizing oligo extension in a ligation-based assay system of the invention.

[00035] FIG. 10 illustrates a second general schematic for assay system utilizing oligo extension in a ligation-based assay system of the invention.

[00036] FIG. 11 illustrates an assay system utilizing a single fixed sequence oligonucleotide.

[00037] FIG. 12 illustrates the genotyping performance that is obtained using one exemplary assay format.

[138] FIG. 13 is a graph illushating the ability of the assay system to determine percent fetal DNA in a maternal sample. 11 2015202048 22 Apr 2015

DEFINITIONS

[00039] The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary ski]] in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit he meaning of such terms unless specifically indicated.

[00040] The term “allele index" refers generally to a series of nucleotides that conesponds to a specific SNP. The allele index may contain additional nucleotides hat allow for the detection of deletion, substitution, or insertion of one or more bases. The index may be combined with any other index to create one index hat provides information for two properties (e.g., sample-identification index, allele-locus index).

[00041] The term “binding pair" means any two molecules that specifically bind to one another using covalent and/or non-covalent binding, and which can be used for attachment of genetic material to a substrate. Examples include, but are not limited to, ligands and their protein binding partners, e.g., biotin and avidin, biotin and srieptavidin, an antibody and its particular epitope, and the like.

[00042] The term “chromosomal abnormality" refers to any genetic variant for all or part of a chromosome. The genetic variants may include but not be limited to any copy number variant such as duplications or deletions, translocations, inversions, and mutations.

[00043] The terms “complementary" or “complementarity" are used in reference to nucleic acid molecules (i.e., a sequence of nucleotides) hat are related by base-pairing rules. Complementary nucleotides are, generally, A and T (or A and U), or c and G. Two single stranded RNA or DNA molecules are said to be 12 2015202048 22 Apr 2015 substantially complementary when the nucleotides of one strand, optimally aligned and with appropriate nucleotide insertions or deletions, pair with at least about 90% to about 95% complementarity, and more preferably from about 98% to about 100% complementarity, and even more preferably with 100% complementarity. Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Selective hybridization conditions include, but are not limited to, stringent hybridization conditions. Stringent hybridization conditions will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM. Hybridization temperatures are generally at least about 2٥c to about 6°c lower than melting temperatures (Tm).

[00044] The term “correction index” refers to an index that may contain additional nucleotides that allow for identification and correction of amplification, sequencing or other experimental errors including the detection of deletion, substitution, or insertion of one or more bases during sequencing as well as nucleotide changes that may occur outside of sequencing such as oligo synthesis, amplification, and any other aspect of he assay.

[00045] The term “diagnostic tool" as used herein refers to any composition or assay of the invention used in combination as, for example, in a system in order to carry out a diagnostic test or assay on a patient sample.

[00046] The term “genetic sample" refers to any sample comprising all or a portion of the genetic information of an organism, including but not limited to vims, bacteria, fungus, plants and animals, and in particular mammals. The genetic information that can be interrogated within a genetic sample includes 13 2015202048 22 Apr 2015 genomic DNA (both coding and non-coding regions), mitochondrial DNA, RNA, and nucleic acid products derived ftom each of these. Such nucleic acid products include cDNA created from mRNA or products of pre-amplification to increase he material for analysis.

[00047] 'fhe term “hybridization" generally means the reaction by which the pairing of complementary strands of nucleic acid occurs. DNA is usually double-srianded, and when the strands are separated they will re-hybridize under the appropriate conditions. Hybrids can form between DNA-DNA, DNA-RNA or RNA-RNA. They can form between a short strand and a long sriand containing a region complementary to the short one. Imperfect hybrids can also form, but the more imperfect they are, the less stable they will be (and the less likely to form).

[00048] The term “identification index" refers generally to a series of nucleotides that are incorporated into a primer for identification purposes, identification index sequences are preferably 6 or more nucleotides in length. In a preferred aspect, he identification index is long enough to have statistical probability of labeling each molecule with a target sequence uniquely. For example, if there are 3000 copies of a particular target sequence, here are substantially more than 3000 identification indexes such hat each copy of a particular target sequence is likely to be labeled with a unique identification index. The identification index may contain additional nucleotides hat allow for identification and correction of sequencing errors including the detection of deletion, substihJtion, or insertion of one or more bases during sequencing as well as nucleotide changes that may occur outside of sequencing such as oligo synthesis, amplification, and any other aspect of the assay. The index may be combined with any other index to create one index 14 2015202048 22 Apr 2015 that provides information for two properties (e.g., sample-identification index, allele-locus index).

[00049] As used herein the term “ligase" refers generally to a class of enzymes, DNA ligases (typically Τ4 DNA ligase), which can link pieces of DNA together. The pieces must have compatible endseiher with boh of hem blunt or with mutually-compatible sticky ends-and the reaction requires ATP. “Ligation” is the process of joining two pieces of DNA together.

[00050] The terms “locus" and “loci” as used herein refer to one or more nucleic acid regions of known location in a genome.

[00051] The term “locus index” refers generally to a series of nucleotides that conespond to a given genomic locus. In a preferred aspect, he locus index is long enough to label each target sequence region uniquely. For instance, if the method uses 192 target sequence regions, there are at least 192 unique locus indexes, each uniquely identifying each target region. The locus index may contain additional nucleotides that allow for identification and correction of sequencing enors including the detection of deletion, substitution, or insertion of one or more bases during sequencing as well as nucleotide changes that may occur outside of sequencing such as oligo synthesis, amplification, and any other aspect of the assay. The index may be combined with any other index to create one index hat provides information for two properties (e.g. sample-identification index, allele-locus index).

[152] The term “maternal sampfe” as used herein refers to any sample taken from a pregnant mammal which comprises boh fetal and maternal cell free DNA. Preferably, maternal samples for use in the invention are obtained *rough 15 2015202048 22 Apr 2015 relatively non-invasive means, e.g., phlebotomy or other standard techniques for extracting peripheral samples from a subject.

[00053] The term “melting temperature" or Tm is commonly defined as the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm = 81.5+16.6(logl0[Na+])0.41(%[G+C])-675/n-1.0m, when a nucleic acid is in aqueous solution having cation concentrations of 0.5 M or less, the (G+C) content is between 30% and 70%, n is the number of bases, and m is the %age of base pair mismatches (see, e.g., Sambrook ل et al.. Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratoiy Press (2001)). Other references include more sophisticated computations, which take stmctural as well as sequence characteristics into account for the calculation of Tm.

[00054] "Microarray" or "array" refers to a solid phase support having a surface, preferably but not exclusively a planar or substantially planar surface, which carries an array of sites containing nucleic acids such hat each site of the array comprises substantially identical or identical copies of oligonucleotides or polynucleotides and is spatially defined and not overlapping with other member sites of tire array; hat is, tire sites are spatially discrete. The array or microarray can also comprise a non-planar interrogatable strtJCture with a surface such as a bead or a well. The oligonucleotides or polynucleotides of tire array may be covalently bound to the solid support, or may be non-covalently bound. Conventional microarray technology is reviewed in, e.g., Schena, Ed., Microarrays: A Practical Approach, IRL Press, Oxford (2000). “Array 16 2015202048 22 Apr 2015 analysis”, “analysis by array” or “analysis by microarray” refers to analysis, such as, e.g., sequence analysis, of one or more biological molecules using a microanay.

[00055] The term “oligonucleotides” or “oligos” as used herein refers to linear oligomers of natural or modified nucleic acid monomers, including deoxyribonucleotides, ribonucleotides, anomeric forms hereof, peptide nucleic acid monomers (PNAs), locked nucleotide acid monomers (LNA), and the like, or a combination thereof, capable of specifically binding to a single-stranded polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 8-12, to several tens of monomeric units, e.g., 100-200 or more. Suitable nucleic acid molecules may be prepared by he phosphoramidite method described by Beaucage and Carruthers (Tetrahedron Lett., 22:1859-1862 (1981)), or by the triester method according to Matteucci, et al. (j. Am. Chem. Soc., 103:3185 (1981)), both incorporated herein by reference, or by other chemical methods such as using a commercial automated oligonucleotide synthesizer.

[00056] As used herein “nucleotide” refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid sequence (DNA and RNA). The term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives hereof. Such derivatives include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives hat 17 2015202048 22 Apr 2015 confer nuclease resistance on fee nucleic acid molecule containing them. The term nucleotide as used herein also refers to dideoxyribonucfeoside triphosphates (ddNTPs) and their derivatives. Illustrated examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

[157] According to fee present invention, a “nucleotide" may be unlabeled or detectably labeled by well known techniques. Fluorescent labels and their attachment to oligonucleotides are described in many reviews, including Ιι%λι1, Handbook of Fluorescent Probes and Research Chemicals, 1 Yi, Molecular Probes, fee., Eugene OR (2002); Keller and Manak, DNA Probes, 2nd Ed., Stockton Press, New York (1993); Eckstein, Ed., Oligonucleotides and Analogues: A Practical Approach, IRL Press, Oxford (1991); Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991); and fee like. Ofeer methodologies applicable to the invention are disclosed in the following sample of references: Fung et al., U.S. Pat. No. 4,757,141; Hobbs, Jr., et al., U.S. Pat. No. 5,151,507; Cmickshank, U.S. Pat. No. 5,091,519; Menchen et al., U.S. Pat. No. 5,188,934; Begot et al., U.S. Pat. No. 5,366,860; Lee et al., U.S. Pat. No. 5,847,162; Khanna et al., U.S. Pat. No. 4,318,846; Lee et al., U.S. Pat. No. 5,800,996; Lee et al., U.S. Pat. No. 5,066,580: Mathies et al., U.S. Pat. No. 5,688,648; and fee like. Labeling can also be canied out wife quantum dots, as disclosed in the following patents and patent publications: U.S. Pat. Nos. 6,322,901; 6,576,291; 6,423,551; 6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392; 2002/0045045; and 2003/0017264. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides 18 2015202048 22 Apr 2015 may include but are not limited fluorescein, 5-carboxyfluorescein (FAM), 2'7'_ dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aiiiinoethyl)aiiiinonaphthalene-1 -sulfonic acid (EDANS). Specific examples of fluroescently labeled nucleotides include [R6G]dUTP, [TAMRAJdUTP, [R110]dCTP, [R6G]dCTP١ [TAMRAJdCTP, [JOE]ddATP, [R6GJddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRAJddGTP, [ROX]ddTTP, [dR6GJddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink FluorX-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, IL; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-υΤΡ, andFluorescein-15-2'-dATP available from Boehringer Mannheim, Indianapolis, IN; and Chromosomee Labeled Nucleotides, B0DIPY-FL-14-UTP, B0DIPY-FL-4-UTP, BODIPY-TMR-14-υΤΡ, B0DIPY-TMR-14-dUTP, B0DIPY-TR-14-UTP, B0DIPY-TR-14-dUTP, Cascade Blue-7-υΤΡ, Cascade Blue-7-dUTP١ fluorescein-12-υΤΡ, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP١ Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP١ and Texas Red-12-dUTP available from Molecular Probes, Eugene, OR.

[00058] ٨s used herein the term “polymerase” refers to an enzyme that links individual nucleotides together into a long strand, using another strand as a 19 2015202048 22 Apr 2015 template. There are two general types of polymerase-DNA polymerases, which synthesize DNA, and RNA polymerases, which synthesize RNA. Within these two classes, there are numerous sub-types of polymerases, depending on what type of nucleic acid can function as template and what type of nucleic acid is formed.

[159] As used herein “polymerase chain reaction" or “PCR" refers to a technique for replicating a specific piece of target DNA in vitro, even in the presence of excess non-specific DNA. Primers are added to the target DNA, where the primers initiate the copying of the target DNA using nucleotides and, typically, Taq polymerase or the like. By cycling the temperature, the target DNA is repetitively denatared and copied. A single copy of the target DNA, even if mixed in with other, random DNA, can be amplified to obtain billions of replicates. The polymerase chain reaction can be used to detect and measure very small amounts of DNA and to create customized pieces of DNA. fn some instances, linear amplification methods may be used as an alternative to PCR.

[00060] The term “polymorphism” as used herein refers to any genetic changes or variants in a loci that may be indicative of that particular loci, including but not limited to single nucleotide polymorphisms (SNPs), methylation differences, short tandem repeats (STRs), and the like.

[00061] Generally, a “primer" is an oligonucleotide used to, e.g., prime DNA extension, ligation and/or synthesis, such as in the synthesis step of he polymerase chain reaction or in the primer extension techniques used in certain sequencing reactions. A primer may also be used in hybridization techniques as a means to provide complementarity of a nucleic acid region to a captare oligonucleoitide for detection of a specific nucleic acid region. 20 2015202048 22 Apr 2015 [162] The term "reseach too]" as used herein refers to any composition or assay of the invention used for scientific enquiry, academic or commercia] in nature, including the development of phamaceutical andor biological therapeutics. The reseach tools of the invention are not intended to be therapeutic or to be subject to regulatory approval," rather, the research tools of the invention ae intended to facilitate research and aid in such development activities, including any activities performed with the intention to produce information to support a regulatory submission.

[00063] ٠e terms ‘‘sequencing’’ as used herein refers generally to any and all biochemical methods that may be used to determine the order of nucleotide bases including but not limited to adenine, guanine, cytosine and thymine, in one or more molecules of DNA. As used herein the term “sequence determination” means using any method of sequencing known in he art to determine the sequence nucleotide bases in a nucleic acid.

[00064] The term “sample index” refers generally to a series of unique nucleotides (i.e., each sample index is unique), and can be used to allow for multiplexing of samples in a single reaction vessel such that each sample can be identified based on its sample index. In a preferred aspect, there is a unique sample index for each sample in a set of samples, and the samples are pooled during sequencing. For example, if twelve samples are pooled into a single sequencing reaction, there are at least twelve unique sample indexes such hat each sample is labeled uniquely. Tire sample index may contain additional nucleotides that allow for identification and correction of sequencing errors including dre detection of deletion, substitution, or insertion of one or more bases during sequencing as well as nucleotide changes that may occur outside of 21 2015202048 22 Apr 2015 sequencing such as oligo synthesis, amplification, and any other aspect of he assay. The index may be combined with any other index to create one index that provides information for two properties (e.g., sample-identification index, allele-locus index).

DETAILED DESCRIPTION OF THE INVENTION

[00065] The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illusriations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. Ι-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual·, Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory Manual·, Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual', Moiv (2.1), Bioinformatics: Sequence and Genome Analysis", ~ ١(!2اأ \\جا؟ا؟ل١ة١ \اعة Condensed Protocols from

Molecular Cloning: A Laboratory Manual·, and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New 22 2015202048 22 Apr 2015

York Ν.Υ.; Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., w. H. Freeman Pub., New York, Ν.Υ.; and Berg et al. (2002) Biochemistry, 5* Ed., W.H. Freeman Pub., New York, Ν.Υ., all of which are herein incorporated in their entirety by reference for all purposes.

[00066] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an allele" refers to one or more copies of allele with various sequence variations, and reference to “the assay system" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[00067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for he purpose of describing and disclosing devices, formulations and methodologies hat may be used in connection with the presently described invention.

[00068] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of hese smaller ranges may independently be included in he smaller ranges, and are also encompassed wilhin the invention, subject to any specifically excluded limit in he stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. 23 2015202048 22 Apr 2015 [00069] In the following description, numerous specific detais are set ίοΛ to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

The Invention in General [00070] The invention provides assay systems to identify copy number variants of nucleic acid regions (including loci, sets of loci and larger genomic regions, e.g.y chromosomes), mutations, and polymorphisms in a genetic sample and/or to select a portion of a genetic sample for re-sequencing in a genetic sample.

[00071] In one aspect, he assay system utilizes methods to selectively identify and/or isolate selected nucleic acid regions ftom two or more genomic regions of interest (e.g., chromosomes or loci) in a genetic sample, and allows determination of an atypical copy number of a particular genomic region based on the comparison between the numbers of detected nucleic acid regions from the two or more chromosomes in the genetic sample or by comparison to one or more reference chromosomes from the same or a different sample.

[00072] More particularly, the assay system utilizes a tandem ligation method comprising the use of first and second non-adjacent oligonucleotides of fixed sequence complement^ to a selected nucleic acid region on a chromosome of interest or a reference chromosome, and one or more short, bridging oligonucleotides (also called “splint" oligos) complementary to the region between and immediately adjacent to the first and second oligonucleotides. 24 2015202048 22 Apr 2015

Hybridization of these three or more oligonucleotides to a selected nucleic acid of interest, followed by ligation of these three or more oligonucleotides, provides a contiguous template for further amplification, detection and quantification of this region. The amplified regions may be quantified directly ftom the amplification reactions, or they are optionally isolated and identified to quantify the number of selected nucleic acid regions in a sample.

[00073] In specific aspects, the tandem ligation metirods use fixed seque oligonucleotides with a set of two or more contiguous, adjacent bridging oligonucleotides that hybridize to the region of the nucleic acid between the region complementary to the fixed sequence oligonucleotides. These bridging oligonucleotides hybridize adjacent to one another and to the fixed sequence oligonucleotides. The contiguous bridging oligonucleotides are ligated during tire ligation reaction with the fixed sequence oligonucleotides and with each other, resulting in a single contiguous template for forther amplification and sequence detemination.

[00074] In other aspects of tire invention, the assay system uses a set of oligonucleotides that bind to non-adjacent regions within a nucleic acid region of interest, and primer extension is utilized to create a contiguous set of hybridized oligos prior to the tandem ligation step. In such aspects, the assay system utilizes a tandem ligation method comprising the use of first and second non-adjacent oligonucleotides of fixed sequence complementary to a selected nucleic acid region on a chromosome of interest or a reference chromosome, and one or more short, bridging oligonucleotides complementary to the region between foe first and second oligonucleotides but not immediately adjacent to one or the other fixed sequence oligonucleotide. Hybridization offoese three or more oligonucleotides to 25 2015202048 22 Apr 2015 a selected nucleic acid of interest is followed by an extension reaction using dNTPs and a polymerase to create a set of adjacent hybridized oligonucleotides, and ligation of he adjacent hybridized oligos. The combination of extension and ligation provides a contiguous template for further amplification, detection and quantification of this region. The amplified regions may be quantified directly from the amplification reactions, or they are optionally isolated and identified to quantify the number of selected nucleic acid regions in a sample.

[175] fn specific aspects, the tandem ligation mehods use fixed sequence oligonucleotides with a set of two or more sequential but non-adjacent bridging oligonucleotides that hybridize to the region of the nucleic acid between the region complementary to the fixed sequence oligonucleotides. The “gap" regions between the fixed sequence oligonucleotides and the bridging oligos and/or between the sequential bridging oligonucleotides are ligated during the ligation reaction, resulting in a single contiguous template for further amplification and sequence determination.

[00076] In preferred aspects of the invention, the nucleic acids fro sample are associated with a substrate, e.g., using binding pairs to attach the genetic material to a substrate surface. Briefly, a first member of a binding pair (e.g., biotin) can be associated with a nucleic acid of interest, and the associated nucleic acid attached to a substrate comprising a second member of a binding pair (e.g., avidin or streptavidin) on its surface. This can be particularly useful in removing any unhybridized oligonucleotides following specific binding of foe fixed sequence oligonucleotides and/or the bridging oligonucleotides to foe nucleic acid of interest. Briefly, the attached nucleic acids can be hybridized to foe oligonucleotides, and die surface preferably heated to remove any 26 2015202048 22 Apr 2015 unhybridized oligonucleotides, e.g., by washing or other removal methods such as degradation of such oligonucleotides as discussed in Willis et al.١ U.S. Pat Nos. 7,700,323 and 6,858,412.

[00077] There are a number of methods drat may be used in the association of a nucleic acid via binding pair interactions, as will be apparent to one skilled in the art upon reading he present specification. For example, numerous methods may be used for labeling he nucleic acids of a genetic sample with biotin, including random photobiotinylation, end-labeling with biotin, replicating with biotinylated nucleotides, and replicating with a biotin-labeled primer.

[00078] In a preferred aspect, the assay system of the invention employs a multiplexed reaction with a set of three or more such oligonucleotides for each selected nucleic acid region. This general aspect is illustrated in FIG. 1. Each set of oligonucleotides preferably contains two oligonucleotides 101, 103 of fixed sequence and one or more bridging oligonucleotides 113. Each of foe fixed sequence oligonucleotides comprises a region complementary to the selected nucleic acid region 105, 107, and preferably universal primer sequences 109, 111, i.e. oligo regions complementary to universal primers. These universal primer sequences 109, 111 are used to amplify the different selected nucleic acid regions following ligation of foe hybridized fixed sequence oligonucleotides and the bridging oligonucleotide. The universal primer sequences are located at or near the ends of the fixed sequence oligonucleotides 101, 103, and thus preserve the nucleic acid-specific sequences in the products of any universal amplification methods. Aniplification products can be detected by determination of the sequence of the products, e.g., through sequence determination or hybridization, e.g„ to an array or a bead-based detection system such as the Luminex™ bead- 27 2015202048 22 Apr 2015 based assay (Invitrogen, Carlsbad, CA) or the BeadXpressTM assay (Illumina, San Diego, CA).

[00079] In one aspect of the assay systems of the invention, the fixed sequence oligonucleotides 101, 103 are introduced 102 to the genetic sample 100 and allowed to specifically bind to the complement^ portions of the nucleic acid region of interest 115. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligonucleotide is then introduced and allowed to bind 104 to the region of the selected nucleic acid region 115 between the first 101 and second 103 fixed sequence oligonucleotides. Alternatively, the bridging oligo can be introduced simultaneously to the fixed sequence oligonucleotides. The bound oligonucleotides are ligated 106 to create a contiguous nucleic acid spanning and complementary to the nucleic acid region of interest. Following ligation, universal primers 117, 119 are inrioduced to amplify 108 he ligated template region to create 110 products 121 hat comprise he sequence of the nucleic acid region of interest. These products 121 are optionally isolated, detected, and quantified to provide information on the presence and amount of the selected nucleic acid region in a genetic sample. Preferably, the products are detected and quantified through sequence determination of the product, and in particular sequence determination of the region of the product corresponding to the selected nucleic acid region.

[00080] The number of selected nucleic acid regions analyzed for each chromosome in the assay system of the invention may vary front 2 - 20,000 or more per chromosome analyzed. In a prefened aspect, the number of targeted regions is between 48 and 480. In another aspect, the number of targeted regions 28 2015202048 22 Apr 2015 is at least 100. In another aspect, the number of targeted regions is at least 400. In another aspect, the number of targeted regions is at least 1000.

[00081] In certain aspects, the bridging oligos can be composed of mixtures of oligos with degeneracy in each of the positions, so that the mixture of randomers used will be compatible with all reactions in the multiplexed assay requiring a bridging of the given length. In another aspect, the bridging oligos can be of various lengths so that the mixture of oligos will be compatible with particular tandem ligation reactions in the multiplexed assay requiring bridging oligos of the given lengths.

[00082] In yet another aspect the bridging oligo can have partial degener the multiplexed tandem ligation reactions are restricted to those that require the specific sequences provided by he degeneracy of the bridging oligos. For example, a set of tandem ligation reactions may require only A and c bases in the bridging oligo, and a mixtare of bridging oligos synthesized with only A and c bases would be provided for these particular tandem ligation reactions in a multiplexed assay.

[183] In yet another aspect, he bridging oligo sequences are designed such hat only hose assays hat have the given specific sequences in the bridging region would be multiplexed in he assay system. In one example the bridging oligo is a randomer, where all combinations of the bridging oligo are synthesized. As an example, in he case where a 5-base oligo is used, he number of unique bridging oligos would be 45 = 1024. This would be independent of he number of targeted regions since all possible bridging oligos would be present in the reaction. 29 2015202048 22 Apr 2015 [00084] In another example the bridging oligo is specific, synthesized to match the sequences in the gap. As an example, in the case where a 5-base oligo is used, the number of unique oligos synthesized would be equal to or less than the number of targeted regions. A number less than the number of targeted regions could be achieved if the gap sequence was shared between two or more targeted regions. In one aspect of this example, one might purposelully choose the targeted sequences and especially the gap sequences such that there was as much identical overlap as possible in he gap sequences, minimizing he number of bridging oligos necessary for the multiplexed reaction.

[00085] In another aspect, the sequences of the bridging oligos are designed and the nucleic acid regions are selected so that all selected nucleic acid regions share the same base(s) at each end of the bridging oligo. For instance, one might choose selected nucleic acids and their gap location such that all of the gaps shared an “A" base at the first position and a "G” base at the last position of he gap. Any combination of a first and last base could be utilized, based upon factors such as the genome investigated, the likelihood of sequence variation in hat area, and the like. In a specific aspect of his example, the bridging oligos can be synthesized by random degeneracy of bases at the internal positions of he bridging oligo, specific addition at the first and last position. In the case of a 5-mer, he second, third and fourth positions would be randomly provided, and two specific nucleotides would be added at the proximal positions. In this case, he number of unique bridging oligos would be 43 = 64.

[186] In the human genome the frequency of the dinucleotide CG is much lower than expected by he respective mononucleotide fiequencies. This presents an opportunity to enhance he specificity of an assay with a particular mixture of 30 2015202048 22 Apr 2015 bridging oligos. In this aspect, the bridging oligos may be selected to have a 5' G and a 3' c. This base selection allows each oligo to have a high frequency in the human genome but makes it a rare event for two bridging oligos to hybridize adjacent to each other. The probability is hen reduced that multiple oligos are ligated in locations of the genome that are not targeted in the assay.

[187] The bridging oligo is preferably added to the reaction after the fixed sequence oligonucleotides have been hybridized, and following the optional removal of all unhybridized fixed sequence oligonucleotides have been washed away. The conditions of the hybridization reaction are preferably optimized near the Tm of the bridging oligo to prevent erroneous hybridization of oligos that are not fully complementary to the nucleic acid region. If the bridging oligos have a Tm significantly lower than the fixed sequence oligonucleotides, the splint oligo is preferably added as a part of the ligase reaction.

[00088] The advantage of using short oligos is that ligation on either end would likely occur only when all bases of the bridging oligo match the gap sequence. A further advantage of short bridging oligos is that he number of different oligos necessary could be less than the number of targeted sites, raising the oligos effective concenriation to allow perfect matches to happen faster. Fewer oligos also has advantages in cost and quality conriol. The advantages of using fixed first and last bases with random bases in between include the ability to utilize longer bridging oligos for better specificity while reducing the number of total bridging oligos in the reaction.

Use of Indices in the Assay Systems of the Invention 31 2015202048 22 Apr 2015 [189] Jn certain aspects, all or a portion of the nucleic acids of interest are directly detected using the described techniques. In certain aspects, however, he nucleic acids of interest are associated with one or more indices that are identifying for a selected nucleic acid region and/or a particular sample being analyzed. The detection of the one or more indices can serve as a sunogate detection mechanism of the selected nucleic acid region, or as confirmation of he presence of a particular selected nucleic acid region if both the index and the sequence of the nucleic acid region itself are determined. These indices are preferably associated with he selected nucleic acids during an amplification step using primers that comprise both the index and sequence regions that specifically hybridize to the nucleic acid region.

[001] In one example, he primers used for amplification of a selected nucleic acid region are designed to provide a locus index between he selected nucleic acid region primer region and a universal amplification region. The locus index is unique for each selected nucleic acid region and representative of a locus on a chromosome of interest or reference chromosome, so that quantification of he locus index in a sample provides quantification data for the locus and the particular chromosome containing he locus.

[00091] In another aspect, the primers used for amplification of the selected nucleic acid regions are designed to provide a random index between the selected nucleic acid region primer region and a universal amplification region. In such an aspect, a sufficient number of identification indices are present to uniquely identify each selected nucleic acid region in he sample. Each nucleic acid region to be analyzed is associated with a unique identification index, so that the identification index is uniquely associated with the selected nucleic acid region. 32 2015202048 22 Apr 2015

Quantification of the identification index in a sample provides quantification data for the associated selected nucleic acid region and he chromosome corresponding to the selected nucleic acid region. The identification locus may also be used to detect any amplification bias that occurs downstteam of he initial isolation of die selected nucleic acid regions from a sample.

[00092] In certain aspects, only the locus index and/or the identification index (if present) are detected and used to quantify die selected nucleic acid regions in a sample. In another aspect, a count of die number of times each locus index occurs with a unique identification index is done to determine die relative frequency of a selected nucleic acid region in a sample.

[00093] The primers are preferably designed so that indices comprising identifying infomation are coded at die ends of the primer flanking die region complementaty to the nucleic acid of interest. The indices are non-complementaty but unique sequences used within the primer to provide infomation relevant to the selective nucleic acid region that is isolated and/or amplified using die primer. The advantage of this is that information on the presence and quantity of die selected nucleic acid region can be obtained without the need to determine die actual sequence itself, although in certain aspects it may be desirable to do so. Generally, however, the ability to identify and quantify a selected nucleic acid region dirough identification of one or more indices will decrease the length of sequencing required as the loci information is caphired at die 3’ or 5’ end of the isolated selected nucleic acid region. Use of indices as a sunogate for identification of selected nucleic acid regions may also reduce enor since longer sequencing reads are more prone to the introduction or error. 33 2015202048 22 Apr 2015 [00094] In addition to locus-specific indices and identification indices, additional indices can be introduced to primers to assist in the multiplexing of samples. In addition, indices which identify sequencing error, which allow for highly multiplexed amplification techniques or which allow for hybridization or ligation or attachment to another surface can be added to the primers. The order and placement of these indices, as well as the length of these indices, can vary.

[00095] The primers used for identification and quantification of a selected nucleic acid region may be associated with regions complementary to the 5’ of the selected nucleic acid region, regions complementary to the 3’ of the selected nucleic acid region, or in certain amplification regimes the indices may be present on one or both of a set of amplification primers complementary to the selected nucleic acid region. The primers can be used to multiplex the analysis of multiple selected nucleic acid regions to be analyzed witltin a sample, and can be used either in solution or on a solid substtate, e.g., on a microarray or on a bead. These primers may be used for linear replication or amplification, or they may create circular constmcts for further analysis.

[001] Thus, in some aspects one or both of the fixed sequence oligonucleotides further contain an index region. This index region may comprise a number of different sequences hat can be used to identify he selected nucleic acid region and/or the sample being analyzed in the assay system. Preferably, he index region corresponds to he selected nucleic acid region, so hat identification of the index region can be used as a surrogate for detection of the actual sequence of the selected nucleic acid region. The index region may optionally comprise a 34 2015202048 22 Apr 2015 sample index to identify the oligo set as being from a particular sample in a multiplexed assay system.

[00097] FIG. 2 illustrates the use of a single index region 221 on a first fixed sequence oligonucleotide 201 in an oligo set for a selected nucleic acid region. The fixed sequence oligonucleotides 201, 203 are introduced 202 to the genetic sample 200 and allowed to specifically bind to the selected nucleic acid region 215. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligo is then introduced and allowed to hybridize 204 to he region of the selected nucleic acid region 215 between he first 201 and second 203 fixed sequence oligonucleotides. The bound oligonucleotides are ligated 206 to create a contiguous nucleic acid spanning and complementary to the nucleic acid region of interest. Following ligation, universal primers 217, 219 are introduced to amplify 208 the ligated template region to create 210 products 223 that comprise the sequence of the nucleic acid region of interest. These products 223 are optionally isolated, detected, and/or quantified to provide information on the presence and amount of the selected nucleic acid region in a genetic sample. Preferably, the products are detected and quantified through sequence determination of the index, thus obviating the need for determining the actual sequences of the selected nucleic acid region. In other aspects, however, it is desirable to determine the product comprising sequences of both the index and the selected nucleic acid region, for example, to provide internal confirmation of the results or where the index provides sample information and is not infomative of the selected nucleic acid region. In another aspect, the index permits unique hybridization to a feature 35 2015202048 22 Apr 2015 on an array, such hybridization Jeading to the detection and quantification of the sequences.

[00098] The use of indices is especially useful in a multiplexed assay setting where two or more different selected nucleic acid regions are being simultaneously detected in a genetic sample. FIG. 3 illustrates an example where two different selected nucleic acid regions are detected in a single tandem reaction assay. Two sets of fixed sequence oligonucleotides (301 and 303, 323 and 325) that specifically hybridize to two different nucleic acid regions 315, 331 are introduced 302 to a genetic sample and allowed to hybridize 304 to the respective nucleic acid regions. Each set comprises an oligonucleotide 301, 323 having a sequence specific region 305, 327, a universal primer region 309 and an index region 321, 335. The other fixed sequence oligonucleotide of the sets comprise a sequence specific region 307, 329 and a universal primer region 311. Following hybridization, he unhybridized fixed sequence oligonucleotides are preferably separated ftom he remainder of he genetic sample (not shown). The bridging oligos 313, 333 are introduced to the hybridized fixed sequence oligonucleotide/nucleic acid regions and allowed to hybridize 306 to these regions. Although shown in FIG. 3 as two different bridging oligos, in fact the same bridging oligo may be suitable for both hybridization events, or they may be two oligos ftom a pool of degenerate oligos that are used with multiple tandem ligation events. The bound oligonucleotides are ligated 308 to create a contiguous nucleic acid spanning and complementary to he nucleic acid region of interest. Following ligation, universal primers 317, 319 are inftoduced to amplifj, 310 the ligated template regions to create 312 amplification products 337, 339 that comprise the sequence of the nucleic acid regions of interest. These products 337, 36 2015202048 22 Apr 2015 339 ae optionally isolated, detected and/or quantified to provide information on the presence and amount of the selected nucleic acid region in a genetic sample.

[001] In multiplexed assay systems, he products are detected and quantified dirough sequence determination of the different indices, thus obviating the need for determining the acfeal sequences of he selected nucleic acid region. In oher aspects, however, the index may be a sample specific index as well as a region specific index, and thus the index may not only identify the nucleic acid region, but it may also provide information of fee nucleic acid region and the genetic sample ftom which he region was obtained. Alternatively, the nucleic acid region of the product may be detected, for example, to provide internal confirmation of the results or where he index provides solely sample information and is not informative of the selected nucleic acid region.

Detection of Polymorphic Regions using the Ligation-based Assay System [000100] In certain aspects, the assay system of the invention detects one regions that comprises a polymorphism. This methodology is not primarily designed to identify a particular allele, e.g., as maternal versus fetal, but rather to ensure that different alleles conesponding to a nucleic acid region of interest are included in the quantification methods of the invention. In certain aspects, however, it may be desirable to both use the information to count all such nucleic acid regions in the genetic sample as well as to use the information on specific polymorphisms, e.g., to calculate the amount of fetal DNA contained within a maternal sample, or identify the percentage of alleles with a particular mutation in a genetic sample from a cancer patient. Thus, the invention is intended to encompass both mechanisms for detection of SNP-containing nucleic acid regions 37 2015202048 22 Apr 2015 for direct determination of copy number variant through quantification as well as detection of SNPs for ensuring overall efficiency of the assay.

[1101] Thus, in a particular aspect of the invention, allele-discrimination is provided through the bridging oligo. In this aspect, the bridging oligo is located over a SNP. In this aspect, the polymorphism is preferably located close enough to one end of a ligation reaction as to provide allele specificity.

[000102] In one example of allele detection, both complementary allele bridging oligo variants are present in the same reaction mixture and allele detection results from subsequent sequencing through the polymorphism of he ligated products or their amplification products. FIG. 4 illustrates this aspect.

[000103] In FIG. 4, two fixed sequence oligonucleotides 401, 403 and bridging oligonucleotides corresponding to the two possible SNPs in the nucleic acid regions of interest 415, 429 are used in detection of the selected nucleic acid region, and preferably to detect the region in a single reaction. Each of the fixed sequence oligonucleotides comprises a region complementary to the selected nucleic acid region 405, 407, and universal primer sequences 409, 411 used to amplify the different selected nucleic acid regions following initial selection and/or isolation of he selected nucleic acid regions from the genetic sample. The universal primer sequences are located at the ends of the fixed sequence oligonucleotides 401, 403, and thus preserve the nucleic acid-specific sequences in he products of any universal amplification methods. The fixed sequence oligonucleotides 401, 403 are inttoduced 402 to he genetic sample 400 and allowed to specifically bind to the selected nucleic acid region 415, 429. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The 38 2015202048 22 Apr 2015 bridging oligos corresponding to an Α/Τ SNP 413 or a G/C SNP 433 are introduced and allowed to bind 404 to the region of the selected nucleic acid region 415, 429 between the first 401 and second 403 fixed sequence oligonucleotides. Alternatively, the bridging oligos 413, 433 can be introduced to he sample simultaneously with the fixed sequence oligonucleotides.

[1104] The bound oligonucleotides are ligated 406 to create a contiguous nucleic acid spanning and complementary to he nucleic acid region of interest. Following ligation, universal primers 417, 419 are introduced to amplify 408 the ligated template region to create 410 products 421, 423 that comprise the sequence of he nucleic acid region of interest representing both SNPs in he selected nucleic acid region. These products 421, 423 are detected and quantified through sequence determination of die product, and in particular the region of die product containing the SNP in die selected nucleic acid region.

[000105] In anodier example, the allele detection results from the sequencing of a locus index or an allele index which is provided in one or both of the fixed sequence nucleic acid region oligonucleotides. The locus index and/or allele index is embedded in either the first or second fixed sequence oligonucleotide used in the set for a selected nucleic acid region containing a polymorphism, and is used with eidier a specific fixed sequence oligo or widi a particular bridging oligo, either of which may be designed to detect the polymorphism. Detection of the locus index and/or the allele index in an amplification product allows detection of the presence, amount or absence of a specific allele present in a genetic sample, as well as the number of counts for the region through addition of the polymorphic regions detected in the sample. Two examples of how this may be performed are described in more detail below. 39 2015202048 22 Apr 2015 [000106] For example, in one aspect of the invention, two or more separate reactions are carried out using a single locus index and different bridging oligos corresponding to the different polymorphisms in the region complementary to the bridging oligos. The reactions are differentiated by the bridging oligo, and the ligation, amplification and detection reactions comprising the different bridging oligos remain separate through the detection step. The total counts for a particular nucleic acid region of interest can be determined mathematically using the locus index by adding the detected numbers of he counts for the nucleic acid region from the separate reactions comprising foe bridging oligos having different polymorphic sequences.

[000107] This aspect may be usefol for, e.g., circumstances in which both infomation on polymorphic ffequency in a sample and information on total loci counts are desirable. Since the reactions are detected separately, only one index may be needed for detection in each of the separate reactions, although separate allele indices may also be used in the separate reactions.

[000108] FIG. 5 illustrates one such aspect of the assay system of the invention. Two fixed sequence oligonucleotides 501, 503 and bridging oligonucleotides conesponding to the two possible SNPs in the selected nucleic acid region 515, 525 are used in detection of a nucleic acid region of interest. Each of the fixed sequence oligonucleotides comprises a region complementary to the selected nucleic acid region. The ligation, amplification, and detection steps of the assay system take place in two separate reactions, with a first reaction utilizing a first bridging oligo 513 and the second reaction utilizing a second bridging oligo 533. Both reactions utilize the same fixed sequence oligos 501, 503 having the same regions complementary to allele-specific regions 505, 507. A single locus index 40 2015202048 22 Apr 2015 521 can be used to detect the amplification products in each reaction so that sequence determination of he actaal sequence of he nucleic acids of interest are not necessarily needed, although they may stdl be determined to identify or provide confirmation of the sequence. The universal primer sequences 509, 511 are located at either end flanking the fixed sequence oligonucleotides 501, 503, and thus preserve the nucleic acid-specific sequences and the indices in the products of any universal amplification methods. The fixed sequence oligonucleotides 501, 503 are inhoduced 502 to the genetic sample 500 and allowed to specifically bind to the selected nucleic acid region 515, 525. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligos conesponding to an Α/Τ SNP 513 or a G/C SNP 533 are introduced in separate reactions and allowed to bind 504 to he region of the selected nucleic acid region 515, 525 between he first 505 and second 507 fixed sequence oligonucleotides. Alternatively, he bridging oligos 513, 533 can be introduced to the sample simultaneously with he fixed sequence oligonucleotides.

[000109] The bound oligonucleotides are ligated 506 to create a contiguous nucleic acid spanning and complementary to he nucleic acid region of interest. Following ligation, universal primers 517, 519 are inhoduced to amplify 508 he ligated template region to create 510 products 527, 529 hat comprise he sequence of the nucleic acid region of interest representing boh SNPs in he selected nucleic acid region. These products 527, 529 are detected and quantified dirough sequence determination of he product, and in particular he locus index combined with the knowledge of which bridging oligo was added to which 41 2015202048 22 Apr 2015 reaction. The counts for the nucieic acid region as a whole can be determined through addition of the detected polymorphic regions in the two reactions.

[000110] A different specific aspect of the invention utilizes alle indentify alleles comprising different polymorphisms as well as to detemine counts of the nucleic acid region of interest. In a multiplexed reaction, locus indices may be combined with allele indices. In this aspect, two or more separate ligation reactions are canied out using two or more different bridging oligos corresponding to the different polymorphisms in the region complement^ to the bridging oligos. The reactions are differentiated by the bridging oligo, and each bridging oligo is used with a fixed sequence oligo comprising an allele index that identifies that particular bridging oligo. Following the ligation step, the reactions can be combined either prior to amplification, since the same universal primers are preferably used, or prior to detection, as the different alleles can be distinguished through identification of he different allele-specific indices. The allele may also be distinguished through sequence determination of he allele index or alternatively from hybridizing of he allele index, and total counts for the nucleic acid region can be determined through the addition of he identified allelic regions.

[000111] In FIG. 6, two fixed sets of sequence oligonucleotides are used which comprise substantially the same sequence-specific regions 605, 607 but which comprise different indices, 621, 623 on one of the fixed sequence oligonucleotides of the set. The ligation reactions are carried out with material from the same genetic sample 600, but in separate tabes with the different allele-specific oligo sets. The bridging oligonucleotides corresponding to tae two possible SNPs in tae selected nucleic acid region 613, 633 are used in detection of the selected nucleic 42 2015202048 22 Apr 2015 acid region in each ligation reaction. Two allele indices 621, 623 hat are indicative of the particular polymorphic alleles can be used to detect die amplification products so that sequence determination of the actual sequence of he nucleic acids of interest are not necessarily needed, although these sequences may still be determined to identify and/or provide confirmation of the sequence. Each of he fixed sequence oligonucleotides comprises a region complementary to he selected nucleic acid region 605, 607, and universal primer sequences 609, 611 used to amplify the different selected nucleic acid regions following initial selection and/or isolation of he selected nucleic acid regions ffom he genetic sample. The universal primer sequences are located at he ends of he fixed sequence oligonucleotides 601, 603, and 623 flanking he indices and he regions complementaty to the nucleic acid of interest, thus preserving the nucleic acid-specific sequences and he allele indices in he products of any universal amplification methods. The fixed sequence oligonucleotides 601, 603, 623 are introduced 602 to an aliquot of the genetic sample 600 and allowed to specifically bind to the selected nucleic acid regions 615 or 625. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of he genetic sample (not shown).

[1112] The bridging oligos corresponding to an Α/Τ SNP 613 or a G/C SNP 633 are inrioduced and allowed to bind 604 to (he region of the selected nucleic acid region 615 or 625 between he first 605 and second 607 nucleic acid-complementary regions of the fixed sequence oligonucleotides. Alternatively, the bridging oligos 613, 633 can be introduced to the sample simultaneously with the fixed sequence oligonucleotides. The bound oligonucleotides are ligated 606 in 43 2015202048 22 Apr 2015 the single reaction ntixtore to create a contiguous nucleic acid spanning and complementary to he nucleic acid region of interest.

[1113] Following ligation, the separate reactions are preferably coniliined for the universal amplification and detection steps. Universal primers 617, 619 are introduced to the combined reactions to amplify 608 the ligated template regions and create 610 products 627, 629 that comprise the sequence of the nucleic acid region of interest representing both SNPs in the selected nucleic acid region. These products 627, 629 are detected and quantified through sequence determination of the product, through the allele index and/or he region of the product containing the SNP in the selected nucleic acid region.

[1114] Preferably, the producte of he FIG. 6 methods are detected and quantified dirough sequence determination of he allele indices, thus obviating he need for detemining the actaal sequences of the selected nucleic acid region. In other aspects, however, it is desirable to detertuine the product comprising sequences of both the index and the selected nucleic acid region, for example, to provide internal confirmation of the results or where the index provides sample information and is not informative of the selected nucleic acid region.

[000115] The indices used with the assay systems of the invention can also be used to identify polymorphisms that are associated with the fixed sequences used for the detection of nucleic acids of interest. Thus, in another exemplar assay system, an allele index is associated with an allele-specific fixed sequence oligonucleotide, and he allele detection results from the sequencing of an allele index or alternatively ftom hybridizing of an allele index which is provided in the nucleic acid region primer. The allele index is embedded in either the allele-specific first or second fixed sequence oligonucleotide used in the set for a 44 2015202048 22 Apr 2015 selected nucleic acid region containing a polymorphism. In specific aspects, an allele index is present on boh he first and second fixed sequence oligonucleotides to detect two or more polymorphisms within the fixed sequence regions. The number of fixed sequence oligonucleotides used in such aspects can correspond to the number of possible alleles being assessed for a selected nucleic acid region, and sequence determination or hybridization of the allele index can detect presence, amount or absence of a specific allele is a genetic sample.

[000116] FIG. 7 illustrates this aspect of the invention. In FIG. 7, three fixed sequence oligonucleotides 701, 703 and 723 are used. Two of the fixed sequence oligonucleotides 701, 723 are allele-specific, comprising a region complementary to an allele in a nucleic acid region comprising for example an Α/Τ or G/C SNP, respectively. Each of the fixed allele-specific oligonucleotides 701, 723 also comprises a conesponding allele index 721, 731 and a universal primer sequence 709. The second fixed sequence oligonucleotide 703 has another universal primer sequence 711, and these universal primer sequences are used to amplify the ١nucleic acid regions following initial selection and/or isolation of the nucleic acid regions from the genetic sample. The universal primer sequences are located at the ends of the fixed sequence oligonucleotides 701, 703, 723 flanking the indices and the nucleic acid regions of interest, and thus preserve the nucleic acid-specific sequences and he indices in he products of any universal amplification methods.

[1117] The fixed sequence oligonucleotides 701, 703, 723 are introduced 702 to the DNA sample 700 and allowed to specifically bind to the selected nucleic acid region 715, 725. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligos 713 are introduced and allowed to bind 45 2015202048 22 Apr 2015 704 to the nucleic acid 715 complementary to the region between the first allele-specific fixed sequence oligonucleotide region 705 and the other fixed sequence oligonucleotide region 707 or to he nucleic acid 725 complementary to the region between the second allele-specific fixed sequence oligonucleotide region 735 and the other fixed sequence oligonucleotide region 707. Alternatively, he bridging oligos 713 can be inttoduced to the sample simultaneously with the sets of fixed sequence oligonucleotides.

[1118] The bound oligonucleotides are ligated 706 to create a contiguous nucleic acid spanning and complementary to the nucleic acid region of interest. The ligation primarily occurs only when the allele-specific ends match. Following ligation, universal primers 717, 719 are introduced to amplifj, 708 the ligated template region to create 710 products 727, 729 that comprise the sequence of the nucleic acid region of interest representing both SNPs in the selected nucleic acid region. These products 727, 729 are detected and quantified dirough sequence detemination of the product, and in particular the region of the product containing the SNP in the selected nucleic acid region. Alternatively the products 727, 729 are detected and quantified through hybridization of the allele index to different features on an array. In this detection method, a fluorescent label is incorporated into the products 727, 729 during the universal amplification by amplifying with primers 717 or 719 that are fluorescently labeled. It is important to note drat the ligation 706 is allele-specific. In order to make the ligation allele-specific, the allele-specifying nucleotide must be close to the ligated end. Typically, the allele-specific nucleotide must be within 5 nucleotides of the ligated end. In a prefened aspect, the allele-specific nucleotide is the teminal base. 46 2015202048 22 Apr 2015 [1119] In another example, the allele detection results ftom he hybridization of a locus index to an array. Each allele is detected through an allele-specific labeling step, where each allele is labeled with a spectrally distinct fluorescent label during he universal amplification. FIG. 8 illushates his aspect of he invention. In FIG. 8, three fixed sequence oligonucleotides 801, 803 and 823 are used. Two of the fixed sequence oligonucleotides 801, 823 are allele-specific comprising a region matching a particular allele in he same selected nucleic acid region, a corresponding locus index 821 and allele-s^cific universal primer sequences 809, 839. The matching fixed sequence oligonucleotide 803 has another universal primer sequence 811. The universal primer sequences are used to amplify the different selected nucleic acid regions following initial selection and/or isolation of he selected nucleic acid regions fiom he genetic sample and incorporate a label into the amplification products that distinguish each allele. The universal primer sequences are located at the ends of the fixed sequence oligonucleotides 801, 803, 823 and thus preserve the nucleic acid-specific sequences and he indices in the products of any universal amplification methods. The fixed sequence oligonucleotides 801, 803, 823 are infioduced 802 to the DNA sample 800 and allowed to specifically bind to the selected nucleic acid region 815, 825. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligos 813 are infioduced and allowed to bind 804 to the region of he selected nucleic acid region 815, 825 between the first 805 and second 807 fixed sequence oligonucleotides and between the first 835 and second 807 fixed sequence oligonucleotides. Alternatively, he bridging oligos 47 2015202048 22 Apr 2015 813 can be introduced to the sample simultaneously with the fixed sequence oligonucleotides.

[000120] The bound oligonucleotides are ligated 806 to create a contiguous nucleic acid spanning and complementary to he nucleic acid region of interest. The ligation primarily occurs only when he allele-specific ends match. Following ligation, universal primers 817, 819, 837 are introduced to amplify 808 the ligated template region to create 810 products 827, 829 hat comprise the sequence of the nucleic acid region of interest representing boh SNPs in the selected nucleic acid region. The universal primers 817 and 837 have spectrally distinct fluorescent labels such that the allele-specific information is retained through these fluorescent labels. These products 827, 829 are detected and quantified through hybridization of the locus index 821 to an array and imaging to quantify the presence of the fluorescent label. It is important to note that the ligation 806 is preferably allele-specific. In order to make the ligation allele-specific, the allele specifying nucleotide must be close to the ligated end. Typically, the allele-specific nucleotide must be within 5 nucleotides of the ligated end. In a preferred aspect, the allele-specific nucleotide is the teminal base.

[000121] In another aspect, an allele index is present on both the first and second fixed sequence oligonucleotides to detect a polymorphism at both ends with a corresponding spectrallj/ distinct fluorescent label for each fixed sequence oligonucleotide for a given allele. The number of fixed sequence oligonucleotides corresponds to the number of possible alleles being assessed for a selected nucleic acid region. In the above figures and examples, the fixed sequence oligonucleotides are represented as two distinct oligonucleotides. In another 48 2015202048 22 Apr 2015 aspect, the fixed sequence oligonucleotides may be opposite ends of the same oligonucleotide.

[000122] In the aspects described above, the bridging oligos used hybridize to regions of the nucleic acid of interest that are adjacent to the regions complementary to the fixed sequence oligonucleotides, so hat when he fixed sequence and bridging oligo(s) specifically hybridize they are directly adjacent to one another for ligation. In other aspects, however, the bridging oligo hybridizes to a region that is not directly adjacent to the region complementary to one or both of the fixed sequence oligos, and an intermediate step requiring extension of one or more of the oligos is necessary prior to ligation.

[1123] For example, as illusriated in FIG. 9, each set of oligonucleotides preferably contains two oligonucleotides 901, 903 of fixed sequence and one or more bridging oligonucleotides 913. Each of he fixed sequence oligonucleotides comprises a region complementary to he selected nucleic acid region 905, 907, and preferably universal primer sequences 909, 911, i.e. oligo regions complementary to universal primers. The universal primer sequences 909, 911 are located at or near the ends of the fixed sequence oligonucleotides 901, 903, and thus preserve the nucleic acid-specific sequences in the products of any universal amplification methods. The fixed sequence oligonucleotides 901, 903 are introduced 902 to the genetic sample 900 and allowed to specifically bind to he complementary portions of the nucleic acid region of interest 915. Following hybridization, the unhybridized fixed sequence oligonucleotides are preferably separated from the remainder of the genetic sample (not shown). The bridging oligonucleotide is then inrioduced and allowed to bind 904 to the region of the selected nucleic acid region 915 between the first 901 and second 903 fixed 49 2015202048 22 Apr 2015 sequence oligonucleotides. Alternatively, the bridging oligo can be introduced simultaneously to the fixed sequence oligonucleotides. In this ر aspect, the bridging oligo hybridizes to a region directly adjacent to the firet fixed sequence oligo region 905, but is separated by one or more nucleotides ftom the complementary region of the second fixed sequence oligonucleotide 907. Following hybridization of the fixed sequence and bridging oligos, the bridging oligo 913 is extended 906, e.g., using a polymerase and dNTPs, to fill the gap between the bridging oligo 913 and the second fixed sequence oligo 903. Following extension, the bound oligonucleotides are ligated 908 to create a contiguous nucleic acid spanning and complementary to the nucleic acid region of interest 915. After ligation, universal primers 917, 919 are inttoduced 910 to amplify the ligated template region to create 912 products 923 that comprise the sequence of the nucleic acid region of interest. These products 923 are optionally isolated, detected, and quantified to provide information on he presence and amount of the selected nucleic acid region in a genetic sample. Preferably, the products are detected and quantified through sequence determination of an identification index 921, or, alternatively, sequence determination of the nucleic acid of interest 915 within the amplification product 923.

[000124] In another aspect, as illustrated in FIG. 10, each set of oligonucleotides preferably contains two oligonucleotides 1001, 1003 of fixed sequence and two or more bridging oligonucleotides 1013, 1033 that bind to non-adjacent regions on a nucleic acid of interest 1015. Each of the fixed sequence oligonucleotides comprises a region complementary to the selected nucleic acid region 1005, 1007, and preferably universal primer sequences 1009, 1011, i.e. oligo regions complementary to universal primers. The universal primer sequences 1009, 1011 50 2015202048 22 Apr 2015 are located at or near the ends of the fixed sequence oligonucleotides 1001, 1003, and thus preserve the nucleic acid-specific sequences in the products of any universal amplification methods. The fixed sequence oligonucleotides 1001, 1003 are introduced 1002 to the genetic sample 1000 and allowed to specifically bind to the complementary portions of the nucleic acid region of interest 1015. Following hybridization, he unhybridized fixed sequence oligonucleotides are preferably separated ftom he remainder of the genetic sample (not shown).

[1125] In FIG. 10, two separate bridging oligonucleotides 1013, 1033 are inttoduced and allowed to bind 1004 to he region of the selected nucleic acid region 1015 between but not immediately adjacent to boh he first 1001 and second 1003 fixed sequence oligonucleotides. Alternatively, he bridging oligos can be inttoduced simultaneously to he fixed sequence oligonucleotides. In this exemplary aspect, the first bridging oligo 1033 hybridizes to a region directly adjacent to the first fixed sequence oligo region 1005, but is separated by one or more nucleotides from trie complementary region of the second bridging oligo 1013. The second bridging oligo 1013 is also separated from the second fixed sequence oligonucleotide 1007 by one or more nucleotides. Following hybridization of the fixed sequence and bridging oligos, both bridging oligos 1013, 1033 are extended 1006, e.g., using a polymerase and dNTPs, to fill the gap between the bridging oligos and the gap between the second bridging oligo 1013 and the second fixed sequence oligo 1003. Following extension, the bound oligonucleotides are ligated 1008 to create a contiguous nucleic acid spanning and complementary to the nucleic acid region of interest 1015. Following ligation, universal primers 1017, 1019 are introduced 910 to amplify he ligated template region to create 1012 products 1023 that comprise he sequence of he nucleic acid 51 2015202048 22 Apr 2015 region of interest. These products 1023 are optionally isolated, detected, and quantified to provide information on the presence and amount of the selected nucleic acid region in a genetic sample. Preferably, the products are detected and quantified *rough sequence determination of an identification index 1021, or, alternatively, sequence determination of the nucleic acid of interest 1015 widtin the amplification product 1023.

[000126] In specific aspects, such as the aspect illustrated in FIG. 11, the single fixed sequence oligonucleotide 1101 is complementary to the selected nucleic acid region 1115 on boh ends. When this single fixed sequence oligonucleotide 1101 hybridizes to he selected nucleic acid region 1115, it forms a pre-circle oligonucleotide 1103 where the ends are separated by several nucleotides. The bridging oligonucleotide 1113 then binds between the complementary regions 1105, 1107 of the pre-circle oligonucleotide 1103 to fill this gap. The oligonucleotide regions 1105, 1107 of the pre-circle oligonucleotide 1103 bound to the genetic sample 1115 are then ligated together with the bridging oligonucleotide 1113, forming a complete circle.

[000127] The circular template is then preferably cleaved, and amplified using one or more of the universal primer sites. In specific aspects, a single universal primer region is used to replicate the template using techniques such as rolling circle replication, as disclosed in Tizardi et al., U.S. Pat. No. 6,558,928. In a prefened aspect, as illustrated in FIG. 11 this fixed sequence oligonucleotide has two universal priming sites 1109, 1111 on the circular template and optionally one or more indices 1121 between the ends that are complementary to the selected nucleic acid region. Preferably, a cleavage site 1123 exists between the two universal priming sites. Once circularized through ligation to the bridging oligo 52 2015202048 22 Apr 2015 1113, a nuclease can be used to remove all or most uncircularized oligonucleotides. After the removal of the uncircularized oligonucleotides, the circularized oligonucleotide is cleaved 1106, preserving and in some aspects exposing the universal priming sites 1109, 1111. Universal primers 1117, 1119 are added 1108 and a universal amplification occurs 1110 to create 1112 products 1125 that comprise the sequence of the nucleic acid region of interest. The products 1125 are detected and quantified through sequence detemination of selected nucleic acid region or alternatively the index, which obviates the need for detemining the actual sequences of the selected nucleic acid region. In oher aspects, however, it is desirable to determine the product comprising sequences of both the index and the selected nucleic acid region, for example, to provide internal confirmation of he results or where the index provides sample infomation and is not informative of he selected nucleic acid region. As mentioned above, his single fixed sequence oligonucleotide methodology may be applied to any of he examples in Figures 1-10.

Resequencing [1128] In a particular aspect, the assay system of he invention can be used to resequence a complex nucleic acid. The tandem ligation methods have been found to be exceptionally efficient, and his high efficiency allows the methodology to be expanded to the use of multiple oligos, preferably 2-100 or even more, that bind to nucleic acid regions of interest.

[1129] In the preferred aspect, the bridging oligos would be short, preferably between 1-10, more preferably between 2-7, even more preferably between 3-5 nucleotides in length, and he number of bridging oligos used in a tandem ligation 53 2015202048 22 Apr 2015 reaction wouid be approximately 10-50. In a prefened aspect, the bridging oligos would be 5 bases in length and there would be approximately 15-30 ligations.

[000130] In one example, the bridging oligos might be selected to provi degeneracy for all possible sequence variants for the particular oligo length, for instance all sequence variations of 5-mers. Following the multiple ligations, the ligated oligos can be amplified using the universal amplification techan be used niques described herein, and sequence detemination of the amplified products to identify the underlying sequence. This multiple ligation assay provides the ability to target multiple sections of the genome simultaneously through universally amplification of tandem ligation products, and determination of heir nucleotide composition.

Universal amplification [000131] In preferred aspects of die invention, universal amplification is used to amplify the ligation products created following hybridization of the fixed sequence oligonucleotides and die bridging oligonucleotides. In a multiplexed assay system, diis is preferably done dirough universal amplification of die various nucleic acid regions to be analyzed using the assay systems of die invention. Universal primer sequences are added to the contiguous ligation products so diat diey may be amplified in a single universal amplification reaction. These universal primer sequences are preferably introduced in die fixed sequence oligonucleotides, although they may also be added to the proximal ends of die contiguous ligation products following ligation. The universal primer regions allow a subsequent controlled universal amplification of all or a portion of selected nucleic acids prior to or during analysis, e.g. by sequence determination. 54 2015202048 22 Apr 2015 [1132] Bias and variability can be introduced during DNA amplification, such as that seen during polymerase chain reaction (PCR). In cases where an amplification reaction is multiplexed, there is the potential that loci will amplify at different rates or efficiency. Part of this may be due to he variety of primers in a multiplex reaction with some having better efficiency (i.e. hybridization) than others, or some working better in specific experimental conditions due to the base composition. Each set of primers for a given locus may behave differently based on sequence context of the primer and template DNA, buffer conditions, and other conditions.

[000133] The whole tandem ligation reaction or an aliquot of he tandem ligation reaction may be used for he universal amplification. Using an aliquot allows different amplification reactions to be undertaken using the same or different conditions (e.g., polymerase, buffers, and the like), e.g., to ensure that bias is not inadvertently introduced due to experimental conditions. In addition, variations in primer concentrations may be used to effectively limit the number of sequence specific amplification cycles.

[000134] In certain aspects, the universal primer regions of fee primers or adapters used in the assay system are designed to be compatible with conventional multiplexed assay mefeods that utilize general priming mechanisms to analyze large numbers of nucleic acids simultaneously. Such "universal” priming methods allow for efficient, high volume analysis of the quantity of nucleic acid regions present in a genetic sample, and allow for comprehensive quantification of the presence of nucleic acid regions within such a genetic sample for the determination of aneuploidy. 55 2015202048 22 Apr 2015 [1135] Examples of such assay methods include, but are not limited to, multiplexing methods used to amplify and/or genotype a variety of samples simultaneously, such as those described in Oliphant et al., US Pat. No. 7,582,420, [000136] Some aspects utilize coupled reactions for multiplex detection o acid sequences where oligonucleotides from an early phase of each process contain sequences which may be used by oligonucleotides from a later phase of the process. Exemplar processes for amplifying and/or detecting nucleic acids in samples can be used, alone or in combination, including but not limited to the methods described below, each of which are incorporated by reference in their entirety.

[000137] In certain aspects, the assay system of the invention utilizes one of the following combined selective and universal amplification techniques: (1) LDR coupled to PCR; (2) primary PCR coupled to secondary PCR coupled to LDR; and (3) primary PCR coupled to secondary PCR. Each of these aspects of he invention has particular applicability in detecting certain nucleic acid characteristics. However, each requires the use of coupled reactions for multiplex detection of nucleic acid sequence differences where oligonucleotides ftom an early phase of each process contain sequences which may be used by oligonucleotides ftom a later phase of the process.

[000138] Barany et al., US Pat Nos. 6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, 6,268,148, 6,054,564, 6,027,889, 5,830,711, 5,494,810, describe the use of he ligase chain reaction (LCR) assay for he detection of specific sequences of nucleotides in a variety of nucleic acid samples.

[000139] Baranyetal., US Pat Nos. 7,807,431,7,455,965, 7,429,453, 7,364,858, 7,358,048, 7,332,285, 7,320,865, 7,312,039, 7,244,831, 7,198,894, 7,166,434, 56 2015202048 22 Apr 2015 7,097,980, 7,083,917, 7,014,994, 6,949,370, 6,852,487, 6,797,470, 6,576,453, 6,534,293, 6,506,594, 6,312,892, and 6,268,148 describe the use of the ligase detection reaction with detection reaction ("LDR") coupled with polymerase chain reaction ("PCR") for nucleic acid detection.

[000140] Barany et al., US Pat No. 7,556,924 and 6,858,412, describe the use of padlock probes (also called “precircle probes" or “multi-inversion probes") with coupled ligase detection reaction ("LDR") and polymerase chain reaction ("PCR") for nucleic acid detection.

[000141] Barany et al., US Pat Nos. 7,807,431, 7,709,201, and 7,198, 814 describe the use of combined endonuclease cleavage and ligation reactions for he detection of nucleic acid sequences.

[000142] Willis et al., US Pat Nos. 7,700,323 and 6,858,412, describe he use of precircle probes in multiplexed nucleic acid amplification, detection and genotyping, including [000143] Ronaghi et al., US Pat. No. 7,622,281 describes amplification techniques for labeling and amplifying a nucleic acid using an adapter comprising a unique primer and a barcode.

[000144] In addition to he various amplification techniques, numerous methods of sequence determination are compatible with the assay systems of the inventions. Preferably, such methods include “next generation" methods of sequencing. Exemplary methods for sequence determination include, but are not limited to, including, but not limited to, hybridization-based methods, such as disclosed in Drmanac, U.S. Pat. Nos. 6,864,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication 2005/0191656, which are incorporated by reference, sequencing by synthesis methods, e.g.y Nyren et al, U.S. Pat. No. 57 2015202048 22 Apr 2015 7,648,824, 7,459,311 and 6,210,891; Balasubramanian, U.S. Pat. Nos. 7,232,656 and 6,833,246; Quake, U.S. Pat. No. 6,911,345; Li et al, Proc. Natl. Acad. Sci., 100: 414-419 (2003); pyrophosphate sequencing as described in Ronaghi et al., U.S. Pat. Nos. 7,648,824, 7,459,311, 6,828,100, and 6,210,891;, and ligation-based sequencing determination methods, e.g., Drmanac et al., U.S. Pat. Appln No. 20100105052, and Church et al, U.S. Pat. Appln Nos. 20070207482 and 20090018024.

[000145] Alternatively, nucleic acid regions of interest can be selected and/or identified using hybridization techniques. Methods for conducting polynucleotide hybridization assays for detection of have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2"٥ Ed. Cold Spring Harbor, Ν.Υ., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference [1146] The present invention also contemplates signal detection of hybridization between ligands in certain preferred aspects. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as 58 2015202048 22 Apr 2015 W099/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

[000147] Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as W099/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Use of Indices in the Assay Systems of the Invention [000148] In certain aspects, all or a portion of the sequences of the nucleic acids of interest are directly detected using the described techniques, e.g., sequence detemination or hybridization. In certain aspects, however, he nucleic acids of interest are associated with one or more indices hat are identifying for a selected nucleic acid region or a particular sample being analyzed. The detection of he one or more indices can serve as a sunogate detection mechanism of the selected nucleic acid region, or as confimation of the presence of a particular selected nucleic acid region if both the sequence of the index and the sequence of he nucleic acid region itself are determined. These indices are preferably associated with the selected nucleic acids during an amplification step using primers that comprise both the index and sequence regions hat specifically hybridize to the nucleic acid region.

[000149] In one example, the primers used for amplification of a selected nucleic acid region are designed to provide a locus index between the selected nucleic 59 2015202048 22 Apr 2015 acid region primer region and a universal amplification region. The locus index is unique for each selected nucleic acid region and representative of a locus on a chromosome of interest or reference chromosome, so that quantification of the locus index in a sample provides quantification data for the locus and the particular chromosome containing the locus.

[1150] In another example, he primers used for amplification of a selected nucleic acid region are designed to provide an allele index between the selected nucleic acid region primer region and a universal amplification region. The allele index is unique for particular alleles of a selected nucleic acid region and representative of a locus variation present on a chromosome of interest or reference chromosome, so that quantification of the allele index in a sample provides quantification data for the allele and the summation of he allelic indices for a particular locus provides quantification data for boh he locus and the particular chromosome containing he locus.

[000151] In another aspect, he primers used for amplification of he selected nucleic acid regions to be analyzed for a genetic sample are designed to provide an identification index between he selected nucleic acid region primer region and a universal amplification region. ئ such an aspect, a sufficient number of identification indices are present to uniquely identify each selected nucleic acid region in he sample. Each nucleic acid region to be analyzed is associated with a unique identification index, so hat he identification index is uniquely associated with he selected nucleic acid region. Quantification of he identification index in a sample provides quantification data for the associated selected nucleic acid region and the chromosome corresponding to the selected nucleic acid region. The identification locus may also be used to detect any amplification bias that occurs 60 2015202048 22 Apr 2015 downstream of die initial isolation of die selected nucleic acid regions from a sample.

[000152] In certain aspects, only the locus index and/or the identification index (if present) are detected and used to quantify the selected nucleic acid regions in a sample. In another aspect, a count of the number of times each locus index occurs with a unique identification index is done to determine the relative frequency of a selected nucleic acid region in a sample.

[000153] In some aspects, indices representative of the sample from which a nucleic acid is isolated are used to identify the source of the nucleic acid in a multiplexed assay system. In such aspects, die nucleic acids are uniquely identified with the sample index. Those uniquely identified oligonucleotides may then be combined into a single reaction vessel with nucleic acids from other samples prior to sequencing. The sequencing data is first segregated by each unique sample index prior to determining he frequency of each target locus for each sample and prior to determining whether here is a chromosomal abnormality for each sample. For detection, he sample indices, he locus indices, and the identification indices (if present), are sequenced.

[1154] In aspects of the invention using indices, he fixed sequence oligonucleotides are preferably designed to comprise he indices. Alternatively, he indices and universal amplification sequences can be added to the selectively amplified nucleic acids following initial amplification. In either case, preferably foe indices are encoded upstream of foe nucleic acid region-specific sequences but downstream of foe universal primers so that they are preserved upon 61 2015202048 22 Apr 2015 amplification, but also require less sequencing to access when using the universal primers for sequence determination.

[000155] The indices are non-complementary but unique sequences used within the primer to provide information relevant to the selective nucleic acid region that is isolated and/or amplified using the primer. The advantage of this is that information on the presence and quantity of the selected nucleic acid region can be obtained without he need to determine the actaal sequence itself, although in certain aspects it may be desirable to do so. Generally, however, the ability to identify and quantify a selected nucleic acid region through identification of one or more indices will decrease the length of sequencing required as the loci information is captared at the 3’ or 5’ end of the isolated selected nucleic acid region. Use of indices identification as a surrogate for identification of selected nucleic acid regions may also reduce error since longer sequencing reads are more prone to the introduction of errors.

[000156] In addition to locus indices, allele indices and identification indices, additional indices can be introduced to primers to assist in the multiplexing of samples. For example, correction indices which identify experimental error (e.g., errors inrioduced during amplification or sequence detemination) can be used to identify potential discrepancies in experimental procedures and/or detection methods in the assay systems. The order and placement of these indices, as well as the length of these indices, can vary, and hey can be used in various combinations.

[1157] The primers used for identification and quantification of a selected nucleic acid region may be associated with regions complementary to he 5’ of the selected nucleic acid region, regions complementary to the 3’ of he selected 62 2015202048 22 Apr 2015 nucleic acid region, or in certain amplification regimes the indices may be present on one or both of a set of amplification primers which comprise sequences complementary to the sequences of the selected nucleic acid region. The primers can be used to multiplex the analysis of multiple selected nucleic acid regions to be analyzed within a sample, and can be used either in solution or on a solid substrate, e.g., on a microanay or on a bead. These primers may be used for linear replication or amplification, or they may create circular constmcts for further analysis.

Detection of Other Agents or Risk Factors [000158] Given the multiplexed nature of the assay systems of the invention, in certain aspects it may be beneficial to utilize the assay to detect other nucleic acids that could pose a risk to the health of the subject(s) or otherwise impact on clinical decisions about the treatment or prognostic outcome for a subject. Such nucleic acids could include but are not limited to indicators of disease or risk such as maternal alleles, polymorphisms, or somatic mutations known to present a risk for maternal or fetal health. Such indicators include, but are not limited to, genes associated with Rh status; mutations or polymorphisms associated with diseases such as diabetes, hyperlipidemia, hypercholesterolemia, blood disorders such as sickle cell anemia, hemophilia or thalassemia, cardiac conditions, etc.; exogenous nucleic acids associated with active or latent infections; somatic mutations or copy number variations associated with autoimmune disorders or malignancies (e.g., breast cancer), or any other health issue that may impact on the subject, and in particular on the clinical options that may be available in foe treatment and/or prevention of health risks in a subject based on the outcome of the assay results. 63 2015202048 22 Apr 2015 [1159] Accordingly, as the preferred assay systems of he invention are highly multiplexed and able to interrogate hundreds or even thousands of nucleic acids within a mixed sample, ئ certain aspects it is desirable to interrogate the sample for nucleic acid markers within the mixed sample, e.g., nucleic acids associated with genetic risk or that identify the presence or absence of infectious organisms. Thus, in certain aspects, the assay systems provide detection of such nucleic acids in conjunction with he detection of nucleic acids for copy number determination within a mixed sample.

[000160] For example, in certain mixed samples of interest, including maternal samples, samples ftom subjects with autoimmune disease, and samples ftom patients undergoing chemotherapy, he immune suppression of he subject may increase the risk for the disease due to changes in he subject’s immune system. Detection of exogenous agents in a mixed sample may be indicative of exposure to and infection by an infectious agent, and this finding have an impact on patient care or management of an infectious disease for which a subject tests positively for such infectious agent.

[000161] Specifically, changes in immunity and physiology during pregnancy may make pregnant women more susceptible to or more severely affected by infectious diseases. In fact, pregnancy itself may be a risk factor for acquiring certain infectious diseases, such as toxoplasmosis, Hansen disease, and listeriosis. In addition, for pregnant women or subjects with suppressed immune systems, certain infectious diseases such as influenza and varicella may have a more severe clinical course, increased complication rate, and higher case-fatality rate. Identification of infectious disease agents may therefore allow better treatment for 64 2015202048 22 Apr 2015 maternal disease during pregnancy, leading to a better overall outcome for both mother and fehis.

[1162] In addition, certain infectious agents can be passed to the fetus via vertical transmission, i.e. spread of infections from mother to baby. These infections may occur while he fetas is still in the utertJS, during labor and delivery, or after delivery (such as while breastfeeding).

[1163] Thus, in some preferred aspects, the assay system may include detection of exogenous sequences, e.g., sequences from infectious organisms that may have an adverse effect on he heallh and/or viability of the fetas or infant, in order to protect maternal, fetal, and or infant health.

[1164] Exemplary infections which can be spread via vertical transmission, and which can be tested for using he assay methods of the invention, include but are not limited to congenital infections, perinatal infections and postnatal infections.

[000165] Congenita] infections are passed in utero by crossing the placenta to infect the fetus. Many infectious microbes can cause congenita] infections, leading to problems in fetal development or even death. TORCH is an acronym for several of the more common congenital infections. These are: toxoplasmosis, other infections (e.g., syphilis, hepatitis B, Coxsackie vims, Epstein-Barr vims, varicella-zoster vims (chicken pox), and human parvovims Β19 (fiflh disease)), rubella, cytomegalovims (CMV), and herpes simplex vims.

[000166] Perinatal infections refer to infections that occur as the baby moves through an infected birth canal or through contamination with fecal matter during delivery. These infections can include, but are not limited to, sexually-transmitted 65 2015202048 22 Apr 2015 diseases (e.g., gonorrhea, chlamydia, herpes simplex virus, human papilloma vims, etc.) CMV, and Group B Stteptococci (GBS).

[000167] Infections spread trom mother to baby following delivery are known as postnatal infections. These infections can be spread during breastfeeding through infectious microbes found in he moher’s breast milk. Some examples of postnatal infections are CMV, Human immunodeficiency vims (HIV), Hepatitis c Vims (HCV), andGBS.

EXAMPLES

[000168] The following examples are put forth so as to provide hose of ordinary skill in the art with a complete disclosure and description of how to make and use he present invention, and are not intended to limit he scope of what he inventors regard as their invention, nor are hey intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in he art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing ftom he spirit or scope of he invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

[000169] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, ete.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperatare is in degrees centigrade, and pressure is at or near atmospheric.

Example 1: Genera. Aspects of the Assay Systems of the Invention 66 2015202048 22 Apr 2015 [1170] A number of assay forinats were tested to demonstrate the ability to perform selective amplification and detection of independent loci to demonstrate multiplexed, ligation-based detection of a large number (e.g., 96 or more) of nucleic acid regions of interest using highly multiplexed formats.

[1171] These assays were designed based on human genomic sequences, and each interrogation consisted of two fixed sequence oligos per selected nucleic acid region interrogated in he assay. The first oligo, complementary to he 3’ region of a genomic region, comprised he following sequential (5’ to 3’) oligo elements: a universal PCR priming sequence common to all assays: TACACCGGCGTTATGCGTCGAGAC (SEQ ID N0:l); a nine nucleotide identification code specific to he selected loci; a 9 base locus- or * specific sequence hat acts as a locus code in the first ٠ set and a locus/allele code in he SNP-specific second set; a hybridization breaking nucleotide which is different from the corresponding base in he genomic locus; and a 20-24 bp sequence complementary to he selected genomic locus. In cases where a SNP is detected in this portion of he selected genomic locus, the allele-specific interrogation set consisted of two first tandem ligation primers, each with a different locus/allele code and a different allele-specific base at the SNP position. These first oligos were designed for each selected nucleic acid to provide a predicted uniform Tm with a two degree variation across all interrogations in he 480 assay set.

[1172] The second fixed sequence oligo, complementary to the 5’ region of he genomic loci, comprised he following sequential (5’ to 3’) elements: a 20-24b sequence complimentary to he 5’ region in the genomic locus; a hybridization breaking nucleotide which was different from the corresponding base in the 67 2015202048 22 Apr 2015 genomic locus; and a universal PCR priming sequence which was common to all third oligos in the assay set: ATTGCGGGGACCGATGATCGCGTC (SEQ ID NO:2).

[000173] In cases where a SNP was detected in his portion of the selected genomic locus, the allele-specific interrogation set consisted of two tandem ligation primers, each with a different locus/allele code and a different allele-specific base at the SNP position. This second fixed sequence oligo was designed for each selected nucleic acid to provide a predicted uniform Tm with a two degree variation across all intenogations in the 480 assay set that was substantially the same Tm range as the first oligo set.

[000174] In certain tested aspects, one or niore bridging oligos were used th were complementary to the genomic locus sequence between the region complementaty to the first and second fixed sequence oligos used for each selected nucleic acid region. In specific aspects tested, more than one bridging oligo was used to span the gap between the fixed sequence oligonucleotides, and the one or more oligo may optionally be designed to identify one or more SNPs in the sequence. The length of the bridging oligonucleotides used in the assay systems varied from 5 to 36 base pairs.

[000175] All oligonucleotides used in the tandem ligation formats were synthesized using conventional solid-phase chemistry. The oligos of the first fixed set and the bridging oligonucleotides were synthesized with 5’ phosphate moieties to enable ligation to 3’ hydroxyl termini of adjacent oligonucleotides.

Example 2: Preparation of DNA for Use in Tandem Ligation Procedures 68 2015202048 22 Apr 2015 [000176] Genomic DNA from a Caucasian male (ΝΑ12801) or a Caucasian female (ΝΑ11995) was obtained from Coriell Cell Repositories (Camden, New Jersey) and fragmented by acoustic shearing (Covaris, Woburn, MA) to a mean fragment size of approximately 200bp.

[000177] The Coriell DNA was biotinylated using standard procedures. Briefly, the Covaris fragmented DNA was end-repaired by generating the following reaction in a 1.5 ml microtube: 5ug DNA, -12 μ] 10Χ Τ4 ligase buffer (Enzymatics, Beverly MA), 50 u Τ4 polynucleotide kinase (Enzymatics, Beverly MA), and ¾0 to 120 pi. This was incubated at 37°c for 30 minutes. The DNA was diluted using 10 mM Tris ImM EDTA pH 8.5 to deshed final concentration of -0.5 ng/ul.

[000178] 5 μΐ DNA was placed in each well of a 96-well plate, and the plate sealed with an adhesive plate sealer and spun for 10 seconds at 250 X g. The plate was then incubated at 95٥c for 3 minutes, and cooled to 25.C, and spun again for 10 seconds at 250 X g. A biotinylation master mix was prepared in a 1.5ml microtube to final concentration of: IX TdT buffer (Enzymatics, Beverly MA), 8٧ TdT (Enzymatics, Beverly MA), 250 μΜ CoCl, 0.01 nmol/μΐ bi0tin-16-dUTP (Roche, Nutley NJ), and ¾0 to 1.5 ml. 15 μΐ of the master mix was aliquoted into each well of a 96 well plate, and the plate sealed with adhesive plate sealer. The plate was spun for 10 seconds at 250 X g and incubated for 37.C for 60 minutes. Following incubation, he plate was spun again for 10 seconds at 250 X g, and 7.5 μΐ precipitation mix (1 ng/μΐ Dextran Blue, 3mM NaOAC) was added to each well.

[1179] The plate was sealed with an adhesive plate sealer and mixed using an IKA plate vortexer for 2 minutes at 3000 rpm. 27.5 μΐ of isopropanol was added 69 2015202048 22 Apr 2015 into each well, the plate sealed with adhesive plate sealer, and vortexed for 5 minutes at 3000 rpm. The plate was spun for 20 minutes at 3000 X g, the supernatant was decanted, and the plate inverted and centrifuged at 10 X g for 1 minute onto an absorbent wipe. The plate was air-dried for 5 minutes, and the pellet resuspended in 10 pi lOmM Tris pH8.0, ImM EDTA.

Example 3: Exemplary Assay Formats using Tandem Ligation [1180] Numerous tandem ligation assay formats using the biotinylated DNA were tested to illusttate proof of concept for the assay systems of the invention, and demonsttated he ability to perform highly multiplexed, targeted detection of a large number of independent loci using the series of different assay formats. The exemplary assay systems of he invention were designed to comprise 96 or more interrogations per loci in a genetic sample, and in cases where SNPs were detected the assay formats utilized 192 or more separate interrogations, each utilizing the detection of different alleles per 96 loci in genetic samples. The examples described for each assay format utilized two different sets of fixed sequence oligonucleotides and/or bridging oligos (as described in Example 1), comprising a total 96 or 192 interrogation reactions for the selected nucleic acid regions depending upon whether SNPs were identified.

[000181] A first exemplaty assay format used locus-specific fixed sequence oligos and bridging oligos, where there was a one base gap between the first fixed sequence oligo and the bridging oligos, and a second one base gap between the bridging oligos and he second fixed sequence oligo. Each of the two gaps encompassed two different SNPs. In this format, a DNA polymerase was used to incorporate each of he SNP bases, and ligase was used to seal the nicks formed hereby. SNP base discrimination derived from the fidelity of base ' 70 2015202048 22 Apr 2015 by the polymerase, and in the event ot mis-incorporation, he tendency ofligase to not seal nicks adjacent to mismatched bases.

[000182] Ihe second exemplary assay format used two locus-specific fixed sequence oligonucleotides without a bridging oligo, where there was a -15-35 base gap between the fixed sequence oligos, and where he gap spanned one or more SNPs. In this format, a polymerase was used to incorporate he missing bases, and a ligase was used to seal he nick formed thereby. SNP base discrimination derived from the fidelity of base incorporation by the polymerase, and in the event of misincorporation, he tendency of ligase to not seal nicks adjacent to mismatched bases.

[000183] A third exemplary assay format used allele-specific first and second fixed sequence oligos without a bridging oligo, where here was a -15-35 base gap between the first and second fixed sequence oligos, and where the gap spanned one or more SNPs. Two separate allele-specific first fixed sequence oligos and two separate allele-specific second fixed sequence oligos were used. A polymerase was used to incorporate the missing bases, and a ligase was used to seal the nick formed thereby. SNP base discrimination derived from hybridization specificity, the tendency of non-proofreading polymerase to not extend annealed primers with mismatches near the 3’ end, and the tendency of the ligase to not seal nicks adjacent to mismatched bases.

[000184] A fourth exemplary format used allele-specific fixed sequence oligos and a locus-specific bridging oligo. In this format, two separate fixed sequence oligos complementary to the 3’end of the loci of interest, the first with a 3’ base specific for one allele of the targeted SNP, and the second with a 3’ base specific for the other allele of the targeted SNP. Similarly, two separate second fixed 71 2015202048 22 Apr 2015 sequence oligos were used, the first with a 5’ base specific for one allele of a second targeted SNP, and the second with a 5’ base specific for the other allele of he second targeted SNP. The bridging oligos were complement^ to the region directly adjacent to the locus regions complementary to the first and second fixed sequence oligos, and thus no polymerase was needed prior to ligation. Ligase was used to seal the nicks between the fixed sequence oligos and the bridging oligo. SNP base discrimination in this assay format derived from hybridization specificity and the tendency of the ligase to not seal nicks adjacent to mismatched bases. This exemplar format was tested using either Τ4 ligase or Taq ligase for creation of the contiguous template, and both were proved effective in the reaction as described below.

[000185] A fifth exemplary format used locus-specific fixed sequence oligos that were complementary to adjacent regions on he nucleic acid of interest, and thus no gap was created by hybridization of these oligos. In his format, no polymerase was required, and a ligase was used to seal the single nick between he oligos.

[000186] A sixth exemplar format used allele-specific fixed sequence oligos and locus-specific bridging oligos, where there was a short base gap of five bases between the loci region complementary to the fixed sequence oligos. The locus-specific bridging oligo in this example was a 5mer complementary to the regions directly adjacent to the regions complementary to the first and second fixed sequence oligos. In this format, no polymerase was required, and a ligase was used to seal the two nicks between the oligos.

[000187] A seventh exemplary fomat used locus-specific fixed sequence o and a locus-specific bridging oligo, where there was a shorter base gap of five 72 2015202048 22 Apr 2015 bases containing a SNP in the region complementary to the bridging oligo. Allele-specific bridging oligos corresponding to the possible SNPs were included in the hybridization and ligation reaction. In this format, no polymerase was required, and a ligase was used to seal the two nicks between the oligos. SNP base discrimination in this assay format derives from hybridization specificity and the tendency of the ligase to not seal nicks adjacent to mismatched bases.

[000188] An eighth exemplary format used locus-specific fixed sequence oligos and two adjacent locus-specific bridging oligos, where there is a 10 base gap between the regions complementary to he first and second fixed sequence oligos. Locus-specific bridging oligos were included in the ligation reaction, with the gap requiring two contiguous 5mers to bridge the gap. In this format, no polymerase is required, and a ligase is used to seal the three nicks between the oligos.

[000189] For each of the above-described assay formats, an equimolar pool (40 nM each) of sets of firet and second loci- or allele-specific fixed oligonucleotides was created from the oligos prepared as set forth in Example 2. A separate equimolar pool (20 μΜ each) of bridging oligonucleotides was likewise created for the assay processes based on the sequences of the selected genomic loci.

[1190] 10pg of strepavidin beads were riansferred into the wells of a 96 well plate, and the supernatant was removed. 60 μΐ ΒΒ2 buffer (lOOmM Tris pH 8.0, lOmM EDTA, 500mM NaC12, 58% formamide, 0.17% Tween-80), 10 pL 40 nM fixed sequence oligo pool and 30 pL of the biotinylated template DNA prepared in Example 2 were added to the beads. The plate was sealed with an adhesive plate sealer and vortexed at 3000 rpm until beads were resuspended. The oligos were annealed to the template DNA by incubation at 70.C for 5 minutes, followed by slow cooling to room temperatare. 73 2015202048 22 Apr 2015 [1191] Tlie plate was placed on a raised bar magnetic plate for 2 minutes to pull the magnetic beads and associated DNA to the side of the wells. The supernatant was removed by pipetting, and was replaced with 50uL of 60% ΒΒ2 (v/v in water). The beads were resuspended by vortexing, placed on the magnet again, and the supernatant was removed. This bead wash procedure was repeated once using 50 uL 60% ΒΒ2, and repeated twice more using 50 uL wash buffer (lOmM Tris pH 8.0, ImM EDTA, 50mM NaC12).

[000192] The beads were resuspended in 37 μ] ligation reaction mix consisting of IX Taq ligase buffer (Enzymatics, Beverly ΜΑ), ίου Taq ligase, and 2 uM bridging oligo pool (depending on the assay format), and incubated at 37.C for one hour. Where appropriate, and depending on die assay format, a non-proofreading themostable polymerase plus 200nM each dNTP was included in this mixture. The plate was placed on a raised bar magnetic plate for 2 minutes to pull the magnetic beads and associated DNA to he side of the wells. The supernatant was removed by pipetting, and was replaced with 50uTwash buffer. The beads were resuspended by vortexing, placed on (he magnet again, and the supernatant was removed. The wash procedure was repeated once.

[1193] To elute the products from the strepavidin beads, 3() μΐ of lOmM Tris ImM EDTA, pH 8.0 was added to each well of 96-well plate. The plate was sealed and mixed using an IKA vortexer for 2 minutes at 3000 rpm to resuspend the beads. The plate was incubated at 95.C for 1 minute, and he supernatant aspirated using an 8-channel pipetter. 25 μΐ of supernatant from each well was transferred into a ftesh 96-well plate for universal amplification.

Example 4: Universal Amplification of Tandem Ligated Products 74 2015202048 22 Apr 2015 [1194] The polymerized and/or ligated nucleic acids were amplified using universal PCR primers complementary to the universal sequences present in the first and second fixed sequence oligos hybridized to the nucleic acid regions of interest. 25 μΐ of each of the reaction mixtures of Example 3 were used in each amplification reaction. A 50 uL universal PCR reaction consisting of 25 uL eluted ligation product plus IX Pfusion buffer (Finnzymes, Finland), 1Μ Betaine, 400nM each dNTP, 1 u Pfusion error-correcting thermostable DNA polymerase, and the following primer pairs: (SEQ ID NO:3) and CCGCAA (SEQ ID NO:4), where X represents one of 96 different sample tags used to uniquely identify individual samples prior to pooling and sequencing. The PCR was carried out under stringent conditions using a BioRad TetradTM thermocycler.

[000195] 10 μ] of universal PCR product from each of the samples were pooled and the pooled PCR product was purified using AMPureTM SPRI beads (Beckman-Coulter, Danvers, MA), and quantified using Quant-iTTM PicoGreen, (Invitrogen, Carlsbad, CA).

Example 5: Detection and Analysis of Selected Loci [000196] The purified PCR products of each assay format were sequenced on a single lane of a slide on an Illumina HiSeq 2000. Sequencing runs typically give rise to -100Μ raw reads, of which -85Μ (85%) mapp to expected assay structures. This translat to an average of -885Κ reads/sample across the 75 2015202048 22 Apr 2015 experiment, and (in the case of an experiment using 96 Joci) 9.2Κ reads/replicate/locus across 96 Joci. The mapped reads were parsed into replicate/locus/allele counts, and various metrics were computed for each condition, incJuding: [000197] YieJd: a meriic of the proportion of input DNA that was queried in sequencing, computed as the average number of unique reads per Jocus (only counting unique identification code reads per replicate/locus) divided by the total number of genomic equivalents contained in the input DNA.

[000198] 80 percentile locus frequency range: a metric of the locus frequency variability in the sequencing data, interpreted as the fold range that encompasses 80% of the loci. It is computed on the distribution of total reads per locus, across all loci, as the 90* percentile of total reads per locus divided by the 10* percentile of the total reads per locus.

[000199] SNP error rate: a metric of the error rate at the SNP position, and computed as he proportion of reads containing a discordant base at he SNP position.

[1200] These results are summarized in Table 1:

Table !:Results Summary of Tandem Ligation Assay Formats

ASSAY TORMAT FKED SEQUENCE OLIGO (1 and/or 2“d) BRIDGING OLIGO USED ENZYME USED YELD 80% LOC FREQ RANGE SNP ERROR RATE 1 LrcUS-SPECIFIC Locus specific poltlig 9.5% 5.3 0.18% 2 MCUS-SPECIFIC No poltlig 1.4% 58.3 0.19% 3 ALLELE-SPECIFIC No poltlig 0.4% 61.7 1.00% 4 ALLELE-SPECIFIC Locus specific Taq lig 5.0% 5.9 0.92% 4 ALLELE-SPECIFIC Locus specific T41ig 5.3% 4.4 0.95% 5 LrcUS-SPECIFIC No Taq lig 22.5% 1.7 NA 6 LrcUS-SPECIFIC locus specific Taq lig 12.5 2.9 NA ٦ MCUS-SPECIFIC Allele specific Taq lig 14.3 2.8 0.20% 8 MCUS-SPECIFIC 2 locus specific Taq lig 18.5% 2.8 NA 76 2015202048 22 Apr 2015 [000201] Table 1 indicates hat the locus-specific tandem ligation assay using a bridging oligo converted template DNA into targeted product with high yield (~10%), with a high proportion of product derived from targeted loci (15% of reads did not contain expected assay structures), with limited locus bias (80% of loci fall within a ~5-fold concentration range), and with high SNP accuracy (0.2% SNP error rate). The locus-specific tandem ligation assay without the use of a bridging oligo produced reduced yields and substantia] locus bias, but still produced high accuracy SNP genotyping data. The allele-specific tandem ligation assay wih a bridging oligo produced intermediate yields compared to the locus-specific assay using boh Τ4 and Taq ligase, but still produced limited locus bias and high accuracy SNP genotyping data. The allele-specific tandem ligation assay wihout a bridging produced reduced yields and substantial locus bias, but still produced high accuracy SNP genotyping data.

[1202] Assay formats five and six showed hat template DNA can be converted into targeted product wih high yield (12-16%), with a high proportion of product derived ftom targeted loci (-76% of reads contained expected assay structures), and wih limited locus bias (80% of loci fall within a 2-3-fold concenttation range). Figure 12 illustrates he genotyping performance that is obtained using assay format seven, comparing the sequence counts for the two alleles of all polymorphic assays observed in a single sample. Note the clear separation of he homozygous and heterozygous clusters, as well as the low background counts observed amongst the homozygous clusters.

Example 6: Determination of Percent Fetal DNA using Tandem Ligation 77 2015202048 22 Apr 2015 [000203] One exemplary assay system of the invention was designed comprising 480 separate intenogations, each utilizing the detection of different loci in a maternal sample. The initial example utilized a determination of percent fetal DNA in subjects carrying a male fetus, and so loci on the Y chromosome were utilized as well as loci containing a paternally-inherited fetal SNP that is different from the maternal sequence.

[000204] Specifically, 480 selected nucleic acids were interrogated using the assay system. The 480 selected nucleic acids comprised 48 sequence-specific intenogations of nucleic acids corresponding to loci on chromosome Y, 192 sequence-specific interrogations of nucleic acids conesponding to loci on chromosome 21, 192 sequence-specific intenogations of selected nucleic acids corresponding to loci on chromosome 18, and 144 sequence-specific interrogations of selected nucleic acids corresponding to polymorphic loci on chromosomes 1-16. These assays were designed based on human genomic sequences, and each interrogation used three oligos per selected nucleic acid interrogated in he assay.

[0105] The first oligo used for each interrogation was complementary to he 3’ region of he selected genomic region, and comprised he following sequential (5’ to 3’) oligo elements: a universal PCR priming sequence common to all assays: TACACCGGCGTTATGCGTCGAGAC (SEQ ID N0:l); an identification code specific to the selected loci comprising nine nucleotides; and a 20-24 bp sequence complementary to he selected genomic locus. This first oligo was designed for each selected nucleic acid to provide a predicted uniform Tin with a two degree variation across all intenogations in the 480 assay set. 78 2015202048 22 Apr 2015 [000206] The second oJigo used for each interrogation was a bridging oJigo complementay to the genomic Jocus sequence directly adjacent to the genomic region complementary to the first oligonucleotide. Based on the selected nucleic acids of interest, the bridging oligos were designed to allow utilization of a total of 12 oligonucleotide sequences that could serve as bridging oligos for all of the 480 intenogations in the assay set.

[0107] The third oligo used for each interrogation was complementary to the 5’ region of the selected genomic locus, comprised the following sequential (5’ to 3’) elements: a 20-24b sequence complimentary to the 5’ region in the genomic locus; a hybridization breaking nucleotide which was different ftom the corresponding base in the genomic locus; and a universal PCR priming sequence which is common to all third oligos in the assay set: ATTGCGGGGACCGATGATCGCGTC (SEQ ID NO:2). This third oligo was designed for each selected nucleic acid to provide a predicted uniform Tm with a two degree variation across all intenogations in the 480 assay set, and the Tm range was substantially the same as the Tm range as the first oligo set.

[000208] All oligonucleotides were synthesized using conventional solid-phase chemistry. The first and bridging oligonucleotides were synthesized with 5’ phosphate moieties to enable ligation to 3’ hydroxy] termini of adjacent oligonucleotides. An equimolar pool of sets of the first and thhd oligonucleotides used for all intenogations in the multiplexed assay was created, and a separate equimolar pool of all bridging oligonucleotides was created to allow for separate hybridization reactions.

[000209] Genomic DNA was isolated ftom 5mT plasma using he Dynal Silane viral NA kit (Invitrogen, Carlsbad, CA). Approximately 12ng DNA was 79 2015202048 22 Apr 2015 processed from each of 37 females, including 7 non-pregnant female subjects, 10 female subjects pregnant with males, and 22 female subjects pregnant with females. The DNA was biotinylated using standard procedures, and the biotinylated DNA was immobilized on a solid surface coated with strepavidin to allow retention of the genomic DNA in subsequent assay steps.

[000210] The immobilized DNA was hybridized to the first pool comprising he first and third oligos for each interrogated sequences under stringent hybridization conditions. The unhybridized oligos in the pool were then washed from the surface of the solid support, and the immobilized DNA was hybridized to the pool comprising the bridging oligonucleotides under stringent hybridization conditions. Once the bridging oligonucleotides were allowed to hybridize to the immobilized DNA, the remaining unbound oligos were washed from the surface and the three hybridized oligos bound to he selected nucleic acid regions were ligated using Τ4 ligase to provide a contiguous DNA template for amplification.

[000211] The ligated DNA was amplified from the solid substrate using an error correcting themostable DNA polymerase, a first universal PCR primer GA (SEQ ID NO:3) and a second universal PCR primer CCCCGCAA (SEQ ID NO:4), where X represents one of 96 different sample indices used to uniquely identify individual samples prior to pooling and sequencing. 10pL of universal PCR product from each of the 37 samples described above were and the pooled PCR product was purified using AMPure 80 2015202048 22 Apr 2015 SPRI beads (Beckman-Coulter, Danvers, MA), and quantified using Quant-iTTM picoGreen, (Invittogen, CarJsbad, CA).

[000212] The purified PCR product was sequenced on 6 lanes of a single slide on an Illumina HiSeqTM 2000. The sequencing run gave rise to 384Μ raw reads, of which 343Μ (89%) mapped to expected genomic loci, resulting in an average of 3.8Μ reads per sample across the 37 samples, and 8Κ reads per sample per locus across the 480 loci. The mapped reads were parsed into sample and locus counts, and two separate metrics of percent fetal DNA were computed as follows.

[000213] Percent male DNA detected by chromosome Y loci corresponds to the relative proportion of reads derived from chromosome Y locus interrogations versus the relative proportion of reads derived from autosomal locus interrogations, and is computed as (number of chromosome Y reads in a test subject/number of autosome reads in test subject)/(number of reads in male control subject/number of autosome reads in he male control subject). This metric was used as a measure of percent fetal DNA in he case of a male fetas using the relative reads of chromosome Y.

[000214] Percent fetal DNA detected by polymorphic loci corresponds to he proportion of reads derived from ηοη-matemal versus maternal alleles at loci where such a distinction can be made, first, for each identified locus, he number of reads for the allele with the fewest counts (the low ftequency allele) was divided by the total number of reads to provide a minor allele frequency (MAF) for each locus. Then, loci with an MAF between 0.075% and 15% were identified as infomative loci. The estimated percent fetal DNA for the sample was calculated as the mean of the minor allele frequency of the 81 2015202048 22 Apr 2015 informative loci multiplied by two, i.e. computed as 2Χ average (MAF) occurrence where 0.075%<MAF<15%.

[0115] FIG. 13 demonstrates the results ftom these computations. As shown in FIG. 13, the percent male loci determined using the above-described chromosome Y metrics (grey circles) can separate pregnancies involving male fetuses ftom pregnancies involving female fetuses (grey diamonds) and non-pregnant samples (black circles). In addition, computation of the percent fetal amount in a sample by polymorphic loci metric can distinguish pregnant samples ftom non-pregnant samples. Finally, there is a correlation between the percent fetal DNA estimates for a sample obtained from chromosome Y and polymorphic loci in pregnancies involving male fetuses. This correlation persists down to quite low percent fetal values.

[000216] While this invention is satisfied by aspects in many different forms, as described in detail in connection with prefened aspects of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific aspects illusftated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the genera] public, to quickly detemine the genera] nature of the invention. In the claims that follow, unless the term “means" is used, none of the 82 2015202048 22 Apr 2015 features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. §112, ،j[6. 83

Claims (19)

1. The claims defining the invention are as follows:

   1. A method for detecting and quantifying nucleic acid regions of interest in a genetic sample comprising maternal and fetal DNA to detect fetal chromosomal copy number variations, comprising the steps of: providing a genetic sample; introducing at least two sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the set of fixed sequence oligonucleotides to specifically hybridize to complementary regions in each nucleic acid region of interest in both the maternal and fetal DNA, wherein the first set of first and second fixed sequence oligonucleotides specifically hybridize to complementary regions in nucleic acid regions of interest in a first chromosome and the second set of first and second fixed sequence oligonucleotides specifically hybridize to complementary regions in nucleic acid regions of interest in a second chromosome; introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid regions of interest complementary to the first and second fixed sequence oligonucleotides of a set, and wherein the one or more bridging oligonucleotides hybridize noncontiguously between the first and second fixed sequence oligonucleotides; extending the hybridized first and second fixed sequence oligonucleotides and/or the hybridized bridging oligonucleotides using a primer extension reaction to create adjacently-hybridized oligonucleotides; ligating the adjacently-hybridized fixed sequence and bridging oligonucleotides to create contiguous ligation products complementary to the nucleic acid regions of interest; amplifying the contiguous ligation products to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and detecting and quantifying the amplification products from the first and second chromosomes, wherein detecting and amplifying the amplification products provides detection and quantification of the nucleic acid regions of interest from the first and second chromosomes in the sample and allows detection of fetal chromosomal copy number variations.
2. 2. The method of claim 1, wherein one or both of the first and second fixed sequence oligonucleotides of the at least two sets of fixed sequence oligonucleotides comprise universal primer regions that are used in amplification of the contiguous ligation products.
3. 3. The method of claim 1 or 2, wherein unhybridized fixed sequence oligonucleotides are removed prior to amplification of the contiguous ligation products.
4. 4. The method of any one of claims 1 to 3, wherein the first and second fixed sequence oligonucleotides are introduced prior to introduction of the bridging oligonucleotides.
5. 5. The method of any one of claims 1 to 4, wherein the hybridization products of the fixed sequence oligonucleotides and the nucleic acid regions of interest to which they hybridize are isolated prior to introduction of the bridging oligonucleotides.
6. 6. The method of any one of claims 1 to 3, wherein the one or more bridging oligonucleotides are introduced simultaneously with the first and second sets of fixed sequence oligonucleotides.
7. 7. The method of any one of claims 1 to 6, wherein the amplification products are quantified by next generation sequencing.
8. 8. The method of any one of claims 1 to 7, wherein the first or second fixed sequence oligonucleotide comprises one or more indices.
9. 9. The method of claim 8, wherein the amplification products are detected and quantified by next generation sequencing of the one or more indicies.
10. 10. The method claim 8, wherein the first or second fixed sequence oligonucleotide comprises a locus index.
11. 11. The method of claim 10, wherein the amplification products are detected and quantified by hybridization of the locus index to an array.
12. 12. The method of claim 8, wherein the one or more indices comprises an allele index and wherein a bridging oligonucleotide complementary for a specific polymorphism is used in the hybridization with the corresponding allele index.
13. 13. The method of any one of claims 1 to 12, wherein the first or second fixed sequence oligonucleotide of at least one set is specific for a polymorphism in a nucleic acid region interest.
14. 14. The method of claim 13, wherein the fixed sequence oligonucleotide specific for a polymorphism in a nucleic acid region of interest further comprises an allele index.
15. 15. A method for detecting nucleic acid regions of interest in a genetic sample comprising maternal and fetal DNA to detect fetal chromosomal copy number variations, comprising the steps of: providing a genetic sample; introducing at least two sets of first and second fixed sequence oligonucleotides to the genetic sample under conditions that allow the set of fixed sequence oligonucleotides to specifically hybridize to complementary regions in each nucleic acid region of interest in both the maternal and fetal DNA, wherein the first set of first and second fixed sequence oligonucleotides specifically hybridize to complementary regions in nucleic acid regions of interest in a first chromosome and the second set of first and second fixed sequence oligonucleotides specifically hybridize to complementary regions in nucleic acid regions of interest in a second chromosome, and wherein the first fixed sequence oligonucleotide of at least one set is specific for a polymorphism in a nucleic acid region of interest; introducing one or more bridging oligonucleotides to the genetic sample under conditions that allow the bridging oligonucleotides to specifically hybridize to complementary regions in the nucleic acid regions of interest, wherein the one or more bridging oligonucleotides are complementary to a region between the region of the nucleic acid regions of interest complementary to the first and second fixed sequence oligonucleotides of a set, and wherein the one or more bridging oligonucleotides hybridize noncontiguously between the first and second fixed sequence oligonucleotides; extending the hybridized first and second fixed sequence oligonucleotides and/or the hybridized bridging oligonucleotides using a primer extension reaction to create adjacently-hybridized oligonucleotides; ligating the adjacently-hybridized fixed sequence and bridging oligonucleotides to create contiguous ligation products complementary to the nucleic acid regions of interest; amplifying the contiguous ligation products to create amplification products that reflect the relative frequency of the nucleic acid regions of interest in the genetic sample; and detecting and quantifying the amplification products from the first and second chromosomes, wherein detecting and amplifying the amplification products provides detection and quantification of the nucleic acid regions of interest from the first and second chromosomes in the sample and allows detection of fetal chromosomal copy number variations.
16. 16. The method of claim 15, wherein one or both of the first or second fixed sequence oligonucleotides of each set comprises universal primer regions.
17. 17. The method of claim 16, wherein the first fixed sequence oligonucleotides specific for a polymorphism in the nucleic acid regions of interest further comprise an allele index and wherein first fixed sequence oligonucleotides that are not specific for a polymorphism in the nucleic acid regions of interest comprise a locus index.
18. 18. The method of claim 16 or 17, wherein the universal primer regions are used to amplify the contiguous ligation products and resulting amplification products are quantified by next generation sequencing of the allele and locus indices.
19. 19. The method of claim 17 or 18, wherein the universal primer regions are sued to amplify the contiguous ligation products and resulting amplification products are quantified by hybridization of the allele and locus indices to an array.