Combinatorial variant detection for rapid genotyping

Abstract

The present disclosure relates to methods of detecting DNA sequences of interest. More specifically, this disclosure relates to multiplex methods for detecting DNA sequences of interest using combinations of probe sets via highly sensitive methods.

Description

Attorney Docket No. 131588-1602COMBINATORIAL VARIANT DETECTION FOR RAPID GENOTYPINGCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No. 63 / 693,014, filed on September 10, 2024, the entire disclosure of which is incorporated by reference herein.TECHNICAL FIELD

[0002] The present disclosure relates to methods of detecting DNA sequences of interest. More specifically, this disclosure relates to multiplex methods for detecting DNA sequences of interest using combinations of probe sets via highly sensitive methods.BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Identification of DNA variants or DNA sequences of interest represents an integral part of diagnostic and personalized medicine. Different DNA variants or sequences of interest may be associated with likelihood of developing a disease, presence of a disease, progression of a disease, and responsiveness of a disease to specific therapeutics. DNA variant detection technology has been used in many contexts, including, but not limited to, pre-natal screening and cancer diagnosis and treatment.

[0005] Two major DNA technologies are currently used to detect sequences of interest. The first is polymerase chain reaction, which can detect as little as a single molecule of interest but has drawbacks including higher rates of false positives due to amplification of off-target molecules and is limited by the number of targets that can be detected in a single experiment. The second is directly sequencing DNA, e.g., via whole genome sequencing, which has a high capacity to detect mutations but is expensive and time intensive. Thus, there is a need for an effective, highly-sensitive, efficient, and rapid method for detecting mutations associated with disease likelihood, phenotypes, outcomes, and responsiveness.SUMMARY14932-1527-2550.1Attorney Docket No. 131588-1602

[0006] The present disclosure provides methods and substrates for combinatorial assays that can detect a particular sequence of interest without the need for sequencing.

[0007] In one aspect, the present disclosure provides methods of detecting a DNA sequence of interest, comprising: (a) obtaining a DNA sample from a subject; (b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three wells; (c) reacting each well with a chemical or enzyme such that a proportion of each probe or probe pair that specifically hybridizes to the DNA sample is modified; and (d) detecting the presence or absence of each different DNA sequence of interest; wherein detection of a given modified probe or probe pair in a sufficient plurality of the wells that contain a given corresponding probe or probe pair indicates the presence of the DNA sequence of interest to which the given corresponding probe or the probe pair hybridized.

[0008] In some embodiments, the methods further comprise amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each different DNA sequence of interest.

[0009] In some embodiments, reacting each well comprises pyrophosphorolysis of the probes. In some embodiments, reacting each well comprises ligation of the probe pairs.

[0010] In some embodiments, each probe or probe pair is detectably labeled.

[0011] In some embodiments, each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs that each hybridize to a different DNA sequence of interest.

[0012] In some embodiments, the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest.24932-1527-2550.1Attorney Docket No. 131588-1602

[0013] In some embodiments, the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different DNA sequences of interest.

[0014] In some embodiments, the subject has cancer, had cancer, or is suspected of having cancer. The cancer may be selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0015] In some embodiments, each different DNA sequence of interest corresponds to a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, a somatic mutation, a germline mutation, or any combination thereof. For example, the sequences of interest may comprise single nucleotide polymorphisms (SNP), insertions / deletions (indels), breakpoints, copy number variants (CNV), etc.

[0016] In some embodiments, each different DNA sequence of interest comprises a different mutation in a single gene. In some embodiments, the single gene is BRCA1 or BRCA2. In some embodiments, the single gene is PALB2.

[0017] In some embodiments, detection of a given modified probe or probe pair comprises using qPCR.

[0018] In another aspect, the present disclosure provides methods of detecting a mutation in a gene of interest, comprising: (a) obtaining a DNA sample from a subject; (b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes, wherein each probe in a well hybridizes to a different34932-1527-2550.1Attorney Docket No. 131588-1602 mutation of the gene of interest, and wherein each probe is present in at least three wells; (c) reacting each well with a pyrophosphorolysis enzyme, thereby digesting each probe, wherein any probe that specifically hybridizes to a mutation in the gene of interest that is present in the DNA sample is digested such that in can be circularized; (d) circularizing any digested probes that specifically hybridized to the mutation in the gene of interest that is present in the DNA sample, thereby forming circularized probes; and (e) detecting the presence or absence of the mutation in the gene of interest; wherein detection of a given circularized probes in a plurality of the wells that contain a given corresponding probe indicates the presence of the mutation in the gene of interest to which the given corresponding probe specifically hybridizes. In some embodiments, the method further comprises amplifying the circularized probes prior to detecting the presence or absence of the mutation in the gene of interest.

[0019] In some embodiments, each probe is detectably labeled.

[0020] In some embodiments, each different combination of probes comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes.

[0021] In some embodiments, the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes that each hybridize to a different mutation of the gene of interest.

[0022] In some embodiments, the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations of interest.

[0023] In some embodiments, the subject has cancer, had cancer, or is suspected of having cancer. The cancer may be selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal44932-1527-2550.1Attorney Docket No. 131588-1602 cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0024] In some embodiments, each different mutation of interest is a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, a somatic mutation, a germline mutation, or any combination thereof. For example, the sequences of interest may comprise single nucleotide polymorphisms (SNP), insertions / deletions (indels), breakpoints, copy number variants (CNV), etc. In some embodiments, the mutation in the gene of interest is a somatic mutation. In some embodiments, the gene of interest s BRCAl or BRCA2 or PALB2.

[0025] In some embodiments, the DNA sample comprises tumor DNA. In some embodiments, the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

[0026] In another aspect, the present disclosure provides methods of detecting a mutation associated with responsiveness or resistance to a therapy, comprising: (a) obtaining tumor DNA from a subject, wherein the subject the subject has cancer, had cancer, or is suspected of having cancer; (b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different mutation associated with responsiveness or resistance to a therapy, wherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled; (c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; and (d) detecting the presence or absence of each mutation associated with responsiveness or resistance to a therapy; wherein detection of a given modified probe or a given modified probe pair in a substantial plurality of the wells that contain a corresponding given probe or a corresponding given probe pair indicates the presence of the mutation associated with responsiveness or resistance to a therapy to which the a corresponding given probe or the a corresponding given probe pair hybridizes. In some embodiments, the method further comprises (d) amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each mutation associated with responsiveness or resistance to a therapy.54932-1527-2550.1Attorney Docket No. 131588-1602

[0027] In some embodiments, reacting each well comprises pyrophosphorolysis of the probes. In some embodiments, reacting each well comprises ligation of the probe pairs.

[0028] In some embodiments, the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

[0029] In some embodiments, each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

[0030] In some embodiments, the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different mutation associated with responsiveness or resistance to a therapy.

[0031] In some embodiments, the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations associated with responsiveness or resistance to a therapy.

[0032] In some embodiments, detection of a given modified probe or probe pair comprises using qPCR.

[0033] In another aspect, the present disclosure provides methods of detecting a mutation in BRCA1 or BRCA2, comprising: (a) obtaining tumor DNA from a subject, wherein the subject the subject has breast cancer, had breast cancer, or is suspected of having breast cancer; (b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes of a different mutation in BRCA1 or BRCA2, wherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled; (c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; and (d) detecting the presence or absence of each mutation in BRCA1 or BRCA2, wherein detection of a modified probe or a modified probe pair in a substantial plurality of the wells that contain a probe or a probe pair that corresponds to the modified probe or probe pair indicates the presence of the mutation in BRCA1 or BRCA2 to which the corresponding probe or probe64932-1527-2550.1Attorney Docket No. 131588-1602 pair hybridized. In some embodiments, the methods further comprise amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each mutation in BRCA1 or BRCA2.

[0034] In some embodiments, reacting each well comprises pyrophosphorolysis of the probes. In some embodiments, reacting each well comprises ligation of the probe pairs.

[0035] In some embodiments, the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

[0036] In some embodiments, the mutation in BRCA1 or BRCA2 is a somatic mutation. In some embodiments, the mutation in BRCA1 or BRCA2 is a germline mutation. The mutation may comprise single nucleotide polymorphisms (SNP), insertions / deletions (indels), breakpoints, copy number variants (CNV), etc.

[0037] In some embodiments, each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

[0038] In some embodiments, the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different mutation in BRCA1 or BRCA2.

[0039] In some embodiments, method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations in BRCA1 or BRCA2.

[0040] In some embodiments, the method may further comprise detecting a mutation in PALB2.

[0041] In another aspect, the present disclosure provides substrates comprising a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes of a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three different wells. In some embodiments, the substrate is a plate or a microfluidic device. In some embodiments, the plurality of wells comprises 48, 96, 384, or 1536 wells. In some74932-1527-2550.1Attorney Docket No. 131588-1602 embodiments, each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. In some embodiments, the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest. In some embodiments, each probe or probe pair is detectably labeled. In some embodiments, each well in the plurality of wells comprises a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof. In some embodiments, the plurality of wells are arranged in rows and columns, and the different combination of probes or probe pairs are distributed such that each probe or probe pair is present in a unique combination of wells.

[0042] In another aspect, the present disclosure provides kit comprising a substrate disclosed herein. In some embodiments, the kit may further comprise a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWING

[0043] The Fig. shows an example of a 96-well plate format arranged with multiplexed probes to detect a particular sequence of interest.DETAILED DESCRIPTION

[0044] Provided herein is a method of detecting variants in a biological sample taken from a patient with a disease, suspected of having a disease, or suspected to be at risk for a disease. The method utilizes combinations of a plurality of highly sequence-specific probes or probe pairs, each of which only recognizes a single nucleotide sequence, to identify mutations in sequences associated with cancer. These probes or probe pairs may be used in Multiplex Ligation-dependent Probe Amplification (MLP A) assays or probes used in pyrophosphorolysis assays. By utilizing unique combinations of the plurality of probes or probe pairs in various wells, it is possible to identify the mutated sequences based on the presence of amplicons and / or double-stranded DNA complexes of interest in the wells that each have the DNA sequence of interest specific to a particular probe or probe pair. This method of detecting mutations can be designed to detect thousands of potential mutated sequences based on the number of highly sequence-specific probes or probe pairs used.84932-1527-2550.1Attorney Docket No. 131588-1602

[0045] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology. It is to be understood that the present disclosure is not limited to particular uses, methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein for the purpose of describing particular embodiments only and is not intended to be limiting.I. Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0047] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0048] As used herein, the term “primer pairs” or “probes” or “probe pairs” shall be understood as being polynucleotide molecules having a sequence identical, complementary, or homologous to the complement of regions of a target polynucleotide, which is to be detected or quantified. Nucleotide analogues may be used in the probe or probe pairs.

[0049] As used herein, the term “cancer” is not limited to any stage, grade, histomorphological feature, aggressivity, or malignancy of an affected tissue or cell aggregation.

[0050] As used herein, the term “sample” or “biological sample” may be derived from or may have been in contact with a biological organism. Examples for biological samples94932-1527-2550.1Attorney Docket No. 131588-1602 include, but are not limited to, cells, tissue, body fluids, lavage fluid, smear samples, biopsy specimens, blood, urine, saliva, plasma, serum, and cell culture supernatant.

[0051] A “tumor sample” is a biological sample containing tumor cells, whether intact or degraded. The sample may be of any biological tissue or fluid. Such samples include, but are not limited to, blood, serum, plasma, blood cells (e.g., white blood cells), tissue, core or fine needle biopsy samples, cell-containing body fluids, urine, peritoneal fluids, tissue biopsies, and cells isolated therefrom. Tumor samples may also include sections of tissues such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular parts thereof. A tumor sample to be analyzed can be tissue material from a neoplastic lesion taken by aspiration or punctuation, excision or by any other surgical method leading to biopsy or resected cellular material. Such comprises tumor cells or tumor cell fragments obtained from the patient. The sample may be a body fluid, including, but not limited to, blood fluids, serum, plasma, lymph, ascitic fluids, and urine.

[0052] A “gene” is a set of segments of nucleic acid that contains the information necessary to produce a functional RNA product. A “gene product” is a biological molecule produced through transcription or expression of a gene, e.g., an mRNA, cDNA, or the translated protein.

[0053] The term “hybridization-based method” as used herein refers to methods imparting a process of combining complementary, single-stranded nucleic acids or nucleotide analogues into double stranded molecules. Nucleotides or nucleotide analogues will bind to their complement under normal conditions, such that two perfectly complementary stands will bind to each other readily. In bioanalytics, single stranded probes are used to find complementary target sequences. If such sequences exist in the sample, the probes will hybridize to said sequences, which can then be used in later steps to detect sequences of interest.

[0054] An oligonucleotide capable of specifically binding sequences of a gene or fragments thereof relates to an oligonucleotide which specifically hybridizes to a gene or gene product, such as the gene’s mRNA or cDNA or to a fragment thereof. As used herein, the term “specifically hybridize” refers to an oligonucleotide that is 100% complementary to a sequence of interest.104932-1527-2550.1Attorney Docket No. 131588-1602

[0055] As used herein, the term “substantial plurality” in the context of detection of modified or amplified probes, primers, or samples means a sufficient quantity of wells to allow an individual practicing the claimed methods to confirm that the detection is not in error (i.e., a false positive or a false negative). In general, it is desirable for all wells with a specified primer or probe to provide a detectable response when the corresponding target sequence is present in sample, but because the possibility of assay failure cannot be ruled out, detection of every well in which the specified primer or probe may not be required. For example, in a hypothetical assay in which 8 wells contain a given primer or probe of interest, if 7 are positive among the 8 wells, those 7 alone may be sufficient for unique identification. In other words, not every well containing a given primer / probe of interest must be positive in order to identify the variant / sequence of interest corresponding to the given primer / probe so long as the pooling technique is sufficiently over-specified, i.e., each variant has probes in x wells and any set of positive wells > y (where y < x) is able to identify the variant.

[0056] It is understood that aspects and variation of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and variations. Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.II. DNA Sequencing to Detect Disease Likelihood, Progression, and Treatment Possibilities

[0057] DNA variants or sequences of interest may be associated with the presence of different diseases, different likelihoods of disease development, different outcomes or progression, or different treatment responsiveness to different therapies. Thus, for instance, by detecting particular sequences of interest (e.g., particular mutations or single nucleotide polymorphisms) in a DNA sample taken from a subject having, suspected of having, or suspected of developing cancer, it may be possible to predict a number of disease-related phenotypes and tailor subsequent treatment or prognosis based on the specific sequences of interest that are identified. Diseases and conditions that could benefit from such detection114932-1527-2550.1Attorney Docket No. 131588-1602(e.g., detection of sequences of interest according to the disclosed methods) include cancer, genetic disorders, and the like.

[0058] Sequencing the full genome of a subject or only the relevant portion of the subject’s genome is expensive and time-intensive. Thus, methods of identifying specific DNA sequences of interest without having to fully sequence the subject’s DNA represent an improvement in the field of personalized medicine, including cancer diagnosis and treatment, as these methods are faster and cheaper to implement than current methods used. The disclosed methods provide such benefits, as they allow for rapid, highly specific detection of specific mutations or sequences of interest at a fraction of the time and cost of sequencingbased methods.III. Combinatorial Methods of Detecting DNA Variants and DNA Sequences of Interest

[0059] Provided herein are multiplexed and combinatorial method of detecting DNA variants and / or DNA sequences of interest in a DNA sample from a cell from a subject. The methods may include obtaining a DNA sample from a subject; distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three independent wells; reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the DNA sample is modified; optionally amplifying each probe or probe pair that was modified; and detecting the presence or absence of each different DNA sequence of interest based on the detection or amplification of a probe or probe pair in a substantial plurality of the wells that contain the probe or probe pair. For the purposes of the disclosed methods, the wells are organized with different combinations of probes or probe pairs such that when a specific probe or probe pair are modified and / or amplified, a specific, singular combination of wells indicate detection, thereby indicating the presence of the DNA sequence of interest to which the probe or the probe pair hybridized.

[0060] The disclosed methods transform prior methods of pooling multiple patient samples to instead rapidly and efficiently determine whether a particular sequence of interest is present in a sample from an individual. Briefly, using a substrate such as a multi-well plate (e.g., a 48, 96, 384, or 1536 well plate), probes that bind to different sequences of interest (e.g.,124932-1527-2550.1Attorney Docket No. 131588-1602 different variants of a single gene or variants of multiple different genes) can be arranged in the wells such that different of probes or probe pairs are each distributed in a unique combination of wells. In this arrangement a positive signal that corresponds to a particular sequence of interest can be determined based on the pattern of wells that are positive. Using this combinatorial approach it would be possible to simultaneously screen for hundreds of different sequences of interest (e.g., different variants of a single gene or variants of multiple different genes) in a 96-well format and determine whether any one of the sequences is present in the sample without the need for sequencing. In other words, using a 96-well format, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, or about 1100 sequences of interest could be simultaneously screened.

[0061] The DNA sample may be collected from a biological sample from the subject. The biological sample refers to any substance containing or presumed to contain nucleic acids. The nucleic acids may be RNA or DNA (e.g., genomic DNA, cell free DNA, circulating tumor DNA). The biological sample may include samples such as biopsy or tissue samples, frozen samples, blood and blood factions or products (e.g., serum, platelets, red blood cells, and the like), tumor samples, sputum, bronchoalveolar lavage, cultured cells (e.g., primary cultures, explants, transformed cells, and the like), stool, urine, bodily fluids, plasma, tears, buccal sample, cavity rinse, ascites, organ rinse, feces, and the like. In some embodiments, the sample is a blood, plasma, or serum sample. In some embodiments, the sample is a tumor tissue sample (e.g., a biopsy). A sample may include, but is not limited to, tissue, blood, plasma, saliva, urine, semen, amniotic fluid, oocytes, skin, hair feces, cheek swabs, or pap smear lysate from a subject. A biopsy refers to the process of removing a tissue sample for diagnostic or prognostic evaluation and to the tissue specimen derived from such a process. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. A bodily fluid includes all fluids obtained from a mammalian body, either processed (e.g., serum) or unprocessed, which can include, for example, blood, plasma, urine, lymph, gastric juices, bile, serum, saliva, sweat, and spinal and brain fluids.

[0062] The sample from the subject may be a sample taken from a patient having or suspected of having cancer. The cancer may be breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease,134932-1527-2550.1Attorney Docket No. 131588-1602 cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. The sample may comprise genomic DNA from tumor cells or circulating tumor DNA (ctDNA). The sample may comprise cell-free DNA (cfDNA). The sample may comprise genomic DNA from non-tumor cells.

[0063] The sample from the patient may be a maternal biological sample. The maternal biological sample may include cfDNA and / or fetal DNA. The fetal DNA may include fetal DNA sequences of interest.

[0064] The DNA sequences of interest may include a mutation associated with a disease. The DNA sequence of interest may include a recessive embryonic lethal mutation. The DNA sequence of interest may be a dominant haploinsufficient mutation. The DNA sequence of interest may include a mutation associated with susceptibility to a therapy. The DNA sequence of interest may include a somatic mutation. The DNA sequence of interest may include a germline mutation. The sequences of interest may comprise any number of mutations or variant sequences, including, but not limited to single nucleotide polymorphisms (SNP), insertions / deletions (indels), breakpoints, copy number variants (CNV), etc. The DNA sequences of interest tested in the combinatorial methods described below may include one or more of the above.

[0065] The DNA sequences of interest may be different mutations in a single gene or multiple genes. In instances in which a single gene is the focus of the analysis, each probe or probe pair may correspond to a different mutation in the gene, such that the method allows for the detection / determination of a specific mutation or variation in the gene. The various mutations may indicate that the subject from which the DNA was obtained is responsive or non-responsive to a particular type of treatment. For example, the different mutations in a144932-1527-2550.1Attorney Docket No. 131588-1602 single gene may be mutations in either BRCA1 or BRCA2 or PALB2, as certain mutations in these genes correspond to responsiveness to specific therapies.

[0066] In general, the frequency of any variant or sequence of interest should be considered because if there are too many positives among the pool of variants being interrogated, all of the wells may appear positive, making the results difficult to interpret. Thus, the DNA sequences of interest may include rare variants of interest that are present at a low frequency in a reference population or the subject being assessed. The reference population may be subjects without a disease or condition of interest. The reference population may be subjects with the disease or condition of interest. The rare variants of interest may be present in less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the reference population.

[0067] In instances in which multiple genes or analyzed, the genes / sequences of interest may be combined in a multiplexed format to provide useful diagnostic, prognostic, or therapeutic information. For example, an assay may comprise a combination of genes / sequences of interest in which each gene or sequence of interest comprises a mutation, variant, or other feature that is informative about a subject’s responsiveness or non-responsiveness to a particular therapy.

[0068] The DNA sample, including the DNA sequence of interest, from the sample from the patient may be combined with one or more probes or probe pairs and with a chemical or enzyme that can modify the probe or probe pairs specifically hybridized to the DNA sample.A . Pyrophosphorlyzing Assay Probes

[0069] The probes may be designed to be used in conjunction with a pyrophosphorolyzing enzyme. The DNA sample may be introduced to a well or a plurality of wells, wherein the DNA sample in each well is combined with a unique combination of probes. Each probe in the unique combination of probes may be used to detect a different DNA sequence of interest in the DNA sample. Each unique combination of probes may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes per well. The total number of probes that is utilized in the plurality of the wells may be at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes.154932-1527-2550.1Attorney Docket No. 131588-1602Thus, the method utilizing these probes may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different DNA sequences of interest.

[0070] Each probe may include a unique identifying or barcode sequence at the probe’s 5’ end or 3’ end or a detectable label. The identifying or barcode sequence for each probe may include universal detection primer binding sites. The identifying or barcode sequences for each probe may include unique detection primer binding sites. The detectable label may be a fluorophore, a colorimetric reagent, a radiolabel, or another moiety that is detectable via laboratory methods.

[0071] Each probe may include a sequence complementary to a specific DNA sequence of interest (e.g., at the probe’s 3’ end of the probe). Each probe should possess sufficient sequence complementarity to hybridize with a specific DNA sequence of interest (e.g., a particular mutation) to form a double-stranded complex. The double-stranded complex between the probe and the DNA sequence of interest may be completely hybridized if the DNA sequence of interest and the probe sequence are fully complementary. The doublestranded complex between the probe and the DNA sequence of interest may not be completely hybridized if the DNA sequence of interest and the probe sequence are not fully complementary.

[0072] The chemical(s) or enzyme(s) that modify the probes or probe pairs may be reach ve / active only when a double-stranded complex is formed between a given probe and the corresponding sequence of interest. One non-limiting example of such an enzyme is a pyrophosphorolysing enzyme. The pyrophosphorolysing enzyme may pyrophosphorolyze a double-stranded complex formed between a probe and the corresponding DNA sequence of interest in the 3’ to 5’ direction from the 3’ end of the probe to generate a partially digested probe strand and the DNA sequence of interest. The pyrophosphorolyzing enzyme may only be able to pyrophosphorolyze the probe strand of double-stranded complex in the 3’ to 5’ direction when the double-stranded complex is hybridized; therefore, the pyrophosphorolyzing enzyme may be unable to pyrophosphorolyze the probe strand of the double-stranded complex when there is a mismatch between the sequence of the probe and the DNA sequence of interest. A mismatch between the probe and the DNA sequence of interest of the double-stranded complex that is not at the 3’ terminus of the probe may result in a partially digested probe strand or undigested probe strand that is longer than the partially164932-1527-2550.1Attorney Docket No. 131588-1602 digested probe strand that is completely complementary to the DNA sequence of interest. The pyrophosphorolyzing enzyme may partially digest any of the double-stranded complexes generated from the hybridization of any of the individual probes introduced to the well and the respective DNA sequences of interest to generate a plurality of partially digested probes based on the degree of hybridization of each of the probes to their respective DNA sequence of interest.

[0073] The chemical(s) or enzyme(s) that modify the probes may also include a ligase and / or a ligation oligonucleotide. A ligation oligonucleotide may specifically bind to one of the plurality of the probes. In some embodiments, the ligation oligonucleotide may comprise a region complementary to the identifying or barcode region of the probe adjacent to a region complementary to the probe sequence adjacent to the region digested by the pyrophosphorolyzing enzyme when the probe sequence complementary to the DNA sequence of interest is identical to the DNA sequence of interest. Hybridization of the two regions of the partially digested probe to the ligation oligonucleotide allows the 5’ and 3’ ends of the partially digested probe to be brought into proximity with one another. When the 5’ and 3’ ends of the partially digested probe are brought into proximity based on their hybridization to the ligation oligonucleotide, the 5’ and 3’ ends may be ligated together by the ligase to create a circularized probe product. If the partially digested probe is not completely digested due to mismatch in the double-stranded complex between the probe and the DNA sequence of interest, the 5’ and 3’ ends of the partially digested probe will not be brought into appropriate proximity to allow for the ligation of the probe, and a circularized probe product will not be formed. In some embodiments, the 5’ end of the oligonucleotide may be ligated to the 3’ end of the partially digested probe to form an intermediate probe product. The intermediate probe product may be further ligated together at its 3’ and 5’ ends to generate a circularized probe product.

[0074] A 5’-3’ exonuclease may be introduced to the wells following such a ligation step. The exonuclease may degrade linear oligonucleotide strands at their 5’ end but may be unable to degrade circularized oligonucleotides. Thus, partially digested probes that are not converted into circularized probe products may be degraded by the exonuclease, and the circularized probe products may not be degraded by the exonuclease. The circularized probe products may then be detected via standard detection techniques, including but not limited to polymerase chain reaction, to amplify and detect the identifying or barcode regions of the174932-1527-2550.1Attorney Docket No. 131588-1602 probes that have been circularized and, therefore, undegraded upon application of the endonuclease.

[0075] In some embodiments, identification of DNA sequences of interest may be identified by identifying wells that have circularized probe products via the detectable label associated with the probe and identifying the specific probes in the combination or plurality of probes that are shared amongst the wells, eliminating the need to sequence all of the different amplification products of the DNA sequences of interest to identify the DNA sequences of interest.

[0076] In some embodiments, identification of DNA sequences of interest may be identified by amplifying the identifying or barcoding regions of the plurality of probes. In some embodiments, the identifying or barcode regions of the plurality of probes may be amplified using universal detection primers that can amplify all of the probe identifying or barcode regions. In some embodiments, the identifying or barcode regions of the plurality of probes may be amplified using amplification primers specific to the plurality of probes of the specific well. The specific DNA sequences of interest may be identified by sequencing the identifying or barcode regions of the circularized probes that were not degraded upon application of the endonuclease, as each of the identifying or barcode regions is unique to a specific DNA sequence of interest detected by the individual probe. The specific DNA sequences of interest may be determined by identifying the detectable label of the probes.

[0077] Determination of which sequences of interest are present in the DNA sample may be done without sequencing of the circularized probe products. Detection may be done by identifying wells that have circularized probe products. Not all wells need to have circularized probe products if none of the sequences of interest detected by the plurality of probes in the well are present in the DNA sample. In some embodiments, not all wells need to be analyzed; merely a plurality of wells that allow for detection of each probe at least twice need to be analyzed in order to ensure that a sequence of interest that permits for the proper processing of each probe are tested more than once. In some embodiments, all of the wells are analyzed in order for conclusive detection of possible variants of interest in the DNA sample. Detection of the presence of any of the circularized probe products indicates that at least one of the plurality of probes that detects a specific sequence of interest is present in the sample. As each specific probe is present in more than one well, it is possible to identify that a certain sequence of interest detected by that probe is present in the DNA sample by184932-1527-2550.1Attorney Docket No. 131588-1602 identifying the probe that is present in all of the wells that have been determined to have a circularized probe product. If there are too many wells that are determined to have circularized probe products such that it is not possible to identify the individual probes that gave rise to the circularized probe products, the plurality of probes that are in the wells that are determined to have circularized probe products may be tested again with a reduced number of probes per well to allow for the determination of which probes are giving rise to the circularized probe products. In some embodiments, if, in the first round of detection, there were n ambiguous probes among the x initial probes in each well, a reduced set of size y, where y is less than x, must include a reconfigured set of probes wherein each set has all n or n-1 of the ambiguous probes in order to allow for different combinations of probes to be present in each well to definitively identify variants of interest. The wells may have one or more probes but fewer probes than were present in each well of the first detection round. This process may be repeated until the number of probes per well is low enough to allow for the determination of which probes are giving rise to the circularized probe products. For example, in a situation in which n is 4, if a new pooling scheme is designed such that cone an definitively identify the presence of 3 of them, then if none of the 3 are observed, then it would be apparent that the fourth was present.

[0078] Additionally or alternatively, if there is ambiguity about which sites are positive after a first round of this pooling experiment (where, for example, there are 20 different probes per well across 48 wells), a preferred approach to address the ambiguity is to go to a “less-pooled solution” where there are, for instance, 10 probes per well across 96 wells, or 5 probes per well across 192 wells. The advantage of such a scheme is that it allows for building a single “less-pooled solution” that should work for any patient and any set of ambiguous sites, whereas other approaches could require a bespoke construction of an assay for a given patient and that patient’s specific ambiguous variants. Accordingly, from an operational perspective, it may be advantageous to have a single “less-pooled” secondary assay rather than needing “less-pooled” assays on demand for each person who may need it.B. Multiplex Ligation-dependent Probe Amplification Assay Probes

[0079] The probes / probe pairs may be designed to be used in a multiplex ligation-dependent probe amplification (MLP A) assay. The DNA sample may be introduced to a well or a plurality of wells, wherein the DNA sample in each well is combined with a unique combination of probe pairs. Each probe pair in the unique combination of probe pairs may be194932-1527-2550.1Attorney Docket No. 131588-1602 used to detect a different DNA sequence of interest in the DNA sample. Each unique combination of probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probe pairs per well. The total number of probe pairs that is utilized in the plurality of the wells may be at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probe pairs. Thus, the method utilizing these probe pairs may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different DNA sequences of interest.

[0080] Each probe pair may include a first probe and a second probe. The first probe of the probe pair may have a first amplification primer binding site, and the second probe of the probe pair may have a second amplification primer binding site. The first probe of the probe pair may hybridize to a region at the 3’ end of the sequence of interest, while the second probe of the probe pair may hybridize to a region immediately adjacent to the 3’ end of the first probe and upstream of the hybridization site of the first probe on the sequence of interest. Thus, when the probe pair is hybridized to the sequence of interest, the arrangement of the probes will be, from 5’ to 3’, the first probe followed by the second probe, making them competent for ligation. The first amplification primer binding site and the second amplification primer binding site may be universal detection primer binding sites. The first amplification primer binding site and the second amplification primer binding site may include unique detection primer binding sites for each probe pair. Each probe pair may include a unique identifying or barcode sequence or a detectable label within the probes and / or at the 5’ end of the first probe of the probe pair and / or the 3’ end of the second probe of the probe pair. The detectable label may be a fhiorophore, a colorimetric reagent, a radiolabel, or another moiety that is detectable via laboratory methods.

[0081] The first and second probe of each probe pair may be designed to hybridize to a specific DNA sequence of interest. The first probe of the probe pair may bind at the 3’ portion of the DNA sequence of interest, and the second probe of the probe pair may bind at the 5’ portion of the DNA sequence of interest. The 3’ terminus of the first probe is adjacent to the 5’ terminus of the second probe when both probes of the probe pair are hybridized to the DNA sequence of interest. If the sequences of either probe of the probe pair are not identical to the DNA sequence of interest, the probes of the probe pair may not hybridize to204932-1527-2550.1Attorney Docket No. 131588-1602 the DNA sequence of interest. In particular, the location of a possible mutation of interest to be detected by the probe pair may be the nucleotide of the DNA sequence of interest corresponding the nucleotide at the 3’ terminus of the first probe pair or the nucleotide at the 5’ terminus of the second probe pair, causing the nucleotides at the termini of the probe pairs to not be immediately adjacent to one another.

[0082] Following hybridizing of the probes of the probe pair to the DNA sequence of interest in the DNA sample, the chemical or enzyme that modifies the probe pairs hybridized to the DNA sequence of interest may modify the probe pair. The chemical or enzyme may be a ligase. When the first probe and the second probe have a sequence completely identical to the DNA sequence of interest, the ligase may be able to ligate the 3’ terminus of the first probe to the 5’ terminus of the second probe, creating a double-stranded complex. If the nucleotide at the 3’ terminus of the first probe or the nucleotide of the 5’ terminus of the second probe are not perfectly complementary to the DNA sequence of interest, the ligase will not be able to ligate the termini of the first and second probe together.

[0083] Hybridization of the probe pairs and ligation of the probe pair termini may result in double-stranded complexes. The resulting double-stranded complexes may be denatured, generating single-stranded ligated probe pair products that have the first and second amplification primer binding sites. Any probe pairs that have not been ligated will result in single-stranded probes that lack both amplification primer binding sites. Following denaturation, the probes in the wells may be subjected to an amplification reaction, wherein the probes that have both the first and second amplification probe binding sites are amplified exponentially while probes that have only the first or second amplification probe binding sites are amplified linearly or not amplified at all.

[0084] The specific DNA sequences of interest may be identified by sequencing the identifying or barcode regions of the probe pairs. The specific DNA sequences of interest may be identified by determining the specific wells that include amplification of the ligated probe pair products and determining which probe pairs in the combination of probe pairs is shared between the wells that have the amplification of the ligated probe pair products, eliminating the need to sequence all of the identifying or barcode regions of the amplified ligated probe pair products to identify the DNA sequences of interest. The specific DNA sequences of interest may be detected using quantitative PCR (qPCR) and related technologies. The specific DNA sequences of interest may be identified by further214932-1527-2550.1Attorney Docket No. 131588-1602 sequencing the amplification products resulting from the amplification of the ligated probe pair products.

[0085] The double-stranded complexes produced when the specific sequence of interest is present in the sample may be detected to determine what sequences of interest are present in a sample. The double-stranded complexes do not include either residual double-stranded DNA in the DNA sample that failed to be denatured or hybridized complexes that failed to ligate and / or amplify. These double-stranded complexes may be determined via detection of the amplification products generated from the double-stranded complexes. These amplification products may be determined via gel electrophoresis, DNA sequencing, detectable label detection, double-stranded DNA dye, or real-tine polymerase chain reaction (PCR). The double-stranded complexes may be determined without amplification. These complexes may be detected via gel electrophoresis, DNA sequencing, detectable label detection, doublestranded DNA dye, or real-time polymerase chain reaction (PCR). For example, when both of the probes of the probe pair have different detectable fluorescent labels, the double-stranded complex may be determined by detecting complexes that display co-localization of the two fluorescent labels. The double-stranded complex of interest may be identified by detecting an abundance of the double-stranded complex generated from amplification that is more than a background level of double-stranded DNA that may result from incomplete denaturation of double-stranded DNA from the initial subject sample or from hybridized complexes that have failed to ligate and / or amplify.

[0086] Determination of which sequences of interest are present in the DNA sample may be done without sequencing of the double-stranded complexes of interest. Detection may be done by identifying wells that have double-stranded complexes of interest. It should be noted, however, that wells may include undenatured complexes that are double stranded, but which are not the double-stranded complex of interest. Not all wells need to be analyzed; the minimum number of wells to be analyzed is the number having each probe represented twice, in order to ensure that a sequence of interest that permits for the proper processing of each probe is tested more than once. Not all wells need to have double-stranded complexes if none of the sequences of interest detected by the plurality of probe pairs in the well are present in the DNA sample. Preferably, not all wells will have double-stranded complexes in order to permit for the disambiguation of which sequences of interest are present in the DNA sample based on the production of double-stranded complexes in only a subset of wells. Detection of224932-1527-2550.1Attorney Docket No. 131588-1602 the presence of any of the double-stranded complexes indicates that at least one of the plurality of probe pairs detects a specific sequence of interest that is present in the sample. As each specific probe pair is present in more than one well, it is possible to identify that a certain sequence of interest detected by that probe pair is present in the DNA sample by identifying the probe pair that is present in all of the wells that have been determined to have a double-stranded complex (i.e., a detectable amount of double-stranded complexes). If there are too many wells that are determined to have double-stranded complexes such that it is not possible to identify the individual probe pairs that gave rise to the double-stranded complexes, the plurality of probe pairs that are in the wells that are determined to have double-stranded complexes may be tested again with a reduced number of probe pairs per well or a greater number of wells with the same number of probes per well but with additional unique combinations of probes to allow for the determination of which probe pairs are giving rise to the double-stranded complexes. Additionally or alternatively, if there are too many wells that are determined to have double-stranded complexes such that it is not possible to identify the individual probe pairs that gave rise to the double-stranded complexes, the plurality of probe pairs that are in the wells that are determined to have double-stranded complexes may be tested again with more wells with fewer probes per well. The wells may have one or more probe pairs but fewer probe pairs than were present in each well of the first detection round. This process may be repeated until the number of probe pairs per well is low enough to allow for the determination of which probe pairs are giving rise to the double-stranded complexes.C. Specific Embodiments of the Combinatorial Methods

[0087] Thus, provided herein is a method of detecting a DNA sequence of interest, comprising: (a) obtaining a DNA sample from a subject; (b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three wells; (c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the DNA sample is modified to produce a modified probe product; (d) amplifying each probe or probe pair that was modified in (c); and (e) detecting the presence of absence of each different DNA sequence of interest, wherein detection of amplification of a specific modified probe product in all of the wells that contain234932-1527-2550.1Attorney Docket No. 131588-1602 the specific probe or probe pair indicates the presence of the DNA sequence of interest to which the specific probe or the probe pair hybridized. In other words, the reaction chemistry is such that only probes that are perfect matches for a specific modification in a specific location. For example, in an embodiment using 50-base probes, in which the 50thbase needs to match perfectly, bases 1-49 need only to match well enough to recruit the probe (e.g., based 20-30 could be mismatched so long as bases 1-19 and 31-49 match well).

[0088] Reacting each well may comprise pyrophosphorolysis of the probes. Reacting each well may comprise ligation of the probe pairs. Reacting each well may comprise an initial extension of the probe pairs followed by ligation of the probe pairs. Each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest. The method may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different DNA sequences of interest. The subject may have cancer, have had cancer, or be suspected of having cancer. The cancer may be selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. Each different DNA sequence of interest may correspond to a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, a somatic mutation, or any combination244932-1527-2550.1Attorney Docket No. 131588-1602 thereof. The sequences of interest may comprise any number of mutations or variant sequences, including, but not limited to single nucleotide polymorphisms (SNP), insertions / deletions (indels), breakpoints, copy number variants (CNV), etc. Each different DNA sequence of interest may comprise a different mutation in a single gene. The single gene can be BRCA1 or BRCA2 or PALB2. The DNA sample may comprise tumor DNA. The tumor DNA may be circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

[0089] Also provided herein is a method of detecting a mutation in a gene of interest, comprising (a) obtaining a DNA sample from a subject; (b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes, wherein each probe in a well hybridizes of a different mutation of the gene of interest, and wherein each probe is present in at least three wells; (c) reacting each well with a pyrophosphorolysis enzyme, thereby digesting each probe, wherein any probe that specifically hybridizes to a mutation in the gene of interest that is present in the DNA sample is digested such that it can be circularized; (d) circularizing any digested probes that specifically hybridized to the mutation in the gene of interest that is present in the DNA sample, thereby forming circularized probes; (e) amplifying the circularized probes; and (f) detecting the presence or absence of the mutation in the gene of interest, wherein amplification of the circularized probes in all of the wells that contain that probe indicates the presence of the mutation in the gene of interest to which the probe specifically hybridizes. Each probe may be detectably labeled. Each different combination of probes can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes that each hybridize to a different mutation of the gene of interest. The method may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations of interest. The subject may have cancer, have had cancer, or be suspected of having cancer. The cancer may be selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational254932-1527-2550.1Attorney Docket No. 131588-1602 trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. Each different mutation of interest may be a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, or any combination thereof. The mutation in the gene of interest may be a somatic mutation. The gene of interest may be BRCA1 or BRCA2 or PALB2. The DNA sample may comprise tumor DNA. The tumor DNA may be circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

[0090] Additionally provided herein is a method of detecting a mutation associated with responsiveness or resistance to a therapy, comprising: (a) obtaining tumor DNA from a subject, wherein the subject has cancer, had cancer, or is suspected of having cancer; (b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different mutation associated with responsiveness or resistance to a therapy, wherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled; (c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; (d) amplifying each probe or probe pair that was modified in (c); and (e) detecting the presence or absence of each mutation associated with responsiveness or resistance to a therapy, wherein detection of an amplified probe or an amplified probe pair that in all of the wells that contain the probe or the probe pair indicates the presence of the mutation associated with responsiveness or resistance to a therapy to which the probe or the probe pair hybridizes. Reacting each well may comprise pyrophosphorolysis of the probes. Reacting each well may comprise ligation of the probe pairs. The tumor DNA may be circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample. Each different combination of probes or probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least264932-1527-2550.1Attorney Docket No. 131588-160214, or at least 15 probes or probe pairs. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different mutation associated with responsiveness or resistance to therapy. The method may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations associated with responsiveness or resistance to a therapy.

[0091] Provided herein is a method of detecting a mutation in BRCA1 or BRCA2, comprising (a) obtaining tumor DNA from a subject, wherein the subject has breast cancer, had breast cancer, or is suspected of having breast cancer; (b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different mutation in BRCA1 or BR( A2.jwherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled; (c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; (d) amplifying each probe or probe pair that was modified in (c); and (e) detecting the presence or absence of each mutation in BRCA1 or BRCA2, wherein detection of an amplified probe or an amplified probe pair that in all of the wells that contain the probe or the probe pair indicates the presence of the mutation in BRCA1 or BRCA2 to which the amplified probe or the amplified probe pair hybridized. Reacting each well may comprise pyrophosphorolysis of the probes. Reacting each well may comprise ligation of the probe pairs. The tumor DNA may be circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample. The mutation in BRCA1 or BRCA2 may be a somatic mutation. Each different combination of probes or probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different mutation in BRCA1 or BRCA2. The method may be capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations in BRCA1 or BRCA2.274932-1527-2550.1Attorney Docket No. 131588-1602

[0092] Additionally provided herein is a substrate comprising a plurality of wells that may be used in the method described above. The substrate may comprise a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three different wells. The substrate may be a plate. The plurality of wells may comprise 48, 96, 384, or 1536 wells. Each different combination of probes or probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest. Each probe or probe pair may be detectably labeled. Each well in the plurality of wells may comprise a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof. The plurality of wells may be arranged in rows and columns, and the different combination of probes or probe pairs may be distributed such that each probe or probe pair may be present in a unique combination of wells.

[0093] Additionally, provided herein is a substrate comprising a microfluidic device that may be used in the method described above. The substrate may comprise a plurality of flow cells, flow channels, or microfluidic device reaction volumes / partitions, wherein each flow cell, flow channel, or microfluidic device reaction volume / partition in the plurality comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three different flow cells. The plurality of flow cells, flow channels, or microfluidic device reaction volumes / partitions may comprise 1, 2, 4, 8, 16, 20, 24, 48, 96, 384, or 1536 flow cells, flow channels, or microfluidic device reaction volumes / partitions. Each different combination of probes or probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. The plurality of flow cells, flow channels, or microfluidic device reaction volumes / partitions may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest. Each probe or probe pair may be detectably labeled. Each flow284932-1527-2550.1Attorney Docket No. 131588-1602 cell, flow channel, or microfluidic device reaction volume / partition in the plurality may comprise a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof.IV. Substrates for Combinatorial Testing

[0094] The disclosed methods may generally be performed on substrates such as multi-well plates, such as 48, 96, 384, or 1536 well plates or microfluidic devices comprising a plurality of flow cells, such as 4, 8, or 16 flow cells.

[0095] A substrate a substrate of the present disclose may comprise, for example, a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes of a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three different wells. In general, the plurality of wells is arranged in rows and columns, and the different combination of probes or probe pairs are distributed such that each probe or probe pair is present in a unique combination of wells. By arranging the wells in such a format, the disclosed methods allow for one to quickly and accurately determine whether a specific sequence of interest, such as a particular mutation or gene variant, is present in a sample without the need for sequencing.

[0096] Each different combination of probes or probe pairs may comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs. The plurality of wells may comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest. Each probe or probe pair can be detectably labeled (e.g., labeled with a fluorophore, a colorimetric reagent, a radiolabel, or another moiety that is detectable via laboratory methods).

[0097] In some embodiments, each well in the plurality of wells comprises a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof. In some embodiments, the pyrophosphorolysis enzyme, the ligase, and the polymerase may be added sequentially to the wells. In some embodiments, the pyrophosphorolysis enzyme, the ligase, the polymerase, or any combination thereof may be added simultaneously to the wells.294932-1527-2550.1Attorney Docket No. 131588-1602

[0098] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates that may need to be independently confirmed.EXAMPLES

[0099] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1 — Determination of BRCA Variants

[0100] BRCA1 and BRCA2 play crucial role in the DNA damage response and repair pathway, a function that is critical in preserving the integrity of the genome. Mutations that interfere with normal cellular function of BRCA not only lead to onset and progression of cancer but also modulate therapy outcome of treatment with platinum drugs. Not only are BRCA mutations known to increase risk of breast cancer, but emerging evidence also indicates that different BRCA mutations increase or decrease a patient’s responsiveness to certain therapies. There are roughly 1000 known BRCA variants that account for 90% of BRCA mutations.

[0101] Using the disclosed methods, a pyrophosphorolysis assay (or a similarly sensitive and specific assay with a binary response) can be multiplexed to determine what particular BRCA variant is present in a sample from a patient. For example, pyrophosphorolysis probes can be arranged in a 96-well plate as shown in the Figure, in which wells A-X are labeled. The table below shows the probes and the corresponding variant sequence of interest, each of which is assigned a number 1-12. For the purposes of this example, each well contains 3 probes, but it should be understood that each well could comprise more probes (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15).304932-1527-2550.1Attorney Docket No. 131588-1602

[0102] The sample is added and the pyrophosphorolysis assay is performed, and wells F, O, and W are positive (i.e., a probe or probe product is amplified and / or detectable). The only sequence of interest present in each of those three wells is sequence 8, and therefore it would be apparent that the variant corresponding to sequence 8 is present in the sample.EQUIVALENTS

[0103] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0104] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent that are not inconsistent with the explicit teachings of this specification.314932-1527-2550.1

Claims

Attorney Docket No. 131588-1602CLAIMSWhat is claimed is:

1. A method of detecting a DNA sequence of interest, comprising:(a) obtaining a DNA sample from a subject;(b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three wells;(c) reacting each well with a chemical or enzyme such that a proportion of each probe or probe pair that specifically hybridizes to the DNA sample is modified; and(d) detecting the presence or absence of each different DNA sequence of interest; wherein detection of a given modified probe or probe pair in a sufficient plurality of the wells that contain a given corresponding probe or probe pair indicates the presence of the DNA sequence of interest to which the given corresponding probe or the probe pair hybridized.

2. The method of claim 1, further comprising amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each different DNA sequence of interest.

3. The method of claim 1 or 2, wherein reacting each well comprises pyrophosphorolysis of the probes.

4. The method of any one of claims 1-3, wherein reacting each well comprises ligation of the probe pairs.

5. The method of any one of claims 1-4, wherein each probe or probe pair is detectably labeled.

6. The method of any one of claims 1-5, wherein each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

7. The method of any one of claims 1-6, wherein the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100,324932-1527-2550.1Attorney Docket No. 131588-1602 at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest.

8. The method of any one of claims 1-7, wherein the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different DNA sequences of interest.

9. The method of any one of claims 1-8, wherein the subject has cancer, had cancer, or is suspected of having cancer.

10. The method of claim 9, wherein the cancer is selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

11. The method of any one of claims 1-10, wherein each different DNA sequence of interest corresponds to a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, a somatic mutation, a germline mutation, or any combination thereof.

12. The method of any one of claims 1-11, wherein each different DNA sequence of interest comprises a different mutation in a single gene.

13. The method of claim 12, wherein the single gene is BRCA1 or BRCA2 or PALB2.

14. The method of any one of claims 1-13, wherein the detection of a given modified probe or probe pair in a sufficient plurality of the wells is detected using qPCR.334932-1527-2550.1Attorney Docket No. 131588-160215. A method of detecting a mutation in a gene of interest, comprising:(a) obtaining a DNA sample from a subject;(b) distributing the DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes, wherein each probe in a well hybridizes to a different mutation of the gene of interest, and wherein each probe is present in at least three wells;(c) reacting each well with a pyrophosphorolysis enzyme, thereby digesting each probe, wherein any probe that specifically hybridizes to a mutation in the gene of interest that is present in the DNA sample is digested such that it can be circularized;(d) circularizing any digested probes that specifically hybridized to the mutation in the gene of interest that is present in the DNA sample, thereby forming circularized probes; and(e) detecting the presence or absence of the mutation in the gene of interest; wherein detection of a given circularized probe in a plurality of the wells that contain a given corresponding probe indicates the presence of the mutation in the gene of interest to which the given corresponding probe specifically hybridizes.

16. The method of claim 15, further comprising amplifying the circularized probes prior to detecting the presence or absence of the mutation in the gene of interest.

17. The method of claim 15 or 16, wherein each probe is detectably labeled.

18. The method of any one of claims 15-17, wherein each different combination of probes comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes.

19. The method of any one of claims 15-18, wherein the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes that each hybridize to a different mutation of the gene of interest.

20. The method of any one of claims 15-19, wherein the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations of interest.

21. The method of any one of claims 15-20, wherein the subject has cancer, had cancer, or is suspected of having cancer.344932-1527-2550.1Attorney Docket No. 131588-160222. The method of claim 21, wherein the cancer is selected from breast cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, a brain / CNS tumor, Castleman disease, cervical cancer, colon or rectum cancer, endometrial cancer, esophagus cancer, a Ewing tumor, eye cancer, gallbladder cancer, a gastrointestinal carcinoid tumor, a gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic Syndrome, nasal cavity or paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, a pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

23. The method of any one of claims 15-22, wherein each different mutation of interest is a mutation associated with a disease, a recessive embryonic lethal mutation, a dominant haploinsufficient mutation, a mutation associated with susceptibility to a therapy, a somatic mutation, a germline mutation, or any combination thereof.

24. The method of any one of claims 15-23, wherein the mutation in the gene of interest is a somatic mutation.

25. The method of any one of claims 15-24, wherein the gene of interest is BRCA1 or BRCA2 or PALB2.

26. The method of any one of claims 15-25, wherein the DNA sample comprises tumor DNA.

27. The method of claim 26, wherein the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

28. A method of detecting a mutation associated with responsiveness or resistance to a therapy, comprising:(a) obtaining tumor DNA from a subject, wherein the subject has cancer, had cancer, or is suspected of having cancer;(b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the354932-1527-2550.1Attorney Docket No. 131588-1602 plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes to a different mutation associated with responsiveness or resistance to a therapy, wherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled;(c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; and(d) detecting the presence or absence of each mutation associated with responsiveness or resistance to a therapy; wherein detection of a given modified probe or a given modified probe pair in a substantial plurality of the wells that contain a corresponding given probe or a corresponding given probe pair indicates the presence of the mutation associated with responsiveness or resistance to a therapy to which the a corresponding given probe or the a corresponding given probe pair hybridizes.

29. The method of claim 28, further comprising (d) amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each mutation associated with responsiveness or resistance to a therapy.

30. The method of claim 28 or 29, wherein reacting each well comprises pyrophosphorolysis of the probes.

31. The method of any one of claims 28-30, wherein reacting each well comprises ligation of the probe pairs.

32. The method of any one of claims 28-31, wherein the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.

33. The method of any one of claims 28-32, wherein each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

34. The method of any one of claims 28-33, wherein the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that364932-1527-2550.1Attorney Docket No. 131588-1602 each hybridize to a different mutation associated with responsiveness or resistance to a therapy.

35. The method of any one of claims 28-34, wherein the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations associated with responsiveness or resistance to a therapy.

36. A method of detecting a mutation in BRCA1 or BRCA2, comprising:(a) obtaining tumor DNA from a subject, wherein the subject the subject has breast cancer, had breast cancer, or is suspected of having breast cancer;(b) distributing the tumor DNA sample to a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes of a different mutation in BRCA1 or BRCA2, wherein each probe or probe pair is present in at least three wells, and wherein each probe or probe pair is detectably labeled;(c) reacting each well with a chemical or enzyme such that each probe or probe pair that specifically hybridizes to the tumor DNA sample is modified; and(d) detecting the presence or absence of each mutation in BRCA1 or BR( A2.iwherein detection of a modified probe or a modified probe pair in a substantial plurality of the wells that contain a probe or a probe pair that corresponds to the modified probe or probe pair indicates the presence of the mutation in BRCA1 or BRCA2 to which the corresponding probe or probe pair hybridized.

37. The method of claim 36, further comprising amplifying each probe or probe pair that was modified in (c) prior to detecting the presence or absence of each mutation in BRCA1 or BRCA2.

38. The method of claim 36 or 37, wherein reacting each well comprises pyrophosphorolysis of the probes.

39. The method of any one of claim 36-38, wherein reacting each well comprises ligation of the probe pairs.

40. The method of any one of claims 36-39, wherein the tumor DNA is circulating tumor DNA (ctDNA) or tumor DNA obtained from a tumor sample.374932-1527-2550.1Attorney Docket No. 131588-160241. The method of any one of claims 36-40, wherein the mutation in BRCA1 or BRCA2 is a somatic mutation.

42. The method of any one of claims 36-41, wherein each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

43. The method of any one of claims 36-42, wherein the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different mutation in BRCA1 or BRCA2.

44. The method of any one of claims 36-43, wherein the method is capable of detecting at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 different mutations in BRCA1 or BRCA2.

45. A substrate comprising a plurality of wells, wherein each well in the plurality of wells comprises a different combination of probes or probe pairs, wherein each probe or probe pair in a well hybridizes of a different DNA sequence of interest, and wherein each probe or probe pair is present in at least three different wells.

46. The substrate of claim 45, wherein the substrate is a plate or a microfluidic device.

47. The substrate of claim 45 or 46, wherein the plurality of wells comprises 48, 96, 384, or 1536 wells.

48. The substrate of any one of claims 45-47, wherein each different combination of probes or probe pairs comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 probes or probe pairs.

49. The substrate of any one of claims 45-48, wherein the plurality of wells comprise a total of at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, or at least 1500 probes or probe pairs that each hybridize to a different DNA sequence of interest.384932-1527-2550.1Attorney Docket No. 131588-160250. The substrate of any one of claims 45-49, wherein each probe or probe pair is detectably labeled.

51. The substrate of any one of claims 45-50, wherein the plurality of wells are arranged in rows and columns, and the different combination of probes or probe pairs are distributed such that each probe or probe pair is present in a unique combination of wells.

52. The substrate of any one of claims 45-51, wherein each well in the plurality of wells comprises a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof.

53. A kit comprising the substrate of any one of claims 45-51.

54. The kit of claim 53, further comprising a pyrophosphorolysis enzyme, a ligase, a polymerase, or any combination thereof.394932-1527-2550.1

Images