Methods of removing cell-free DNA from buffy coat DNA

Abstract

Disclosed herein are methods for detecting clonal hematopoietic (CH) or germline gene mutation(s), as well as systems and non-transitory computer readable storage media related thereto. In some embodiments, the methods comprise detecting a mutation in one or more genes from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA).

Description

METHODS OF REMOVING CELL-FREE DNA FROM BUFFY COAT DNACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 538,717, filed September 15, 2023, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] Disclosed herein are methods for detecting clonal hematopoietic (CH) or germline gene mutation(s), as well as systems and non-transitory computer readable storage media related thereto. In some embodiments, the methods comprise detecting a mutation in one or more genes from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA).BACKGROUND

[0003] Cell-free DNA (cfDNA) molecules are circulating, double stranded DNA molecules (dsDNA or duplex DNA) found in the blood stream, typically as the result of cell apoptosis or necrosis, particularly in the context of disease, such as cancer. These degraded linear DNA fragments are typically less than 1000 base pairs base pairs in length, with the majority around 100-300 base pairs in length.

[0004] Clonal hematopoiesis (CH) is characterized by the over-representation of blood cells derived from a single clone, for example a clone with a different genetic pattern than other blood cells due to one or more genetic mutations. This process can occur, e.g., among hematopoietic cancer cells wherein the one or more genetic mutations confer increased survival and / or proliferation to the cell. The incidence for CH is less than 1% of the population under age 40 but approximately 10-20% of the population over age 70. While CH does not definitively confirm the presence of a disease, e.g., blood cancer, in an individual, CH does suggest a higher- than- average risk for diseases including blood cancers and heart disease. See, e.g., Jaiswal and Ebert Science 2019;366(6465):eaan4673.

[0005] To obtain the most accurate identification of genomic alterations derived from circulating tumor DNA (ctDNA) from solid tumors using liquid biopsy, the removal ofbackground alterations in the cell-free DNA (cfDNA) from clonal hematopoietic (CH) and germline alterations may be required. The sequencing of DNA extracted from the buffy coat material matching the source of plasma from which the cfDNA is obtained enables the identification of CH and germline alterations from the cfDNA samples, based on seeing the alterations in the buffy coat DNA (bcDNA). However, because cfDNA is present in the buffy coat material, one may observe ctDNA alterations (z.e., present in the cfDNA) in the bcDNA sequencing, resulting in the ctDNA alterations being misidentified as a CH or germline alteration and as a result filtered out of the results.

[0006] Therefore, a need exists for methods that allow for removal of cfDNA from bcDNA such that any mutations present in contaminating cfDNA are not misidentified as originating from bcDNA, e.g., as CH or germline alterations. This may find use, e.g., in preventing incorrect filtering of ctDNA alterations present in cfDNA rather than bcDNA.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.SUMMARY OF THE INVENTION

[0008] The present disclosure provides, inter alia, methods for detecting one or more clonal hematopoietic (CH) or germline gene mutations from a mixture of nucleic acid molecules, e.g., comprising some nucleic acid molecules from nucleated cells, and some nucleic acid molecules from cfDNA. Advantageously, these methods allow the purification of nucleic acid molecules from nucleated cells (e.g., bcDNA) through a size selection that removes contaminating nucleic acid molecules from cfDNA (e.g., ctDNA) in the bcDNA. This provides more accurate detection of CH and / or germline mutations present in the bcDNA, which can then be filtered out during processes to detect mutations present in cfDNA (e.g., ctDNA) and / or used to provide information on CH and / or germline mutations per se.

[0009] In some aspects, provided herein is a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising: detecting a mutation in one or more genes from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), thereby determining that the detected mutation is a CH or germline mutation. In some embodiments, the method further comprises prior to thedetection, performing a size selection on the mixture of nucleic acid molecules, wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture.

[0010] In some aspects, provided herein is a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising: performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size-selected plurality of nucleic acid molecules; and detecting, by one or more processors, a mutation in one or more genes in the size- selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation. In some embodiments, the method further comprises after performing the size selection: detecting, by the one or more processors, a mutation in one or more genes in the second plurality of nucleic acid molecules. In some embodiments, the method further comprises analyzing a mutation in one or more genes detected in the size- selected plurality of nucleic acid molecules; and analyzing a mutation in one or more genes detected in the second plurality of nucleic acid molecules. In some embodiments, the method further comprises based on the analyzing: identifying a mutation in one or more genes as being from cfDNA when it is represented in the second plurality of nucleic acid molecules but not the size-selected plurality of nucleic acid molecules. In some embodiments, the method further comprises detecting, by one or more processors, a mutation in one or more genes in a plurality of cfDNA molecules. In some embodiments, the method further comprises analyzing a mutation in one or more genes detected in the size-selected plurality of nucleic acid molecules; and analyzing a mutation in one or more genes detected in the plurality of cfDNA molecules. In some embodiments, the method further comprises based on the analyzing: identifying a mutation in one or more genes as a CH or germline mutation when it is represented in the size-selected plurality of nucleic acid molecules.

[0011] In some aspects, provided herein is a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising detecting a mutation in one or more genes by: performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of themixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size-selected plurality of nucleic acid molecules; optionally, ligating one or more adapters onto nucleic acid molecules in the size- selected plurality of nucleic acid molecules, thereby generating ligated nucleic acid molecules; optionally, amplifying the ligated nucleic acid molecules; optionally, capturing, from the amplified nucleic acid molecules, nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes; sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the one or more genes; analyzing, by one or more processors, the plurality of sequence reads; and based on the analysis, detecting a mutation in the one or more genes. In some embodiments, the method further comprises receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes. In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules. In some embodiments, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the sequencer comprises a next generation sequencer. In some embodiments, the method further comprises, e.g., after performing the size selection and prior to adapter ligation: subjecting the size-selected plurality of nucleic acid molecules to fragmentation.

[0012] In some aspects, provided herein is a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising: performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size-selected plurality of nucleic acid molecules; preparing a nucleic acid sequencing library from the size- selected plurality of nucleic acid molecules; amplifying said library; selectively enriching for a plurality of nucleic acid molecules from the amplified library, said plurality of nucleic acid molecules comprising nucleotide sequence(s) corresponding to the one or more genes, thereby producing an enriched plurality of nucleic acid molecules; sequencing, by a sequencer, the enrichedplurality of nucleic acid molecules, thereby producing a plurality of sequence reads; analyzing, by one or more processors, the plurality of sequence reads for presence or absence of a mutation in the one or more genes; and detecting, based on the analysis, presence or absence of a mutation in the one or more genes. In some embodiments, the selective enrichment comprises: combining one or more bait molecules with the amplified library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules. In some embodiments, the methods further comprise receiving, at the one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes. In some embodiments, amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, the sequencer implements a massively parallel sequencing (MPS) technique, a whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencer implements a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS).

[0013] In some embodiments according to any of the embodiments described herein, the size selection separates substantially all nucleic acid molecules of the second plurality of nucleic acid molecules from the first plurality of nucleic acid molecules. In some embodiments, the size selection is performed using one or more of: gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), DNA chromatography, DNA precipitation followed by centrifugation, spin column-based DNA purification, ion exchange chromatography, or solid-phase reversible immobilization (SPRI). In some embodiments, the gel electrophoresis comprises use of agarose or polyacrylamide gel electrophoresis (PAGE). In some embodiments, the HPLC is ion-pair reversed-phase HPLC (IP RP HPLC). In some embodiments, the size selection comprises contacting the mixture of nucleic acid molecules with the SPRI beads.

[0014] In some aspects, provided herein is a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising: performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a firstplurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), wherein the size selection comprises contacting the mixture of nucleic acid molecules with solid-phase reversible immobilization (SPRI) beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0, thereby generating a size-selected plurality of nucleic acid molecules; and detecting a mutation in one or more genes in the size-selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.

[0015] In some embodiments according to any of the embodiments described herein, the SPRI beads comprise paramagnetic SPRI beads. In some embodiments, the size selection comprises contacting the mixture of nucleic acid molecules with the SPRI beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0, between about 0.2 and about 0.8, between about 0.2 and about 0.6, between about 0.4 and about 1.0, between about 0.4 and about 0.8, between about 0.4 and about 0.6, or between about 0.45 and about 0.55. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is any of about 0.2, about 0.3, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.0. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is about 0.5. In some embodiments, the size selection comprises contacting a volume of about 55pL of the mixture of nucleic acid molecules with a volume of about 27.5 pL of SPRI beads. In some embodiments, the method further comprises after contacting the mixture of nucleic acid molecules with the SPRI beads: washing the mixture of nucleic acid molecules under conditions in which the mixture of nucleic acid molecules is bound to the SPRI beads; and eluting the size- selected plurality of nucleic acid molecules from the SPRI beads.

[0016] In some embodiments according to any of the embodiments described herein, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more, about 5000 base pairs or more, about 10,000 base pairs or more, about 15,000 base pairs or more, about 20,000 base pairs or more, about 25,000 base pairs or more, about 30,000 base pairs or more, about 35,000 base pairs or more, about 40,000 base pairs or more, about 45,000 base pairs or more, about 50,000 base pairs or more, about 55,000 base pairs or more, about 60,000 base pairs or more, about 65,000 base pairs or more, about 70,000 base pairs or more, about 75,000 base pairs or more, about 80,000 base pairs or more, about 85,000 base pairs or more, about 90,000 base pairs or more, about 95,000 base pairs or more, or about 100,000 base pairs or more. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragmentsthereof with a length of up to about 200,000 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 1000 base pairs and about 200,000 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 50,000 base pairs or more. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs. In some embodiments, the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than about 1000 base pairs. In some embodiments, the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 999 base pairs or less, about 900 base pairs or less, about 800 base pairs or less, about 700 base pairs or less, about 600 base pairs or less, about 500 base pairs or less, about 400 base pairs or less, about 300 base pairs or less, about 200 base pairs or less, or about 100 base pairs or less. In some embodiments, the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 50 base pairs or more, about 75 base pairs or more, about 100 base pairs or more, about 125 base pairs or more, about 150 base pairs or more, about 175 base pairs or more, about 200 base pairs or more, about 225 base pairs or more, about 250 base pairs or more, about 275 base pairs or more, about 300 base pairs or more, about 325 base pairs or more, about 350 base pairs or more, about 375 base pairs or more, about 400 base pairs or more, about 425 base pairs or more, about 450 base pairs or more, about 475 base pairs or more, or about 500 base pairs or more. In some embodiments, the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and about 1000 base pairs. In some embodiments, the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 100 base pairs and about 300 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between 1000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than 1000 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than 1000 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between 1000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and 1000 base pairs. In some embodiments, the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and about 1000 base pairs.

[0017] In some embodiments according to any of the embodiments described herein, a mutation in the one or more genes is detected by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence- specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing. In some embodiments, sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique, optionally, wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS). In some embodiments, the methods further comprise selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size-selected plurality of nucleic acid molecules, thereby producing an enriched plurality of nucleic acid molecules. In some embodiments, the selective enrichment comprises: combining one or more bait molecules with the size- selected plurality of nucleic acid molecules, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules. In some embodiments, the selective enrichment comprises amplifying one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size- selected plurality of nucleic acid molecules, thereby producing the enriched plurality of nucleic acid molecules. In someembodiments, the methods further comprise sequencing the enriched plurality of nucleic acid molecules to detect the mutation in the one or more genes. In some embodiments, the method further comprises optionally, ligating one or more adapters onto nucleic acid molecules in the size- selected plurality of nucleic acid molecules, thereby generating ligated nucleic acids; optionally, amplifying nucleic acids from the ligated nucleic acids; optionally, capturing or enriching from the amplified nucleic acids a plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes; sequencing, by a sequencer, the plurality of nucleic acids to obtain a plurality of sequence reads corresponding to the one or more genes; analyzing the plurality of sequence reads; and based on the analysis, detecting the mutation in the one or more genes. In some embodiments, the plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes is captured from the amplified nucleic acids by hybridization with one or more bait molecules. In some embodiments, the capturing comprises: combining the one or more bait molecules with the amplified nucleic acids, thereby hybridizing the one or more bait molecules to nucleic acids comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids. In some embodiments, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the method further comprises, e.g., prior to adapter ligation, subjecting the size-selected plurality of nucleic acid molecules to fragmentation. In some embodiments, the sequencer comprises a next generation sequencer. In some embodiments, amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique. In some embodiments, sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the methods further comprise receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes. In some embodiments, the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more genes. Insome embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent. In some embodiments, the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker. In some embodiments, the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule. In some embodiments, the size-selected plurality of nucleic acid molecules comprises a ratio of nucleic acid molecules from nucleated cells to nucleic acid molecules from cfDNA of at least about 20:1.

[0018] In some embodiments according to any of the embodiments described herein, the size selection recovers at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of nucleic acid molecules from the first plurality of nucleic acid molecules. In some embodiments, the size selection recovers at least 50%, at least 60%, or at least 62% of nucleic acid molecules from the first plurality of nucleic acid molecules. In some embodiments, the size selection recovers at least about 60% of nucleic acid molecules from the first plurality of nucleic acid molecules. In some embodiments, the size selection recovers less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% of nucleic acid molecules from the second plurality of nucleic acid molecules. In some embodiments, the size selection recovers less than about 3% of nucleic acid molecules from the second plurality of nucleic acid molecules. In some embodiments, the first plurality of nucleic acid molecules comprises genomic DNA from the nucleated cells. In some embodiments, the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells. In some embodiments, the second plurality of nucleic acid molecules comprises circulating tumor DNA (ctDNA).

[0019] In some embodiments according to any of the embodiments described herein, the mixture of nucleic acid molecules is from a liquid biopsy sample obtained from an individual. In some embodiments, the method further comprises obtaining the liquid biopsy sample from the individual. In some embodiments, the liquid biopsy is from whole blood, plasma, serum, urine, saliva, amniotic fluid, breast milk, pleural effusions or ascites, synovial fluid, orcerebrospinal fluid. In some embodiments, the liquid biopsy is from or comprises whole blood. In some embodiments, the mixture of nucleic acid molecules is obtained from a buffy coat sample derived from the liquid biopsy. In some embodiments, the mixture of nucleic acid molecules is obtained by extracting buffy coat DNA (bcDNA) from the buffy coat sample. In some embodiments, the method further comprises extracting buffy coat DNA (bcDNA) from the buffy coat sample in order to generate the mixture of nucleic acid molecules. In some embodiments, the buffy coat sample is derived by centrifugation of a whole blood liquid biopsy from the individual. In some embodiments, the method further comprises centrifuging a whole blood liquid biopsy from the individual to generate the buffy coat sample. In some embodiments, the buffy coat sample is derived by microfluidic fractionation of a whole blood liquid biopsy from the individual. In some embodiments, the method further comprises subjecting a whole blood sample (e.g., a liquid biopsy sample) from the individual to microfluidic fractionation in order to generate the buffy coat sample. In some embodiments, the buffy coat sample comprises one or more nucleated cells, optionally wherein the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells. In some embodiments, the size selection is performed on bcDNA.

[0020] In some embodiments according to any of the embodiments described herein, the method further comprises generating a molecular profile for the individual based at least in part on detecting the mutation in the one or more genes. In some embodiments, the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test. In some embodiments, the method further comprises selecting a treatment, administering a treatment, or applying a treatment to the individual based at least in part on the generated molecular profile. In some embodiments, the method further comprises generating a report indicating the presence or absence of the mutation in the one or more genes in the sample. In some embodiments, the method further comprises transmitting the report to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third- party payer, an insurance company, or a government office. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection. In some embodiments, detection of the mutation in the one or more genes is used to diagnose orconfirm a diagnosis of disease in an individual. In some embodiments, the mutation in the one or more genes is determined to be caused by clonal hematopoiesis of indeterminate potential (CHIP). In some embodiments, the disease is a cardiovascular or hematological disease. In some embodiments, the individual has, is suspected of having, or is being tested for CHIP. In some embodiments, the disease is a cancer. In some embodiments, the individual has, is suspected of having, or is being tested for cancer. In some embodiments, the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a B cell cancer (multiple myeloma), a melanoma, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of an oral cavity, cancer of a pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, a cancer of hematological tissue, an adenocarcinoma, an inflammatory myofibroblastic tumor, a gastrointestinal stromal tumor (GIST), colon cancer, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia Vera, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), soft-tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, head and neck cancer, small cell cancer, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia, chronic eosinophilic leukemia, neuroendocrine cancers, or a carcinoidtumor. In some embodiments, the cancer comprises acute lymphoblastic leukemia (Philadelphia chromosome positive), acute lymphoblastic leukemia (precursor B-cell), acute myeloid leukemia (FLT3+), acute myeloid leukemia (with an IDH2 mutation), anaplastic large cell lymphoma, basal cell carcinoma, B-cell chronic lymphocytic leukemia, bladder cancer, breast cancer (HER2 overexpressed / amplified), breast cancer (HER2+), breast cancer (HR+, HER2-), cervical cancer, cholangiocarcinoma, chronic lymphocytic leukemia, chronic lymphocytic leukemia (with 17p deletion), chronic myelogenous leukemia, chronic myelogenous leukemia (Philadelphia chromosome positive), classical Hodgkin lymphoma, colorectal cancer, colorectal cancer (dMMR / MSI-H), colorectal cancer (KRAS wild type), cryopyrin-associated periodic syndrome, a cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, a diffuse large B-cell lymphoma, fallopian tube cancer, a follicular B-cell nonHodgkin lymphoma, a follicular lymphoma, gastric cancer, gastric cancer (HER2+), gastroesophageal junction (GEJ) adenocarcinoma, a gastrointestinal stromal tumor, a gastrointestinal stromal tumor (KIT+), a giant cell tumor of the bone, a glioblastoma, granulomatosis with polyangiitis, a head and neck squamous cell carcinoma, a hepatocellular carcinoma, Hodgkin lymphoma, juvenile idiopathic arthritis, lupus erythematosus, a mantle cell lymphoma, medullary thyroid cancer, melanoma, a melanoma with a BRAF V600 mutation, a melanoma with a BRAF V600E or V600K mutation, Merkel cell carcinoma, multicentric Castleman's disease, multiple hematologic malignancies including Philadelphia chromosome-positive ALL and CML, multiple myeloma, myelofibrosis, a non-Hodgkin’s lymphoma, a nonresectable subependymal giant cell astrocytoma associated with tuberous sclerosis, a non-small cell lung cancer, a non-small cell lung cancer (ALK+), a non-small cell lung cancer (PD-L1+), a non-small cell lung cancer (with ALK fusion or ROS1 gene alteration), a non-small cell lung cancer (with BRAF V600E mutation), a non-small cell lung cancer (with an EGFR exon 19 deletion or exon 21 substitution (L858R) mutations), a non- small cell lung cancer (with an EGFR T790M mutation), ovarian cancer, ovarian cancer (with a BRCA mutation), pancreatic cancer, a pancreatic, gastrointestinal, or lung origin neuroendocrine tumor, a pediatric neuroblastoma, a peripheral T-cell lymphoma, peritoneal cancer, prostate cancer, a renal cell carcinoma, rheumatoid arthritis, a small lymphocytic lymphoma, a soft tissue sarcoma, a solid tumor (MSI-H / dMMR), a squamous cell cancer of the head and neck, a squamous non-small cell lung cancer, thyroid cancer, a thyroid carcinoma, urothelial cancer, a urothelial carcinoma, or Waldenstrom's macroglobulinemia. In some embodiments, the method further comprises recommending an anti-cancer therapy for the individual based at least in part on detecting the mutation in the one or more genes. Insome embodiments, the method further comprises selecting an anti-cancer therapy for the individual based, at least in part, on detecting the mutation in the one or more genes. In some embodiments, the method further comprises generating a report comprising one or more anticancer therapies identified for the individual based, at least in part, on detecting the mutation in the one or more genes. In some embodiments, the method further comprises administering an anti-cancer therapy to the individual based, at least in part, on detecting the mutation in the one or more genes. In some embodiments, the anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)- based therapy. In some embodiments, the nucleic acid comprises a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In some embodiments, the anti-cancer therapy comprises chemotherapy, radiation therapy, immunotherapy, a targeted therapy, or surgery. In some embodiments, the anti-cancer therapy comprises a targeted anti-cancer therapy. In some embodiments, the anti-cancer therapy comprises abemaciclib (Verzenio), abiraterone acetate (Zytiga), acalabrutinib (Calquence), ado-trastuzumab emtansine (Kadcyla), afatinib dimaleate (Gilotrif), aldesleukin (Proleukin), alectinib (Alecensa), alemtuzumab (Campath), alitretinoin (Panretin), alpelisib (Piqray), amivantamab-vmjw (Rybrevant), anastrozole (Arimidex), apalutamide (Erleada), asciminib hydrochloride (Scemblix), atezolizumab (Tecentriq), avapritinib (Ayvakit), avelumab (Bavencio), axicabtagene ciloleucel (Yescarta), axitinib (Inlyta), belantamab mafodotin-blmf (Blenrep), belimumab (Benlysta), belinostat (Beleodaq), belzutifan (Welireg), bevacizumab (Avastin), bexarotene (Targretin), binimetinib (Mektovi), blinatumomab (Blincyto), bortezomib (Velcade), bosutinib (Bosulif), brentuximab vedotin (Adcetris), brexucabtagene autoleucel (Tecartus), brigatinib (Alunbrig), cabazitaxel (Jevtana), cabozantinib (Cabometyx), cabozantinib (Cabometyx, Cometriq), canakinumab (Haris), capmatinib hydrochloride (Tabrecta), carfilzomib (Kyprolis), cemiplimab-rwlc (Libtayo), ceritinib(LDK378 / Zykadia), cetuximab (Erbitux), cobimetinib (Cotellic), copanlisib hydrochloride (Aliqopa), crizotinib (Xalkori), dabrafenib (Tafinlar), dacomitinib (Vizimpro), daratumumab (Darzalex), daratumumab and hyaluronidase-fihj (Darzalex Faspro), darolutamide (Nubeqa), dasatinib (Sprycel), denileukin diftitox (Ontak), denosumab (Xgeva), dinutuximab (Unituxin), dostarlimab-gxly (Jemperli), durvalumab (Imfinzi), duvelisib (Copiktra), elotuzumab (Empliciti), enasidenib mesylate (Idhifa), encorafenib (Braftovi), enfortumab vedotin-ejfv (Padcev), entrectinib (Rozlytrek), enzalutamide (Xtandi), erdafitinib (Balversa), erlotinib (Tarceva), everolimus (Afinitor), exemestane (Aromasin), fam-trastuzumab deruxtecan-nxki (Enhertu), fedratinib hydrochloride (Inrebic), fulvestrant (Faslodex), gefitinib (Iressa), gemtuzumab ozogamicin (Mylotarg), gilteritinib (Xospata), glasdegib maleate (Daurismo), hyaluronidase-zzxf (Phesgo), ibrutinib (Imbruvica), ibritumomab tiuxetan (Zevalin), idecabtagene vicleucel (Abecma), idelalisib (Zydelig), imatinib mesylate (Gleevec), infigratinib phosphate (Truseltiq), inotuzumab ozogamicin (Besponsa), iobenguane 1131 (Azedra), ipilimumab (Yervoy), isatuximab-irfc (Sarclisa), ivosidenib (Tibsovo), ixazomib citrate (Ninlaro), lanreotide acetate (Somatuline Depot), lapatinib (Tykerb), larotrectinib sulfate (Vitrakvi), lenvatinib mesylate (Lenvima), letrozole (Femara), lisocabtagene maraleucel (Breyanzi), loncastuximab tesirine-lpyl (Zynlonta), lorlatinib (Lorbrena), lutetium Lu 177-dotatate (Lutathera), margetuximab-cmkb (Margenza), midostaurin (Rydapt), mobocertinib succinate (Exkivity), mogamulizumab-kpkc (Poteligeo), moxetumomab pasudotox-tdfk (Lumoxiti), naxitamab-gqgk (Danyelza), necitumumab (Portrazza), neratinib maleate (Nerlynx), nilotinib (Tasigna), niraparib tosylate monohydrate (Zejula), nivolumab (Opdivo), obinutuzumab (Gazyva), ofatumumab (Arzerra), olaparib (Lynparza), olaratumab (Lartruvo), osimertinib (Tagrisso), palbociclib (Ibrance), panitumumab (Vectibix), panobinostat (Farydak), pazopanib (Votrient), pembrolizumab (Keytruda), pemigatinib (Pemazyre), pertuzumab (Perjeta), pexidartinib hydrochloride (Turalio), polatuzumab vedotin-piiq (Polivy), ponatinib hydrochloride (Iclusig), pralatrexate (Folotyn), pralsetinib (Gavreto), radium 223 dichloride (Xofigo), ramucirumab (Cyramza), regorafenib (Stivarga), ribociclib (Kisqali), ripretinib (Qinlock), rituximab (Rituxan), rituximab and hyaluronidase human (Rituxan Hycela), romidepsin (Istodax), rucaparib camsylate (Rubraca), ruxolitinib phosphate (Jakafi), sacituzumab govitecan-hziy (Trodelvy), seliciclib, selinexor (Xpovio), selpercatinib (Retevmo), selumetinib sulfate (Koselugo), siltuximab (Sylvant), sipuleucel-T (Provenge), sirolimus protein-bound particles (Fyarro), sonidegib (Odomzo), sorafenib (Nexavar), sotorasib (Lumakras), sunitinib (Sutent), tafasitamab-cxix (Monjuvi), tagraxofusp-erzs (Elzonris), talazoparib tosylate (Talzenna),tamoxifen (Nolvadex), tazemetostat hydrobromide (Tazverik), tebentafusp-tebn (Kimmtrak), temsirolimus (Torisel), tepotinib hydrochloride (Tepmetko), tisagenlecleucel (Kymriah), tisotumab vedotin-tftv (Tivdak), tocilizumab (Actemra), tofacitinib (Xeljanz), tositumomab (Bexxar), trametinib (Mekinist), trastuzumab (Herceptin), tretinoin (Vesanoid), tivozanib hydrochloride (Fotivda), toremifene (Fareston), tucatinib (Tukysa), umbralisib tosylate (Ukoniq), vandetanib (Caprelsa), vemurafenib (Zelboraf), venetoclax (Venclexta), vismodegib (Erivedge), vorinostat (Zolinza), zanubrutinib (Brukinsa), ziv-aflibercept (Zaltrap), or any combination thereof. In some embodiments, the method further comprises determining an effective amount of the anti-cancer therapy to administer to the individual based, at least in part, on detecting the mutation in the one or more genes. In some embodiments, the mutation in the one or more genes comprises one or more of: a point mutation; an insertion-deletion (indel); an in-frame deletion of one or more codons; an intragenic deletion; an intragenic insertion; a deletion of a full gene; an inversion; an interchromosomal or intrachromosomal translocation; a tandem duplication; a gene fusion; a genomic rearrangement that comprises an intron sequence; and / or a gene amplification or duplication. In some embodiments, the one or more genes comprise ABL1, ACVR1B, AKT1, AKT2, AKT3, ALK, AL0X12B, AMER1, APC, AR, ARAF, ARFRP1, ARID1A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BCOR, BCORL1, BCR, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BTG1, BTG2, BTK, CALR, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD22, CD274, CD70, CD74, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC, CREBBP, CRKL, CSF1R, CSF3R, CTCF, CTNNA1, CTNNB1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DNMT3A, DOT1L, EED, EGFR, EMSY (Cllorf30), EP300, EPHA3, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC4, ERG, ERRFI1, ESRI, ETV4, ETV5, ETV6, EWSR1, EZH2, EZR, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GATA3, GATA4, GATA6, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2D (MLL2), KRAS, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED12, MEF2B,MEN1, MERTK, MET, MITF, MKNK1, MLH1, MPL, MRE11A, MSH2, MSH3, MSH6, MST1R, MTAP, MTOR, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NT5C2, NTRK1, NTRK2, NTRK3, NUTM1, P2RY8, PALB2, PARK2, PARP1, PARP2, PARP3, PAX5, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CA, PIK3CB, PIK3R1, PIM1, PMS2, POLDI, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PRKAR1A, PRKCI, PTCHI, PTEN, PTPN11, PTPRO, QKI, RAC1, RAD21, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAFI, RARA, RBI, RBM10, REL, RET, RICTOR, RNF43, ROS1, RPTOR, RSPO2, SDC4, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SGK1, SLC34A2, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STK11, SUFU, SYK, TBX3, TEK, TERC, TERT, TET2, TGFBR2, TIPARP, TMPRSS2, TNFAIP3, TNFRSF14, TP53, TSC1, TSC2, TYRO3, U2AF1, VEGFA, VHL, WHSCI, WHSC1L1, WT1, XPO1, XRCC2, ZNF217, ZNF703, or any combination thereof. In some embodiments, the one or more genes comprise ABL, ALK, ALL, B4GALNT1, BAFF, BCL2, BRAF, BRCA, BTK, CD19, CD20, CD3, CD30, CD319, CD38, CD52, CDK4, CDK6, CML, CRACC, CS1, CTLA-4, dMMR, EGFR, ERBB1, ERBB2, FGFR1-3, FLT3, GD2, HD AC, HER1, HR, IDH2, IL-ip, IL-6, IL-6R, JAK1, JAK2, JAK3, KIT, KRAS, MEK, MET, MSI-H, mTOR, PARP, PD-1, PDGFR, PDGFRa, PDGFRp, PD-L1, PI3K5, PIGF, PTCH, RAF, RANKL, RET, ROS1, SLAMF7, VEGF, VEGFA, VEGFB, or any combination thereof.

[0021] In some aspects, provided herein is a system for detecting one or more clonal hematopoietic (CH) or germline gene mutations (e.g., in a sample), comprising: a memory configured to store one or more program instructions, and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to perform the method according to any one of the above embodiments. In some embodiments, the one or more program instructions when executed by the one or more processors are configured to obtain a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA); analyze the plurality of sequence reads for the presence of a mutation in one or more genes; and detect,based on the analyzing, the mutation in the one or more genes in the sample, thereby detecting a clonal hematopoietic (CH) or germline gene mutation. In some embodiments, the one or more program instructions when executed by the one or more processors are further configured to generate, based at least in part on the detecting, a molecular profile for a sample from which the mixture of nucleic acid molecules was obtained.

[0022] In some aspects, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method according to any one of the above embodiments for detecting one or more clonal hematopoietic (CH) or germline gene mutations. In some embodiments, the method comprises obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA); analyzing, using the one or more processors, the plurality of sequence reads for the presence of a mutation in one or more genes; and detecting, using the one or more processors and based on the analyzing, the mutation in the one or more genes, thereby detecting a clonal hematopoietic (CH) or germline gene mutation. In some embodiments, the method further comprises generating, based at least in part on the detecting, a molecular profile for a sample from which the mixture of nucleic acid molecules was obtained.

[0023] In some embodiments according to any of the embodiments described herein, the plurality of sequence reads is obtained by sequencing; optionally wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and further optionally wherein the massively parallel sequencing technique comprises next generation sequencing (NGS). In some embodiments, the sample is obtained from an individual, and wherein the individual is administered a treatment based at least in part on the molecular profile. In some embodiments, the molecular profile further comprises (a) results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof; and / or (b) results from a nucleic acid sequencing-based test. In some embodiments, the sample is a liquid biopsy sample.

[0024] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Various aspects of the disclosed methods, devices, and systems are set forth with particularity in the appended claims. A better understanding of the features and advantages of the disclosed methods, devices, and systems will be obtained by reference to the following detailed description of illustrative embodiments and the accompanying drawings, of which:

[0026] FIG. 1 shows the recovery of cell free DNA (cfDNA) and buffy coat DNA (bcDNA) fragments after elution from solid-phase reversible immobilization (SPRI) beads using the indicated SPRI bead ratios, as described in Example 1 herein. The DNA fragments were analyzed using a fragment analyzer. “Original sample” refers to a non-purified DNA sample with 50% cfDNA (50 - 1000 base pairs [bp]) and 50% bcDNA (1000 bp - 150 kilobase pairs [kb]). Each peak in FIG. 1 represents DNA fragments of the specific molecular weight (z.e., size in bp) as indicated on the x-axis, and the peak height indicates the relative abundance of DNA molecules.

[0027] FIG. 2 shows the concentration of bcDNA and cfDNA (ng / pL), as well as the percent recovery of bcDNA and cfDNA after elution from SPRI beads using the indicated bead ratios (“CleanUp Ratio”) according the automated SPRI clean-up workflow process described in Example 1 herein. The results in FIG. 2 were obtained using a Fragment Analyzer (FA). The “50%cfDNA / 50%bcDNA” sample refers to a non-purified DNA sample with 50% cfDNA (50 - 1000 bp) and 50% bcDNA (1000 bp - 150 kb).

[0028] FIG. 3 shows a graphical representation of bcDNA and cfDNA recovery (ng / pE; y- axis) after elution from AMPure SPRI beads using the SPRI bead ratios indicated on the x- axis. Results from up to four replicates are shown. The results were obtained using a Fragment Analyzer (FA). “Stock” refers to a non-purified DNA sample with 50% cfDNA (50 - 1000 bp) and 50% bcDNA (1000 bp - 150 kb).

[0029] FIG. 4 shows an exemplary process for the purification of high molecular weight (HMW) bcDNA and removal of low molecular weight (EMW) cfDNA using an SPRI bead ratio of 0.5x (i.e., 55 pF DNA sample and 27.5 pF beads). In step 1, buffy coat material isseparated from the rest of a blood sample, e.g., by centrifugation; in step 2, bcDNA from the buffy coat material is extracted; in step 3, bcDNA is purified to remove cfDNA by mixing 55 pL bcDNA (step 3a) with 27.5 pL AMPure SPRI beads (step 3b), followed by binding of DNA to the beads, washing, eluting and recovering the purified bcDNA (steps 3c-3d); in step 4, the eluted and recovered bcDNA is quantified (e.g., using a fragment analyzer); in step 5, bcDNA is fragmented for downstream processes such as sequencing library preparation, amplification, and / or sequencing.

[0030] FIG. 5 depicts an exemplary device, in accordance with some embodiments.

[0031] FIG. 6 depicts an exemplary system, in accordance with some embodiments.

[0032] FIGS. 7A & 7B depict a block diagram of an exemplary process for detecting one or more CH or germline mutations, in accordance with some embodiments.DETAILED DESCRIPTION

[0033] Described herein, inter alia, are methods for detecting one or more clonal hematopoietic (CH) or germline gene mutations from a mixture of nucleic acid molecules, e.g., comprising some nucleic acid molecules from nucleated cells, and some nucleic acid molecules from cfDNA. In some embodiments, the mixture of nucleic acid molecules can be from a sample, for example a liquid biopsy sample, e.g., a whole blood sample or a buffy coat sample isolated from whole blood. In some embodiments, the methods use nucleic acid size selection to remove the nucleic acid contaminants in order to isolate a predominantly or nearly pure gDNA sample from buffy coat to accurately detect and filter out one or more CH or germline gene mutations, e.g., for downstream detection of a disease, for example a cancer, or clonal hematopoiesis of indeterminate potential (CHIP).

[0034] Current methods of nucleic acid extraction from liquid biopsies, e.g., whole blood or buffy coat isolated from whole blood, have a high likelihood of containing cfDNA, e.g., ctDNA. These nucleic acid molecules may contain mutations that are not present in the germline and therefore can confound the sequencing results for CH and germline gene mutations. As a result, the interpretation of the sequencing analysis may include false positives, wherein subjects (e.g., human patients) are identified to present with one or more specific CH or germline gene mutation(s) inaccurately, or wherein the results are interpreted to be caused by contamination and the sample identified as unusable. The methods described herein provide for the reduction and / or elimination of these contaminating nucleic acids from the sample and to prevent the loss of subject information and / or the misidentification of CH and / or germline gene mutation(s) in a subject.General Techniques

[0035] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J.B. Lippincott Company, 1993).Definitions

[0036] Unless otherwise defined, all of the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field to which this disclosure belongs.

[0037] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0038] ‘ ‘About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0039] As used herein, the terms "comprising" (and any form or variant of comprising, such as "comprise" and "comprises"), "having" (and any form or variant of having, such as "have" and "has"), "including" (and any form or variant of including, such as "includes" and "include"), or "containing" (and any form or variant of containing, such as "contains" and "contain"), are inclusive or open-ended and do not exclude additional, un-recited additives, components, integers, elements, or method steps.

[0040] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0041] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers.

[0042] The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive as referred to herein.

[0043] “Polynucleotide,” “nucleic acid,” or “nucleic acid molecule” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double- stranded DNA, DNA including single- and double- stranded regions, single- and double- stranded RNA, and RNA including single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or include single- and double- stranded regions. In addition, the term “polynucleotide” as used herein refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide”specifically includes cDNAs. In some embodiments, nucleic acid molecules of the present disclosure include DNA (e.g., gDNA or cfDNA).

[0044] Nucleic acid molecules (and pluralities thereof) “from nucleated cells” can refer to nucleic acid molecules (e.g., DNA, such as gDNA) that exist in nucleated cells prior to obtaining a sample, purifying or otherwise manipulating (e.g., centrifuging or fractionating) the sample, and / or extracting nucleic acid molecules from the sample. In some embodiments, nucleic acid molecules from nucleated cells are present in a mixture of nucleic acid molecules comprising (a) nucleic acid molecules from nucleated cells and (b) nucleic acid molecules from cfDNA. In some embodiments, the mixture is present in buffy coat, e.g., a sample of bcDNA after initial DNA extraction but before a size selection of the present disclosure. In some embodiments, unless clearly indicated otherwise by context (e.g., if size selection is performed on the bcDNA), references herein to “bcDNA” refer to bcDNA resulting from a size selection of the present disclosure (e.g., a size-selected plurality of nucleic acid molecules of the present disclosure), e.g., bcDNA from nucleated cells that substantially lacks cfDNA.

[0045] Nucleic acid molecules (and pluralities thereof) “from cfDNA” can refer to nucleic acid molecules (e.g., DNA) that exists in cell-free form prior to obtaining a sample, purifying or otherwise manipulating (e.g., centrifuging or fractionating) the sample, and / or extracting nucleic acid molecules from the sample. In some embodiments, nucleic acid molecules from cfDNA may be specifically ctDNA (i.e., DNA shed specifically from a tumor). Although cfDNA ultimately originates from nucleated cells (e.g., cancer cells), cfDNA is not considered a nucleic acid molecule from nucleated cells of the present disclosure because it is present in cell-free form prior to obtaining a sample, purifying or otherwise manipulating (e.g., centrifuging or fractionating) the sample, and / or extracting nucleic acid molecules (e.g., DNA, such as gDNA) from the sample. In some embodiments, nucleic acid molecules from cfDNA are present in a mixture of nucleic acid molecules comprising (a) nucleic acid molecules from nucleated cells and (b) nucleic acid molecules from cfDNA. In some embodiments, the mixture is present in buffy coat, e.g., a sample of bcDNA after initial DNA extraction but before a size selection of the present disclosure.

[0046] The term “detection” includes any means of detecting, including direct and indirect detection.

[0047] “Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. Forexample, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and / or sequence errors that occur during amplification.

[0048] The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and / or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid.

[0049] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject and / or individual of interest that contains a cellular and / or other molecular entity that is to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and / or molecular entity that is to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, cerebro- spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof. In some instances, the sample is a whole blood sample, a plasma sample, a buffy coat sample, a serum sample, or a combination thereof.

[0050] The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, for instance, by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).

[0051] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating” , and the like) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0052] As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, e.g., a mammal (including such non-human animals as, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non- human primates) for which treatment is desired. In particular embodiments, the individual, patient, or subject herein is a human.

[0053] As used herein, “administering” (and grammatical variations thereof such as “administration” or “administer”, and the like) refers to a method of giving a dosage of an agent or a pharmaceutical composition (e.g., a pharmaceutical composition including the agent) to a subject (e.g., a patient). Administering can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules, including, but not limited to, single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0054] When a range of values is provided, it is to be understood that each intervening value between the upper and lower limit of that range, and any other stated or intervening value in that states range, is encompassed within the scope of the present disclosure. Where the statedrange includes upper or lower limits, ranges excluding either of those included limits are also included in the present disclosure.

[0055] The section headings used herein are for organization purposes only and are not to be construed as limiting the subject matter described. The description is presented to enable one of ordinary skill in the art to make and use the invention and is provided in the context of a patent application and its requirements. Various modifications to the described embodiments will be readily apparent to those persons skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiment shown but is to be accorded the widest scope consistent with the principles and features described herein.Methods

[0056] In some aspects, the present disclosure provides methods for detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample.

[0057] In some embodiments, the methods comprise detecting a mutation in one or more genes from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), thereby determining that the detected mutation is a CH or germline mutation. In some embodiments, the methods further comprise, prior to the detection, performing a size selection of the present disclosure on the mixture of nucleic acid molecules, wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture.

[0058] In some embodiments, the methods comprise performing a size selection of the present disclosure on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules; and detecting (e.g., by one or more processors) a mutation in one or more genes in the size-selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.

[0059] In some embodiments, the methods further comprise (e.g., after size selection) detecting (e.g., by one or more processors) a mutation in one or more genes in the second plurality of nucleic acid molecules. In some embodiments, the methods further comprise (e.g., after detecting a mutation) analyzing a mutation in one or more genes detected in a size- selected plurality of nucleic acid molecules of the present disclosure; and analyzing a mutation in one or more genes detected in cfDNA. In some embodiments, the methods further comprise (e.g., based on the analysis) identifying a mutation in one or more genes as being a CH or germline mutation, e.g., when it is represented in the size-selected plurality of nucleic acid molecules.

[0060] In some embodiments, a mutation is detected in cfDNA from the mixture from which the size-selected plurality of nucleic acid molecules is isolated. In some embodiments, the methods further comprise (e.g., based on the analysis) identifying a mutation in one or more genes as being from cfDNA, e.g., when it is represented in the second plurality of nucleic acid molecules but not the size- selected plurality of nucleic acid molecules. In some embodiments, the mutation is detected in cfDNA from the same sample from which the mixture is obtained. For example, the cfDNA can be from plasma, whereas the mixture can be obtained from buffy coat. In some embodiments, the mutation is detected in cfDNA from a different sample from which the mixture is obtained.

[0061] In some embodiments, the methods comprise performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell- free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size-selected plurality of nucleic acid molecules; optionally, ligating one or more adapters onto nucleic acid molecules in the size-selected plurality of nucleic acid molecules, thereby generating ligated nucleic acid molecules; optionally, amplifying the ligated nucleic acid molecules; optionally, capturing, from the amplified nucleic acid molecules, nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes; sequencing (e.g., by a sequencer) the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the one or more genes; analyzing, by one or more processors, the plurality of sequence reads; and based on the analysis, detecting a mutation in the one or more genes, thereby determining that the detected mutation is a CH or germline mutation. 1

[0062] In some embodiments, the methods further comprise receiving (e.g., at one or more processors) sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying (e.g., using one or more processors) the presence or absence of sequence reads comprising a mutation in the one or more genes. In some embodiments, amplified nucleic acid molecules are captured by hybridization with one or more bait molecules. In some embodiments, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences. In some embodiments, the sequencer comprises a next generation sequencer.

[0063] In some embodiments, the methods further comprise (e.g., after performing size selection and / or prior to adapter ligation) subjecting a size-selected plurality of nucleic acid molecules of the present disclosure to fragmentation. Methods for nucleic acid (e.g., DNA) fragmentation (e.g., as part of or prior to library preparation) are known in the art and include without limitation physical shearing (e.g., sonication, nebulization, focused acoustic shearing, etc.) and enzymatic shearing (e.g., via endonuclease or a cocktail of endonucleases). In some embodiments, the methods further comprise (e.g., after fragmentation) end repair, A-tailing, and / or adapter ligation.

[0064] In some instances, a size- selected plurality of nucleic acid molecules of the present disclosure can be used to construct a library (e.g., a nucleic acid sequencing library as described herein). In some instances, the nucleic acids are fragmented using any of the methods described herein, optionally subjected to repair of chain end damage, and optionally attached, e.g., ligated, to synthetic adapters, primers, and / or barcodes (e.g., amplification primers, sequencing adapters, flow cell adapters, substrate adapters, and / or sample barcodes or indices), size-selected (e.g., by preparative gel electrophoresis), and / or amplified (e.g., using PCR, a non-PCR amplification technique, or an isothermal amplification technique). In some embodiments, a sample index is included in the sequencing adapter, making the incorporation of a sample index in a downstream amplification process unnecessary. In some instances, the sequencing adaptors can include a molecular barcode, for example a molecular barcode that can be used to create a unique molecular identifier (UMI). These UMIs can allow for (e.g.) pairing the first strand (top) and the second strand (bottom) of double stranded nucleic acid duplexes. UMIs may be comprised of i) unique exogenous molecular barcodes alone that are attached to nucleic acid molecules during, e.g., ligation, ii) endogenous information within each individual nucleic acid molecule (e.g., molecular coordinates in relation to a reference genome, sequence information representing a portion ofthe molecule, or topological information about the molecule (such as length, methylation status, other physical characteristics)), or iii) a combination of non-unique or unique molecular barcodes and endogenous molecular information. In some instances, the fragmented and adapter-ligated group of nucleic acids is used without explicit size selection or amplification prior to hybridization-based selection of target sequences. In some instances, the nucleic acid is amplified by any of a variety of specific or non-specific nucleic acid amplification methods known to those of skill in the art. In some instances, the nucleic acids are amplified, e.g., by a whole-genome amplification method such as random-primed stranddisplacement amplification. Examples of nucleic acid library preparation techniques for next-generation sequencing are described in, e.g., van Dijk, et al. (2014), Exp. Cell Research 322:12 - 20, and Illumina’s genomic DNA sample preparation kit.

[0065] In some instances, the resulting nucleic acid sequencing library may contain all or substantially all of the complexity of the genome. The term “substantially all” in this context refers to the possibility that there can in practice be some unwanted loss of genome complexity during the initial steps of the procedure. The methods described herein also are useful in cases where the nucleic acid library comprises a portion of the genome, e.g., where the complexity of the genome is reduced by design. In some instances, any selected portion of the genome can be used with a method described herein. For example, in certain embodiments, the entire exome or a subset thereof is isolated. In some embodiments, the library may include at least any of 95%, 90%, 80%, 70%, 60%, or 50% of the genomic DNA. In some embodiments, the library may consist of cDNA copies of genomic DNA that includes copies of at least any of 95%, 90%, 80%, 70%, 60%, or 50% of the genomic DNA. In certain instances, the amount of nucleic acid used to generate the nucleic acid library may be less than 5 micrograms, less than 1 microgram, less than 500 ng, less than 200 ng, less than 100 ng, less than 50 ng, less than 10 ng, less than 5 ng, or less than 1 ng. In some embodiments, the library comprises less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% of nucleic acid molecules from cell-free DNA.

[0066] In some instances, a library (e.g., a nucleic acid sequencing library) includes a collection of nucleic acid molecules. As described herein, the nucleic acid molecules of the library can include a target nucleic acid molecule (e.g., a tumor nucleic acid molecule, a reference nucleic acid molecule, and / or a control nucleic acid molecule; also referred to herein as a first, second, and / or third nucleic acid molecule, respectively). The nucleic acidmolecules of the library can be from a single subject or individual. In some instances, a library can comprise nucleic acid molecules derived from more than one subject (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 or more subjects). For example, two or more libraries from different subjects can be combined to form a library having nucleic acid molecules from more than one subject (where the nucleic acid molecules derived from each subject are optionally ligated to a unique sample barcode corresponding to a specific subject). In some instances, the subject is a human having, or at risk of having, CHIP, a cancer, or tumor.

[0067] In some instances, the library (or a portion thereof) may comprise one or more subgenomic intervals. In some instances, a subgenomic interval can be a single nucleotide position, e.g., a nucleotide position for which a variant at the position is associated (positively or negatively) with a CHIP, cancer, or tumor phenotype. In some instances, a subgenomic interval comprises more than one nucleotide position. Such instances include sequences of at least about any of 2, 5, 10, 50, 100, 150, 250, or more than 250 nucleotide positions in length. Subgenomic intervals can comprise, e.g., one or more entire genes (or portions thereof), one or more exons or coding sequences (or portions thereof), one or more introns (or portion thereof), one or more micro satellite region (or portions thereof), or any combination thereof. A subgenomic interval can comprise all or a part of a fragment of a naturally occurring nucleic acid molecule, e.g., a genomic DNA molecule. For example, a subgenomic interval can correspond to a fragment of genomic DNA which is subjected to a sequencing reaction. In some instances, a subgenomic interval is a continuous sequence from a genomic source. In some instances, a subgenomic interval includes sequences that are not contiguous in the genome, e.g., subgenomic intervals in cDNA can include exon-exon junctions formed as a result of splicing. In some instances, the subgenomic interval comprises a tumor nucleic acid molecule. In some instances, the subgenomic interval comprises a non-tumor nucleic acid molecule.

[0068] In some embodiments, the methods comprise performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell- free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size-selected plurality of nucleic acid molecules; preparing a nucleic acid sequencing library from the size- selected plurality of nucleic acid molecules; amplifying said library; selectively enriching for a plurality of nucleic acid molecules from the amplified library, said plurality of nucleic acid molecules comprising nucleotidesequence(s) corresponding to the one or more genes, thereby producing an enriched plurality of nucleic acid molecules; sequencing (e.g., by a sequencer) the enriched plurality of nucleic acid molecules, thereby producing a plurality of sequence reads; analyzing (e.g., by one or more processors) the plurality of sequence reads for presence or absence of a mutation in the one or more genes; and detecting (e.g., based on the analysis) the presence or absence of a mutation in the one or more genes, thereby determining that the detected mutation is a CH or germline mutation.

[0069] Various methods for selective enrichment are known in the art and described herein, such as specific amplification (e.g., by PCR) or hybrid capture. For example, in some embodiments, the selective enrichment comprises combining one or more bait molecules with the amplified library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules. In some embodiments, the methods further comprise receiving (e.g., at one or more processors) sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises identifying (e.g., using one or more processors) the presence or absence of sequence reads comprising a mutation in the one or more genes.

[0070] Various techniques for amplification of nucleic acids or a sequencing library thereof are known in the art and described herein. In some embodiments, the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique.

[0071] Various sequencers and techniques for sequencing nucleic acids or a sequencing library thereof are known in the art and described herein. In some embodiments, the sequencer implements a massively parallel sequencing (MPS) technique, a whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the sequencer implements a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS).

[0072] A flow chart illustrating an exemplary and non-limiting workflow for performing a method of the present disclosure in accordance with some embodiments is provided in FIG.4. In some embodiments, workflow 400 is used to detect one or more clonal hematopoietic (CH) or germline gene mutations. At 410, a sample of the present disclosure (e.g., a whole blood sample) is fractionated (e.g., using centrifugation or microfluidics as described herein)to generate a buffy coat fraction (containing a mixture of nucleic acid molecules of the present disclosure) and optionally a plasma fraction (containing cfDNA). At 420, a mixture of nucleic acid molecules of the present disclosure (e.g., containing a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cfDNA) is obtained by extracting the mixture of nucleic acid molecules (e.g., DNA) from the buffy coat fraction. At 430, a size selection of the present disclosure is performed on the mixture of nucleic acid molecules of the present disclosure to generate a size-selected plurality of nucleic acid molecules of the present disclosure (e.g., enriched for bcDNA and substantially lacking cfDNA). In embodiments where SPRI is used to perform the size selection, this can include, e.g., applying a volume of the extracted mixture of nucleic acid molecules to a well of a plate (see 432); contacting the mixture with SPRI beads (see 434); binding the nucleic acids of the mixture to the SPRI beads, washing the beads, and eluting nucleic acid molecules from the beads (see 436); and recovering a size-selected plurality of nucleic acid molecules of the present disclosure (e.g., bcDNA; see 438). At 440, the size- selected plurality of nucleic acid molecules of the present disclosure (e.g., bcDNA) is quantified. At 450, the size-selected plurality of nucleic acid molecules of the present disclosure (e.g., bcDNA) is subjected to fragmentation. Optionally, after 450, the methods can further comprise end repair, A-tailing, adapter ligation, library preparation, amplification, selective enrichment, and / or sequencing, e.g., as described herein.Size Selection

[0073] In some embodiments, the methods of the present disclosure comprise a size selection, e.g., a size exclusion, by which nucleic acid molecules from nucleated cells (e.g., a first plurality of the present disclosure) are selected from a mixture comprising nucleic acid molecules from nucleated cells (e.g., a first plurality of the present disclosure) and nucleic acid molecules from cfDNA (e.g., a second plurality of the present disclosure). In some embodiments, the size selection comprises separating the first plurality of nucleic acid molecules of the mixture (e.g., bcDNA) from the second plurality of nucleic acid molecules of the mixture (e.g., cfDNA or ctDNA), thereby generating a size- selected plurality of nucleic acid molecules. In some embodiments, the first plurality of nucleic acid molecules comprises genomic DNA from the nucleated cells (e.g., bcDNA) such as lymphocytes, monocytes, granulocytes, and / or nucleated blood cells. In some embodiments, the second plurality of nucleic acid molecules comprises circulating tumor DNA (ctDNA).

[0074] In some embodiments, the size selection separates all or substantially all nucleic acid molecules of the second plurality of nucleic acid molecules away from the first plurality of nucleic acid molecules. In some embodiments, the size selection separates all or substantially all cfDNA from bcDNA. For example, in some embodiments, the size selection produces a size- selected plurality of nucleic acid molecules comprising a ratio of nucleic acid molecules from nucleated cells (e.g., bcDNA) to nucleic acid molecules from cfDNA (e.g., cfDNA or ctDNA) of at least about 10:1, at least about 11:1, at least about 12:1, at least about 13:1, at least about 14:1, at least about 15:1, at least about 16:1, at least about 17:1, at least about 18:1, at least about 19:1, or at least about 20:1.

[0075] In some embodiments, the size selection recovers at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of nucleic acid molecules from the first plurality of nucleic acid molecules (e.g., bcDNA). In some embodiments, the size selection recovers at least 50%, at least 60%, or at least 62% of nucleic acid molecules from nucleated cells (e.g., bcDNA). In some embodiments, the size selection recovers at least about 60% of nucleic acid molecules from nucleated cells (e.g., bcDNA).

[0076] In some embodiments, the size selection recovers less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% of nucleic acid molecules from the second plurality of nucleic acid molecules (e.g., cfDNA or ctDNA). In some embodiments, the size selection recovers less than about 3% of nucleic acid molecules from the second plurality of nucleic acid molecules (e.g., cfDNA or ctDNA).

[0077] In some embodiments, the size selection is performed using one or more of: gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), DNA chromatography, DNA precipitation followed by centrifugation, spin column-based DNA purification, ion exchange chromatography, or solid-phase reversible immobilization (SPRI).

[0078] In some embodiments, the gel electrophoresis comprises use of agarose or polyacrylamide gel electrophoresis (PAGE).

[0079] In some embodiments, the HPLC is ion-pair reversed-phase HPLC (IP RP HPLC).

[0080] In some embodiments, the methods of the present disclosure comprise performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), wherein the size selection comprises contacting the mixture of nucleic acid molecules with solid-phase reversible immobilization (SPRI) beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0, thereby generating a size-selected plurality of nucleic acid molecules; and detecting a mutation in one or more genes in the size-selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation. In some embodiments, the SPRI comprises use of SPRI beads, for example paramagnetic SPRI beads. In some embodiments, the size selection comprises contacting a mixture of nucleic acid molecules of the present disclosure (e.g., comprising a first and a second plurality of nucleic acid molecules of the present disclosure) with SPRI beads. SPRI uses a solid phase such as beads, e.g., paramagnetic beads, to selectively bind nucleic acids by type and size, and the SPRI beads are used for high-performance nucleic acid isolation and / or purification. This technology allows for tunable fragment size selection. The paramagnetic beads are coated with carboxyl molecules, which bind to nucleic acid molecules due to the charge groups. Nucleic acids are immobilized on the paramagnetic beads, and a magnetic field is then used to pull the microparticles, or contaminants, out of solution to yield a nucleic acid solution with high purity. The ratio of SPRI beads to nucleic acid molecules may be relevant to the overall retention of nucleic acid molecules that are present in the sample contacted by the SPRI beads for isolation and / or purification. This technology can be utilized in a manual and / or an automated fashion.

[0081] In some embodiments, the size selection comprises contacting a mixture of nucleic acid molecules of the present disclosure (e.g., comprising a first and a second plurality of nucleic acid molecules of the present disclosure) with SPRI beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.4 and about 0.6, or between about 0.45 and about 0.55. For example, the ratio of volume of beads to volume of nucleic acid molecules may be between about 0.4 and about 0.55, between about 0.35 and about 0.5, between about 0.45 and about 0.6, between about 0.5 and about 0.65, between about 0.4 and about 0.55, or between about 0.5 and about 0.55. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is any of about 0.4, about 0.45, about 0.5, about 0.55, or about 0.6. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is about 0.5. For example, in some embodiments, the size selection comprisescontacting a volume of about 55|aL of a mixture of nucleic acid molecules of the present disclosure (e.g., comprising a first and a second plurality of nucleic acid molecules of the present disclosure) with a volume of about 27.5 pL of SPRI beads.

[0082] In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is between about 0.45 and about 0.55. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is any of about 0.4, about 0.45, about 0.5, about 0.55, or about 0.6. In some embodiments, the ratio of volume of beads to volume of nucleic acid molecules is about 0.5. For example, in some embodiments, the size selection comprises contacting a volume of about 27.5 pL of SPRI beads with a volume of a mixture of nucleic acid molecules of the present disclosure (e.g., comprising a first and a second plurality of nucleic acid molecules of the present disclosure) of about 55pL.

[0083] In some embodiments, the methods of the present disclosure further comprise (e.g. after contacting a mixture of nucleic acid molecules with SPRI beads) washing the mixture of nucleic acid molecules under conditions in which the mixture of nucleic acid molecules is bound to the SPRI beads; and eluting the size-selected plurality of nucleic acid molecules from the SPRI beads. Methods for using SPRI beads are known in the art and exemplified herein; see, e.g., instructions for use in the AMPure XP SPRI Reagent (Beckman Coulter).

[0084] Size selection of the present disclosure can used to enrich for nucleic acids of a desired length. The length of the nucleic acid fragment can be indicative of the molecule origin; for example fragments greater than 1,000 nucleotides in length may have originated from or been isolated from nucleated cells, whereas nucleic acids that are shorter than 1,000 nucleotides in length may be examples of cell-free DNA (cfDNA).

[0085] In some embodiments, a first plurality of nucleic acid molecules or a plurality of size- selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more, about 5000 base pairs or more, about 10,000 base pairs or more, about 15,000 base pairs or more, about 20,000 base pairs or more, about 25,000 base pairs or more, about 30,000 base pairs or more, about 35,000 base pairs or more, about 40,000 base pairs or more, about 45,000 base pairs or more, about 50,000 base pairs or more, about 55,000 base pairs or more, about 60,000 base pairs or more, about 65,000 base pairs or more, about 70,000 base pairs or more, about 75,000 base pairs or more, about 80,000 base pairs or more, about 85,000 base pairs or more, about 90,000 base pairs or more, about 95,000 base pairs or more, or about 100,000 base pairs or more. For example, in some embodiments, a first plurality of nucleic acid molecules or aplurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 1000 base pairs to about 10,000 base pairs, between about 5000 base pairs to about 15,000 base pairs, between about 10,000 base pairs to about 20,000 base pairs, between about 15,000 base pairs to about 25,000 base pairs, between about 20,000 base pairs to about 30,000 base pairs, between about 25,000 base pairs to about 35,000 base pairs, between about 30,000 base pairs to about 40,000 base pairs, between about 35,000 base pairs to about 45,000 base pairs, between about 40,000 base pairs to about 50,000 base pairs, between about 45,000 base pairs to about 55,000 base pairs, between about 50,000 base pairs to about 65,000 base pairs, between about 60,000 base pairs to about 70,000 base pairs, between about 65,000 base pairs to about 75,000 base pairs, between about 70,000 base pairs to about 80,000 base pairs, between about 75,000 base pairs to about 85,000 base pairs, between about 80,000 base pairs to about 90,000 base pairs, between about 85,000 base pairs to about 95,000 base pairs, between about 90,000 base pairs to about 100,000 base pairs, between about 95,000 base pairs to about 105,000 base pairs, between about 100,000 base pairs to about 110,000 base pairs, between about 105,000 base pairs to about 115,000 base pairs, between about 110,000 base pairs to about 120,000 base pairs, between about 115,000 base pairs to about 125,000 base pairs, between about 120,000 base pairs to about 130,000 base pairs, between about 125,000 base pairs to about 135,000 base pairs, between about 130,000 base pairs to about 140,000 base pairs, between about 135,000 base pairs to about 145,000 base pairs, between about 140,000 base pairs to about 150,000 base pairs, between about 145,000 base pairs to about 155,000 base pairs, between about 150,000 base pairs to about 165,000 base pairs, between about 160,000 base pairs to about 170,000 base pairs, between about 165,000 base pairs to about 175,000 base pairs, between about 170,000 base pairs to about 180,000 base pairs, between about 175,000 base pairs to about 185,000 base pairs, between about 180,000 base pairs to about 190,000 base pairs, between about 185,000 base pairs to about 195,000 base pairs, between about 190,000 base pairs to about 200,000 base pairs, or between about 195,000 base pairs to about 205,000 base pairs.

[0086] In some embodiments, a first plurality of nucleic acid molecules or a plurality of size- selected nucleic acid molecules of the present disclosure e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of up to about 200,000 base pairs. In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid moleculesof the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of about 1,000 base pairs or more. In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 1,000 base pairs and about 200,000 base pairs. In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of about 50,000 base pairs or more. In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs.

[0087] In some embodiments, the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of less than about 1,000 base pairs. In some embodiments, the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of about 999 base pairs or less, about 900 base pairs or less, about 800 base pairs or less, about 700 base pairs or less, about 600 base pairs or less, about 500 base pairs or less, about 400 base pairs or less, about 300 base pairs or less, about 200 base pairs or less, or about 100 base pairs or less.

[0088] In some embodiments, the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of about 50 base pairs or more, about 75 base pairs or more, about 100 base pairs or more, about 125 base pairs or more, about 150 base pairs or more, about 175 base pairs or more, about 200 base pairs or more, about 225 base pairs or more, about 250 base pairs or more, about 275 base pairs or more, about 300 base pairs or more, about 325 base pairs or more, about 350 base pairs or more, about 375 base pairs or more, about 400 base pairs or more, about 425 base pairs or more, about 450 base pairs or more, about 475 base pairs or more, or about 500 base pairs or more. In some embodiments, the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length from about 50 base pairs to less than about 1,000 base pairs.

[0089] In some embodiments, the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs to about 150 base pairs, between about 100 base pairs to about 200 base pairs, between about 150 base pairs to about 250 base pairs, between about 100 base pairs to about 300 base pairs, between about 200 base pairs to about 300 base pairs, between about 250 base pairs to about 350 base pairs, between about 300 base pairs to about 400 base pairs, between about 350 base pairs to about 450 base pairs, between about 400 base pairs to about 500 base pairs, between about 450 base pairs to about 550 base pairs, between about 500 base pairs to about 600 base pairs, between about 550 base pairs to about 650 base pairs, between about 600 base pairs to about 700 base pairs, between about 650 base pairs to about 750 base pairs, between about 700 base pairs to about 800 base pairs, between about 750 base pairs to about 850 base pairs, between about 800 base pairs to about 900 base pairs, between about 850 base pairs to about 950 base pairs, between about 900 base pairs to about 1000 base pairs, or between about 950 base pairs to about 1,050 base pairs.

[0090] In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 1,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of less than about 1,000 base pairs.

[0091] In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules of the present disclosure (e.g., cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of less than about 1,000 base pairs.

[0092] In some embodiments, the first plurality of nucleic acid molecules or a plurality of size-selected nucleic acid molecules of the present disclosure (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) comprises nucleic acid molecules or fragments thereof with a length of between about 1,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules of the present disclosure (e.g.,cfDNA or ctDNA) comprises nucleic acid molecules or fragments thereof with a length of less than about 1,000 base pairs.

[0093] In some embodiments, the nucleic acid molecules from nucleated cells comprise nucleic acid fragments with a length of between about 1,000 base pairs and about 200,000 base pairs, and the nucleic acid molecules from cell-free DNA comprise nucleic acid fragments with a length between about 50 base pairs and about 1,000 base pairs.

[0094] In some embodiments, the nucleic acid molecules from nucleated cells comprise nucleic acid fragments with a length of between about 50,000 base pairs and about 200,000 base pairs, and the nucleic acid molecules from cell-free DNA comprise nucleic acid fragments with a length between about 50 base pairs and about 1,000 base pairs.

[0095] Various assays may be used in order to detect the presence of a mutation in one or more genes. For example, in some embodiments, the mutation in the one or more genes is detected by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence- specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing. In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique, optionally, wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS). Various sequencing techniques suitable for the methods of the present disclosure are known in the art and described in greater detail infra.

[0096] A nucleic acid hybridization assay involves the bonding of short, complementary nucleic acid probes to a target sequence, wherein the probe is labeled, for example with a radioisotope, an enzyme, a chemiluminescent reporter, or a fluorescent reporter that can be detected as a readout of probe binding. Fluorescence in situ hybridization (FISH) is an example of a nucleic acid hybridization assay using a fluorescent reporter to detect and locate a specific nucleic acid sequence, e.g., on a chromosome, including multicolor FISH (mFISH) to identify and observe more than one target sequence within the same experimental run. An amplification-based assay works by creating many copies e.g., 2ncopies, wherein n is the number of rounds of amplification) of a target sequence, e.g., to boost the signal and increase the likelihood of correctly identifying a target sequence. An example of an amplification-based assay includes real-time PCR, which measures the accumulation of an amplicon during the reaction. A polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) assay is an assay wherein restriction enzymes are used after DNA is amplified, e.g., in a PCR reaction, to digest the amplified DNA in order to assess the presence of different DNA fragment lengths that can be indicative of, e.g., a polymorphism. Spectral karyotype (SKY) is a karyotype in which the homologous pairs of chromosomes are manipulated in such a way that they have distinctive colors. The SKY technique makes it easier for scientists to detect chromosomal abnormalities, as compared with a conventional karyotype. High-performance liquid chromatography (HPLC) is used to separate out and quantify the presence of chemical compounds in a liquid sample, for example to identify the degree to which a DNA sample may be modified, e.g., methylated or acetylated. HPLC can be used in combination with another assay, for example an amplification-based assay followed by restriction enzyme digestion to generate DNA fragments of various lengths, sizes, and / or weights. Mass- spectrometric genotyping involves amplification of a nucleic acid sequence, e.g., a genomic region, to be analyzed, which is then digested by restriction enzyme and analyzed by electrospray ionization mass spectrometry. Sequencing techniques, as described in more detail in the present application, can be used to identify the specific nucleic acid sequence of one or more target sequences or gene loci.

[0097] In some embodiments, the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique, optionally, wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

[0098] In some embodiments, the methods of the present disclosure further comprise selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in a size-selected plurality of nucleic acid molecules of the present disclosure, thereby producing an enriched plurality of nucleic acid molecules. Various methods for selective enrichment are known in the art and described herein, such as specific amplification e.g., by PCR) or hybrid capture. For example, in some embodiments, the selective enrichment comprises combining one or more bait molecules with the size-selected plurality of nucleic acid molecules, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules. In otherembodiments, the selective enrichment comprises amplifying one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size- selected plurality of nucleic acid molecules (e.g., using one or more gene- specific primer pairs), thereby producing the enriched plurality of nucleic acid molecules. In some embodiments, the methods further comprise sequencing the enriched plurality of nucleic acid molecules to detect the mutation in the one or more genes, e.g., using a sequencing technique of the present disclosure.

[0099] In some embodiments, the methods of the present disclosure further comprise optionally, ligating one or more adapters onto nucleic acid molecules in the size-selected plurality of nucleic acid molecules, thereby generating ligated nucleic acids; optionally, amplifying nucleic acids from the ligated nucleic acids; optionally, capturing or enriching from the amplified nucleic acids a plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes; sequencing, by a sequencer, the plurality of nucleic acids to obtain a plurality of sequence reads corresponding to the one or more genes; analyzing the plurality of sequence reads; and based on the analysis, detecting the mutation in the one or more genes. In some embodiments, the plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes is captured from the amplified nucleic acids by hybridization with one or more bait molecules.

[0100] Exemplary methods and aspects of hybrid capture e.g., using bait molecule(s) of the present disclosure) are described herein. In some embodiments, capturing or enriching from the amplified nucleic acids a plurality of nucleic acids comprises combining one or more bait molecules with amplified nucleic acids, thereby hybridizing the one or more bait molecules to nucleic acids comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids. In some embodiments, one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more genes. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides. In some embodiments, the one or more bait molecules are conjugated to an affinity reagent (including but not limited to an antibody, an antibody fragment, or biotin) or to a detection reagent (including but not limited to a fluorescent marker). In some embodiments, the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.

[0101] In some embodiments, the methods of the present disclosure (e.g., size selection) further involve the recovery of the nucleic acid molecules, such that the recovery process will yield a specific percentage of the original sample input of nucleic acid molecules from nucleated cells, up to about 100% recovery. Further, the recovery process will yield a specific percentage of the original sample input of nucleic acid molecules from cell-free DNA, as low as about 0%.

[0102] For example, in some embodiments, the size selection recovers at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more of nucleic acid molecules from nucleated cells.

[0103] In some embodiments, the size selection recovers at least about 50% of nucleic acid molecules from nucleated cells. In some embodiments, the size selection recovers at least about 60% of nucleic acid molecules from nucleated cells. In some embodiments, the size selection recovers at least about 62% of nucleic acid molecules from nucleated cells.

[0104] In some embodiments, the size selection recovers less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% or less of nucleic acid molecules from cell-free DNA.

[0105] In some embodiments, the size selection recovers less than about 3% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers less than about 2% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers less than about 1% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers less than about 0.5% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers less than about 0.25% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers less than about 0.1% of nucleic acid molecules from cell-free DNA. In some embodiments, the size selection recovers about 0% of nucleic acid molecules from cell-free DNA.Samples

[0106] In some embodiments, a mixture of nucleic acid molecules of the present disclosure is from a liquid sample obtained from an individual. In some embodiments, the liquid sample isa liquid biopsy sample. In some embodiments, the methods further comprise obtaining the sample from the individual.

[0107] The disclosed methods can be used with any of a variety of samples (also referred to herein as specimens) comprising nucleic acids (e.g., DNA) that are collected from a subject (e.g., a human patient). Examples of a sample (e.g., a liquid sample) include, but are not limited to, a liquid biopsy sample, a blood sample (e.g., a peripheral whole blood sample), a blood plasma sample, a blood serum sample, a lymph sample, a saliva sample, a sputum sample, a urine sample, a gynecological fluid sample, a circulating tumor cell (CTC) sample, a cerebrospinal fluid (CSF) sample, a pericardial fluid sample, a pleural fluid sample, an ascites (peritoneal fluid) sample, a feces (or stool) sample, or other body fluid, secretion, and / or excretion sample (or sample derived therefrom). In some embodiments, the nucleic acid molecules are obtained from a liquid biopsy sample or a blood sample (e.g., a whole blood sample comprising plasma and a buffy coat). In some embodiments, the sample is a liquid sample, and may comprise, e.g., whole blood, plasma, serum, urine, saliva, amniotic fluid, breast milk, pleural effusions or ascites, synovial fluid, or cerebrospinal fluid. In some embodiments, the liquid sample is from whole blood.

[0108] In some embodiments, the mixture comprises nucleic acids from the individual that are obtained from a buffy coat sample. In some embodiments, the buffy coat sample is derived by centrifugation of a whole blood liquid sample from the individual. In some embodiments, the buffy coat sample comprises one or more nucleated cells, optionally wherein the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells. In some embodiments, the nucleic acid molecules from nucleated cells comprise DNA from nucleated cells, optionally genomic DNA.

[0109] In some embodiments, a mixture of nucleic acid molecules of the present disclosure is obtained from a buffy coat sample, e.g., derived from a liquid biopsy sample. In some embodiments, the mixture of nucleic acid molecules is obtained by extracting buffy coat DNA (bcDNA) from the buffy coat sample (and optionally the extracted bcDNA is fragmented). In some embodiments, the methods of the present disclosure further comprise extracting buffy coat DNA (bcDNA) from the buffy coat sample (and optionally fragmenting the extracted bcDNA) in order to generate the mixture of nucleic acid molecules. In some embodiments, size selection of the present disclosure is performed on the bcDNA, for example to enrich for nucleic acids from nucleated cells (e.g., bcDNA). Examples of DNA from the sample or extracted from buffy coat that may be suitable for analysis by thedisclosed methods include, but are not limited to, genomic DNA, mitochondrial DNA, and fragments and / or combinations thereof.

[0110] In some embodiments, the buffy coat sample is derived by centrifugation of a whole blood sample (e.g., a liquid biopsy sample) from the individual. In some embodiments, the methods of the present disclosure further comprise centrifuging a whole blood liquid biopsy from the individual to generate the buffy coat sample.

[0111] In other embodiments, the buffy coat sample is derived by microfluidic fractionation of a whole blood liquid biopsy from the individual. In some embodiments, the methods of the present disclosure further comprise subjecting a whole blood sample (e.g., a liquid biopsy sample) from the individual to microfluidic fractionation in order to generate the buffy coat sample. Microfluidic approaches to blood fractionation are known in the art; see, e.g., Kuan, D.H. et al. (2018) Sci Rep 8:15345.

[0112] In some embodiments, a second plurality of nucleic acid molecules of the present disclosure comprises cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA). In some embodiments, the nucleic acid molecules from cell-free DNA comprise circulating tumor DNA (ctDNA). Cell-free DNA (cfDNA) is comprised of fragments of DNA that are released from normal and / or cancerous cells during apoptosis and necrosis and circulate in the blood stream and / or accumulate in other bodily fluids. Circulating tumor DNA is comprised of fragments of DNA that are released from cancerous cells and tumors that circulate in the blood stream and / or accumulate in other bodily fluids.

[0113] DNA may be extracted from liquid samples, including but not limited to blood, plasma, cerebrospinal fluid, sputum, stool, urine, or saliva or other bodily fluid samples using any of a variety of techniques known to those of skill in the art (see, e.g., Example 1 of International Patent Application Publication No. WO 2012 / 092426; Tan, et al. (2009), “DNA, RNA, and Protein Extraction: The Past and The Present”, J. Biomed. Biotech. 2009:574398; the technical literature for the Maxwell® 16 LEV Blood DNA Kit (Promega Corporation, Madison, WI); and the Maxwell 16 Buccal Swab LEV DNA Purification Kit Technical Manual (Promega Literature #TM333, January 1, 2011, Promega Corporation, Madison, WI)).

[0114] A typical DNA extraction procedure, for example, comprises (i) collection of the fluid sample from which DNA is to be extracted, (ii) treatment of the fluid sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate out the precipitated proteins, lipids, and RNA, and (iii) purification of DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during previoussteps. The DNA sample optionally may be further treated with an RNase for digestion of RNA in the sample.

[0115] Examples of suitable techniques for DNA purification include, but are not limited to,(i) precipitation in ice-cold ethanol or isopropanol, followed by centrifugation (precipitation of DNA may be enhanced by increasing ionic strength, e.g., by addition of sodium acetate),(ii) phenol-chloroform extraction, followed by centrifugation to separate the aqueous phase containing the nucleic acid from the organic phase containing denatured protein, and (iii) solid-phase chromatography where the nucleic acids adsorb to the solid phase (e.g., silica or other) depending on the pH and salt concentration of the buffer.

[0116] In some instances, cellular and histone proteins bound to the DNA may be removed either by adding a protease or by having precipitated the proteins with sodium or ammonium acetate, or through extraction with a phenol-chloroform mixture prior to a DNA precipitation step.

[0117] In some instances, DNA may be extracted using any of a variety of suitable commercial DNA extraction and purification kits. Examples include, but are not limited to, the QIAamp (for isolation of genomic DNA from human samples) and DNAeasy (for isolation of genomic DNA from animal or plant samples) kits from Qiagen (Germantown, MD) or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI).

[0118] After isolation, the nucleic acids are typically dissolved in a slightly alkaline buffer, e.g., Tris-EDTA (TE) buffer, or in ultra-pure water.Subjects

[0119] In some instances, the sample is obtained (e.g., collected) from a subject (e.g., patient) with a condition or disease (e.g., a hyperproliferative disease or a non-cancer indication), suspected of having the condition or disease, or at risk of developing the condition or disease. In some embodiments, detection of a mutation in one or more genes (e.g., as disclosed herein) is used to diagnose or confirm a diagnosis of disease in an individual.

[0120] In some embodiments, a mutation in the one or more genes (e.g., detected by the methods of the present disclosure, such as a CH mutation of the present disclosure) is determined to be caused by clonal hematopoiesis of indeterminate potential (CHIP). In some embodiments, the individual has, is suspected of having, or is being tested for CHIP. In some embodiments, the disease is a cardiovascular or hematological disease.

[0121] In some embodiments, the disease is a cancer. In some instances, the cancer is a solid tumor or a metastatic form thereof. In some embodiments, the cancer is a carcinoma, asarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma. In some instances, the cancer is a hematological cancer, e.g., a leukemia or lymphoma.

[0122] In some embodiments, the subject has a cancer or is at risk of having a cancer. For example, in some instances, the subject has a genetic predisposition to a cancer (e.g., having one or more genetic mutation(s) that increases his or her baseline risk for developing a cancer). In some embodiments, detection of the mutation in the one or more genes is used to diagnose or confirm a diagnosis of disease in the individual. In some instances, the subject has been exposed to an environmental perturbation (e.g., radiation or a chemical) that increases his or her risk for developing a cancer.

[0123] In some instances, the subject may benefit from being monitored for development of a cancer. In some instances, the subject may benefit from being monitored for cancer progression or regression, e.g., after being treated with an anti-cancer therapy (or anti-cancer treatment). In some instances, the subject may benefit from being monitored for relapse of cancer. In some instances, the subject may benefit from being monitored for minimal residual disease (MRD). In some instances, the subject has been treated or is being treated for cancer. In some instances, the subject has not been treated with an anti-cancer therapy (or anti-cancer treatment).

[0124] In some instances, the subject (e.g., a patient) is being treated or has been previously treated with one or more targeted therapies. In some instances, e.g., for a patient who has been previously treated with a targeted therapy, a post-targeted therapy sample (e.g., specimen) is obtained (e.g., collected). In some instances, the post-targeted therapy sample is a sample obtained after the completion of the targeted therapy.

[0125] In some instances, the patient has not been previously treated with a targeted therapy. In some instances, e.g., for a patient who has not been previously treated with a targeted therapy, the sample comprises a resection, e.g., an original resection, or a resection following recurrence (e.g., following a disease recurrence post- therapy).

[0126] In some instances, the sample is acquired from a subject having a cancer. Exemplary cancers include, but are not limited to, B cell cancer (multiple myeloma), a melanoma, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of an oral cavity, cancer of a pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, a cancerof hematological tissue, an adenocarcinoma, an inflammatory myofibroblastic tumor, a gastrointestinal stromal tumor (GIST), colon cancer, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia Vera, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), soft-tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, head and neck cancer, small cell cancer, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia, chronic eosinophilic leukemia, neuroendocrine cancers, carcinoid tumors, and the like.

[0127] In some instances, the cancer comprises acute lymphoblastic leukemia (Philadelphia chromosome positive), acute lymphoblastic leukemia (precursor B-cell), acute myeloid leukemia (FLT3+), acute myeloid leukemia (with an IDH2 mutation), anaplastic large cell lymphoma, basal cell carcinoma, B-cell chronic lymphocytic leukemia, bladder cancer, breast cancer (HER2 overexpressed / amplified), breast cancer (HER2+), breast cancer (HR+, HER2- ), cervical cancer, cholangiocarcinoma, chronic lymphocytic leukemia, chronic lymphocytic leukemia (with 17p deletion), chronic myelogenous leukemia, chronic myelogenous leukemia (Philadelphia chromosome positive), classical Hodgkin lymphoma, colorectal cancer, colorectal cancer (dMMR and MSLH), colorectal cancer (KRAS wild type), cryopyrin- associated periodic syndrome, a cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, a diffuse large B-cell lymphoma, fallopian tube cancer, a follicular B-cell nonHodgkin lymphoma, a follicular lymphoma, gastric cancer, gastric cancer (HER2+), a gastroesophageal junction (GEJ) adenocarcinoma, a gastrointestinal stromal tumor, a gastrointestinal stromal tumor (KIT+), a giant cell tumor of the bone, a glioblastoma,granulomatosis with polyangiitis, a head and neck squamous cell carcinoma, a hepatocellular carcinoma, Hodgkin lymphoma, juvenile idiopathic arthritis, lupus erythematosus, a mantle cell lymphoma, medullary thyroid cancer, melanoma, a melanoma with a BRAF V600 mutation, a melanoma with a BRAF V600E or V600K mutation, Merkel cell carcinoma, multicentric Castleman's disease, multiple hematologic malignancies including Philadelphia chromosome-positive ALL and CML, multiple myeloma, myelofibrosis, a non-Hodgkin’s lymphoma, a nonresectable subependymal giant cell astrocytoma associated with tuberous sclerosis, a non-small cell lung cancer, a non-small cell lung cancer (ALK+), a non-small cell lung cancer (PD-L1+), a non-small cell lung cancer (with ALK fusion or ROS1 gene alteration), a non-small cell lung cancer (with BRAF V600E mutation), a non-small cell lung cancer (with an EGFR exon 19 deletion or exon 21 substitution (L858R) mutations), a non- small cell lung cancer (with an EGFR T790M mutation), ovarian cancer, ovarian cancer (with a BRCA mutation), pancreatic cancer, a pancreatic, gastrointestinal, or lung origin neuroendocrine tumor, a pediatric neuroblastoma, a peripheral T-cell lymphoma, peritoneal cancer, prostate cancer, a renal cell carcinoma, rheumatoid arthritis, a small lymphocytic lymphoma, a soft tissue sarcoma, a solid tumor (MSLH / dMMR), a squamous cell cancer of the head and neck, a squamous non-small cell lung cancer, thyroid cancer, a thyroid carcinoma, urothelial cancer, a urothelial carcinoma, or Waldenstrom's macroglobulinemia.

[0128] In some instances, the cancer is a hematologic malignancy (or premalignancy). As used herein, a hematologic malignancy refers to a tumor of the hematopoietic or lymphoid tissues, e.g., a tumor that affects blood, bone marrow, or lymph nodes. Exemplary hematologic malignancies include, but are not limited to, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, acute monocytic leukemia (AMoL), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), or large granular lymphocytic leukemia), lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma (e.g., classical Hodgkin lymphoma or nodular lymphocyte-predominant Hodgkin lymphoma), mycosis fungoides, non-Hodgkin lymphoma (e.g., B-cell non-Hodgkin lymphoma (e.g., Burkitt lymphoma, small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, or mantle cell lymphoma) or T- cell non-Hodgkin lymphoma (mycosis fungoides, anaplastic large cell lymphoma, or precursor T-lymphoblastic lymphoma)), primary central nervous system lymphoma, Sezary syndrome, Waldenstrom macroglobulinemia), chronic myeloproliferative neoplasm,Langerhans cell histiocytosis, multiple myeloma / plasma cell neoplasm, myelodysplastic syndrome, or myelodysplastic / myeloproliferative neoplasm.Targeting gene loci for analysis

[0129] The methods described herein can be used in combination with, or as part of, a method for evaluating a plurality or set of subject intervals (e.g., target sequences), e.g., from a set of genomic loci (e.g., gene loci or fragments thereof), as described herein.

[0130] In some embodiments, the set of genomic loci evaluated by the disclosed methods comprises a plurality of, e.g., genes, which in mutant form are associated with an effect on cell division, growth, or survival, or are associated with CHIP or a cancer, e.g., a cancer described herein.

[0131] In some embodiments, the set of gene loci evaluated by the disclosed methods comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or more than 100 gene loci.

[0132] In some embodiments, the selected gene loci (also referred to herein as target gene loci or target sequences), or fragments thereof, may include subject intervals comprising noncoding sequences, coding sequences, intragenic regions, or intergenic regions of the subject genome. For example, the subject intervals can include any of a non-coding sequence or fragment thereof (e.g., a promoter sequence, enhancer sequence, 5’ untranslated region (5’ UTR), 3’ untranslated region (3’ UTR), or a fragment thereof), a coding sequence of fragment thereof, an exon sequence or fragment thereof, and an intron sequence or a fragment thereof.

[0133] In some embodiments, the selected gene loci comprise one or more genes, further optionally comprising one or more clonal hematopoietic (CH) or germline gene mutations. In some embodiments, the mutation in the one or more genes comprises one or more of: a point mutation; an insertion-deletion (indel); an in-frame deletion of one or more codons; an intragenic deletion; an intragenic insertion; a deletion of a full gene; an inversion; an interchromosomal or intrachromosomal translocation; a tandem duplication; a gene fusion; a genomic rearrangement that comprises an intron sequence; and / or a gene amplification or duplication.

[0134] In some embodiments, the one or more genes comprise ABL1, ACVR1B, AKT1, AKT2, AKT3, ALK, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID1A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1,BCL2L2, BCL6, BCOR, BCORL1, BCR, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BTG1, BTG2, BTK, CALR, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD22, CD274, CD70, CD74, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC, CREBBP, CRKL, CSF1R, CSF3R, CTCF, CTNNA1, CTNNB1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DNMT3A, DOT1L, EED, EGFR, EMSY (Cllorf30), EP300, EPHA3, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC4, ERG, ERRFI1, ESRI, ETV4, ETV5, ETV6, EWSR1, EZH2, EZR, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GATA3, GATA4, GATA6, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2D (MLL2), KRAS, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MERTK, MET, MITF, MKNK1, MLH1, MPL, MRE11A, MSH2, MSH3, MSH6, MST1R, MTAP, MTOR, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NT5C2, NTRK1, NTRK2, NTRK3, NUTM1, P2RY8, PALB2, PARK2, PARP1, PARP2, PARP3, PAX5, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CA, PIK3CB, PIK3R1, PIM1, PMS2, POLDI, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PRKAR1A, PRKCI, PTCHI, PTEN, PTPN11, PTPRO, QKI, RAC1, RAD21, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAFI, RARA, RBI, RBM10, REL, RET, RICTOR, RNF43, ROS1, RPTOR, RSPO2, SDC4, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SGK1, SLC34A2, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STK11, SUFU, SYK, TBX3, TEK, TERC, TERT, TET2, TGFBR2, TIPARP, TMPRSS2, TNFAIP3, TNFRSF14, TP53, TSC1, TSC2, TYRO3, U2AF1, VEGFA, VHL, WHSCI, WHSC1L1, WT1, XPO1, XRCC2, ZNF217, ZNF703, or any combination thereof.

[0135] In some embodiments, the one or more genes comprise ABL, ALK, ALL, B4GALNT1, BAFF, BCL2, BRAF, BRCA, BTK, CD19, CD20, CD3, CD30, CD319, CD38, CD52, CDK4, CDK6, CML, CRACC, CS1, CTLA-4, dMMR, EGFR, ERBB1, ERBB2, FGFR1-3, FLT3, GD2, HDAC, HER1, HR, IDH2, IL-ip, IL-6, IL-6R, JAK1, JAK2, JAK3,KU, KRAS, MEK, MET, MSI-H, mTOR, PARP, PD-1, PDGFR, PDGFRa, PDGFRp, PD- Ll, PI3K5, PIGF, PTCH, RAF, RANKE, RET, ROS1, SLAMF7, VEGF, VEGFA, VEGFB, or any combination thereof.

[0136] The methods described herein may comprise contacting a nucleic acid library with a plurality of target capture reagents in order to select and capture a plurality of specific target sequences (e.g., nucleic acids that comprise gene sequences or fragments thereof) for analysis. In some instances, a target capture reagent (z.e., a molecule which can bind to and thereby allow capture of a target molecule) is used to select the subject intervals to be analyzed. For example, a target capture reagent can be a bait molecule, e.g. , a nucleic acid molecule (e.g., a DNA molecule or RNA molecule) which can hybridize to (i.e., is complementary to) a target molecule, and thereby allows capture of the target nucleic acid. In some instances, the target capture reagent, e.g., a bait molecule (or bait sequence), is a capture oligonucleotide (or capture probe). In some instances, the target nucleic acid is a genomic DNA molecule, an RNA molecule, a DNA / RNA molecule, a cDNA molecule derived from an RNA molecule, a micro satellite DNA sequence, or the like. In some embodiments, the target nucleic acid is from bcDNA, e.g., gDNA. In some instances, the target capture reagent is suitable for solution-phase hybridization to the target. In some instances, the target capture reagent is suitable for solid-phase hybridization to the target. In some instances, the target capture reagent is suitable for both solution-phase and solid-phase hybridization to the target. The design and construction of target capture reagents is described in more detail in, e.g., International Publication No. WO2020 / 236941.

[0137] The methods described herein provide for optimized sequencing of a large number of genomic loci (e.g., genes or gene products (e.g., mRNA), micro satellite loci, etc.) from samples (e.g., whole blood samples, liquid biopsy samples, and the like) from one or more subjects by the appropriate selection of target capture reagents to select the target nucleic acid molecules to be sequenced. In some instances, a target capture reagent may hybridize to a specific target locus, e.g., a specific target gene locus or fragment thereof. In some instances, a target capture reagent may hybridize to a specific group of target loci, e.g., a specific group of gene loci or fragments thereof. In some instances, a plurality of target capture reagents comprising a mix of target- specific and / or group -specific target capture reagents may be used.

[0138] In some instances, the number of target capture reagents (e.g., bait molecules) in the plurality of target capture reagents (e.g., a bait set) contacted with a nucleic acid library to capture a plurality of target sequences for nucleic acid sequencing is greater than 10, greaterthan 50, greater than 100, greater than 200, greater than 300, greater than 400, greater than 500, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1,000, greater than 1,250, greater than 1,500, greater than 1,750, greater than 2,000, greater than 3,000, greater than 4,000, greater than 5,000, greater than 10,000, greater than 25,000, or greater than 50,000.

[0139] In some instances, the overall length of the target capture reagent sequence can be between about 10 nucleotides and 1000 nucleotides. In one instance, the target capture reagent length is between about 100 and 300 nucleotides, 110 and 200 nucleotides, or 120 and 170 nucleotides, in length. In addition to those mentioned above, intermediate oligonucleotide lengths of about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 400, 500, 600, 700, 800, and 900 nucleotides in length can be used in the methods described herein. In some embodiments, oligonucleotides of about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, or 230 bases can be used. In some embodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.

[0140] In some instances, each target capture reagent sequence can include: (i) a targetspecific capture sequence (e.g., a gene locus or micro satellite locus-specific complementary sequence), (ii) an adapter, primer, barcode, and / or unique molecular identifier sequence, and (iii) universal tails on one or both ends. As used herein, the term "target capture reagent" can refer to the target- specific target capture sequence or to the entire target capture reagent oligonucleotide including the target- specific target capture sequence.

[0141] In some instances, the target- specific capture sequences in the target capture reagents are between about 10 nucleotides and about 1000 nucleotides in length. In some instances, the target- specific capture sequence is between about 70 nucleotides and about 300 nucleotides in length. In some instances, the target- specific sequence is between about 100 nucleotides and about 200 nucleotides in length. In yet other instances, the target- specific sequence is between about 120 nucleotides and about 170 nucleotides in length, typically 120 nucleotides in length. Intermediate lengths in addition to those mentioned above also can be used in the methods described herein, such as target- specific sequences of about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 300, 400, 500, 600, 700, 800, and 900 nucleotides in length, as well as target- specific sequences of lengths between the above-mentioned lengths. In someembodiments, the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 100 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.

[0142] In some instances, the target capture reagent may be designed to select a subject interval containing one or more rearrangements, e.g., an intron containing a genomic rearrangement. In such instances, the target capture reagent is designed such that repetitive sequences are masked to increase the selection efficiency. In those instances where the rearrangement has a known juncture sequence, complementary target capture reagents can be designed to recognize the juncture sequence to increase the selection efficiency.

[0143] In some instances, the disclosed methods may comprise the use of target capture reagents designed to capture two or more different target categories, each category having a different target capture reagent design strategy. In some instances, the hybridization-based capture methods and target capture reagent compositions disclosed herein may provide for the capture and homogeneous coverage of a set of target sequences, while minimizing coverage of genomic sequences outside of the targeted set of sequences. In some instances, the target sequences may include the entire exome of genomic DNA or a selected subset thereof. In some instances, the target sequences may include, e.g., a large chromosomal region (e.g., a whole chromosome arm). The methods and compositions disclosed herein provide different target capture reagents for achieving different sequencing depths and patterns of coverage for complex sets of target nucleic acid sequences.

[0144] Typically, DNA molecules are used as target capture reagent sequences, although RNA molecules can also be used. In some instances, a DNA molecule target capture reagent can be single stranded DNA (ssDNA) or double-stranded DNA (dsDNA). In some instances, an RNA-DNA duplex is more stable than a DNA-DNA duplex and therefore provides for potentially better capture of nucleic acids. In some embodiments, the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.

[0145] In some instances, the disclosed methods comprise providing a selected set of nucleic acid molecules (e.g., a library catch) captured from one or more nucleic acid libraries. For example, the method may comprise: providing one or a plurality of nucleic acid libraries, each comprising a plurality of nucleic acid molecules (e.g., a plurality of target nucleic acid molecules and / or reference nucleic acid molecules) extracted from one or more samples (e.g., liquid sample(s)) from one or more subjects (e.g., human patient(s)); contacting the one or aplurality of libraries (e.g. , in a solution-based hybridization reaction) with one, two, three, four, five, or more than five pluralities of target capture reagents (e.g., oligonucleotide target capture reagents) to form a hybridization mixture comprising a plurality of target capture reagent / nucleic acid molecule hybrids; separating the plurality of target capture reagent / nucleic acid molecule hybrids from said hybridization mixture, e.g. , by contacting said hybridization mixture with a binding entity that allows for separation of said plurality of target capture reagent / nucleic acid molecule hybrids from the hybridization mixture, thereby providing a library catch (e.g., a selected or enriched subgroup of nucleic acid molecules from the one or a plurality of libraries).

[0146] In some embodiments, the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules. In some embodiments, the plurality of nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes is captured from the amplified nucleic acids by hybridization with one or more bait molecules. In some embodiments, the capturing comprises: (a) combining the one or more bait molecules with the amplified nucleic acids, thereby hybridizing the one or more bait molecules to nucleic acids comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and (b) isolating the nucleic acid hybrids.

[0147] In some instances, the disclosed methods may further comprise amplifying the library catch (e.g., by performing PCR). In other instances, the library catch is not amplified.

[0148] In some instances, the target capture reagents can be part of a kit which can optionally comprise instructions, standards, buffers, enzymes, or other reagents.

[0149] As noted above, the methods disclosed herein may include the step of contacting the library (e.g., the nucleic acid library) with a plurality of target capture reagents to provide a selected library target nucleic acid sequences (i.e., the library catch). The contacting step can be effected in, e.g., solution-based hybridization. In some instances, the method includes repeating the hybridization step for one or more additional rounds of solution-based hybridization. In some instances, the method further includes subjecting the library catch to one or more additional rounds of solution-based hybridization with the same or a different collection of target capture reagents.

[0150] In some instances, the contacting step is effected using a solid support, e.g., an array. Suitable solid supports for hybridization are described in, e.g., Albert, T.J. et al. (2007) Nat. Methods 4(11):903-5; Hodges, E. et al. (2007) Nat. Genet. 39(12): 1522-7; and Okou, D.T. et al. (2007) Nat. Methods 4(11):907-9.

[0151] Hybridization methods that can be adapted for use in the methods herein are described in the art, e.g., as described in International Publication No. WO2012 / 092426. Methods for hybridizing target capture reagents to a plurality of target nucleic acid molecules are described in more detail in, e.g., International Publication No. WO2020 / 236941.

[0152] The methods and systems disclosed herein can be used in combination with, or as part of, a method or system for sequencing nucleic acids (e.g., a next-generation sequencing system) to generate a plurality of sequence reads that overlap one or more gene loci within a subgenomic interval in the sample and thereby determine, e.g., gene allele sequences at a plurality of gene loci. “Next-generation sequencing” (or “NGS”) as used herein may also be referred to as “massively parallel sequencing” (or “MPS”), and refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., as in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a high throughput fashion (e.g., wherein greater than 103, 104, 105, or more than 105molecules are sequenced simultaneously).

[0153] Next-generation sequencing methods are known in the art, and are described in, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46. Other examples of sequencing methods suitable for use when implementing the methods and systems disclosed herein are described in, e.g., International Publication No. WO2012 / 092426. In some instances, the sequencing may comprise, for example, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, or direct sequencing. In some instances, sequencing may be performed using, e.g., Sanger sequencing. In some instances, the sequencing may comprise a paired-end sequencing technique that allows both ends of a fragment to be sequenced and generates high-quality, alignable sequence data for detection of, e.g., genomic rearrangements, repetitive sequence elements, gene fusions, and novel transcripts.

[0154] The disclosed methods and systems may be implemented using sequencing platforms such as the Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, Pacific Bioscience, Helicos, and / or the Polonator platform. In some instances, sequencing may comprise Illumina MiSeq sequencing. In some instances, sequencing may comprise Illumina HiSeq sequencing. In some instances, sequencing may comprise Illumina NovaSeq sequencing. Optimized methods for sequencing a large number of target genomic loci in nucleic acids extracted from a sample are described in more detail in, e.g., International Publication No. WO2020 / 236941.

[0155] In certain instances, the disclosed methods comprise one or more of the steps of: (a) acquiring a library comprising a plurality of normal and / or tumor nucleic acid moleculesfrom a liquid sample from an individual (e.g., a human patient); (b) simultaneously or sequentially contacting the library with one, two, three, four, five, or more than five pluralities of target capture reagents under conditions that allow hybridization of the target capture reagents to the target nucleic acid molecules, thereby providing a selected set of captured normal and / or tumor nucleic acid molecules (z.e., a library catch); (c) separating the selected subset of the nucleic acid molecules (e.g., the library catch) from the hybridization mixture, e.g., by contacting the hybridization mixture with a binding entity that allows for separation of the target capture reagent / nucleic acid molecule hybrids from the hybridization mixture, (d) sequencing the library catch to acquire a plurality of reads (e.g., sequence reads) that overlap one or more subject intervals (e.g., one or more target sequences) from said library catch that may comprise a mutation (or alteration), e.g., a variant sequence comprising a CH or germline gene mutation; (e) aligning said sequence reads using an alignment method as described elsewhere herein; and / or (f) assigning a nucleotide value for a nucleotide position in the subject interval (e.g., calling a mutation using, e.g., a Bayesian method or other method described herein) from one or more sequence reads of the plurality.

[0156] In some instances, acquiring sequence reads for one or more subject intervals may comprise sequencing at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, at least 950, at least 1,000, at least 1,250, at least 1,500, at least 1,750, at least 2,000, at least 2,250, at least 2,500, at least 2,750, at least 3,000, at least 3,500, at least 4,000, at least 4,500, or at least 5,000 loci, e.g., genomic loci, gene loci, micro satellite loci, etc. In some instances, acquiring a sequence read for one or more subject intervals may comprise sequencing a subject interval for any number of loci within the range described in this paragraph, e.g., for at least 2,850 gene loci.

[0157] In some instances, acquiring a sequence read for one or more subject intervals comprises sequencing a subject interval with a sequencing method that provides a sequence read length (or average sequence read length) of at least 20 bases, at least 30 bases, at least 40 bases, at least 50 bases, at least 60 bases, at least 70 bases, at least 80 bases, at least 90 bases, at least 100 bases, at least 120 bases, at least 140 bases, at least 160 bases, at least 180 bases, at least 200 bases, at least 220 bases, at least 240 bases, at least 260 bases, at least 280 bases, at least 300 bases, at least 320 bases, at least 340 bases, at least 360 bases, at least 380 bases, or at least 400 bases. In some instances, acquiring a sequence read for the one or more subject intervals may comprise sequencing a subject interval with a sequencing method that providesa sequence read length (or average sequence read length) of any number of bases within the range described in this paragraph, e.g., a sequence read length (or average sequence read length) of 56 bases.

[0158] In some instances, acquiring a sequence read for one or more subject intervals may comprise sequencing with at least lOOx or more coverage (or depth) on average. In some instances, acquiring a sequence read for one or more subject intervals may comprise sequencing with at least lOOx, at least 150x, at least 200x, at least 250x, at least 500x, at least 750x, at least l,000x, at least 1,500 x, at least 2,000x, at least 2,500x, at least 3,000x, at least 3,500x, at least 4,000x, at least 4,500x, at least 5,000x, at least 5,500x, or at least 6,000x or more coverage (or depth) on average. In some instances, acquiring a sequence read for one or more subject intervals may comprise sequencing with an average coverage (or depth) having any value within the range of values described in this paragraph, e.g., at least 160x.

[0159] In some instances, acquiring a read for the one or more subject intervals comprises sequencing with an average sequencing depth having any value ranging from at least lOOx to at least 6,000x for greater than about 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% of the gene loci sequenced. For example, in some instances acquiring a read for the subject interval comprises sequencing with an average sequencing depth of at least 125x for at least 99% of the gene loci sequenced. As another example, in some instances acquiring a read for the subject interval comprises sequencing with an average sequencing depth of at least 4,100x for at least 95% of the gene loci sequenced.

[0160] In some instances, the relative abundance of a nucleic acid species in the library can be estimated by counting the relative number of occurrences of their cognate sequences (e.g., the number of sequence reads for a given cognate sequence) in the data generated by the sequencing experiment.

[0161] In some instances, the disclosed methods and systems provide nucleotide sequences for a set of subject intervals (e.g., gene loci), as described herein. In certain instances, the sequences are provided without using a method that includes a matched normal control (e.g., a wild-type control) and / or a matched tumor control (e.g., primary versus metastatic).

[0162] In some instances, the level of sequencing depth as used herein (e.g., an X-fold level of sequencing depth) refers to the number of reads (e.g., unique reads) obtained after detection and removal of duplicate reads (e.g., PCR duplicate reads). In other instances, duplicate reads are evaluated, e.g., to support detection of copy number alteration (CNAs).

[0163] In some embodiments, the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generationsequencing (NGS), optionally further comprising receiving, at one or more processors, sequence read data for the plurality of sequence reads. In some embodiments, analyzing the plurality of sequence reads comprises detecting, using the one or more processors, the presence or absence of sequence reads comprising a mutation in the one or more genes, determining if the mutation is a CH or germline gene mutation, and filtering out the mutation when the mutation is determined to be a CH or germline gene mutation, optionally further comprising generating a molecular profile for the individual, based, at least in part, on detecting the mutation in the one or more genes.

[0164] Alignment is the process of matching a read with a location, e.g., a genomic location or locus. In some instances, NGS reads may be aligned to a known reference sequence (e.g., a wild-type sequence). In some instances, NGS reads may be assembled de novo. Methods of sequence alignment for NGS reads are described in, e.g., Trapnell, C. and Salzberg, S.L. Nature Biotech., 2009, 27:455-457. Examples of de novo sequence assemblies are described in, e.g., Warren R., et al., Bioinformatics, 2007, 23:500-501; Butler, J. et al., Genome Res., 2008, 18:810-820; and Zerbino, D.R. and Birney, E., Genome Res., 2008, 18:821-829. Optimization of sequence alignment is described in the art, e.g., as set out in International Publication No. WO2012 / 092426. Additional description of sequence alignment methods is provided in, e.g., International Publication No. WO2020 / 236941.

[0165] Misalignment (e.g., the placement of base-pairs from a short read at incorrect locations in the genome), e.g., misalignment of reads due to sequence context (e.g., the presence of repetitive sequence) around an actual cancer mutation can lead to reduction in sensitivity of mutation detection, can lead to a reduction in sensitivity of mutation detection, as reads for the alternate allele may be shifted off the histogram peak of alternate allele reads. Other examples of sequence context that may cause misalignment include short-tandem repeats, interspersed repeats, low complexity regions, insertions - deletions (indels), and paralogs. If the problematic sequence context occurs where no actual mutation is present, misalignment may introduce artifactual reads of “mutated” alleles by placing reads of actual reference genome base sequences at the wrong location. Because mutation-calling algorithms for multigene analysis should be sensitive to even low-abundance mutations, sequence misalignments may increase false positive discovery rates and / or reduce specificity.

[0166] In some instances, the methods and systems disclosed herein may integrate the use of multiple, individually tuned, alignment methods or algorithms to optimize base-calling performance in sequencing methods, particularly in methods that rely on massively parallel sequencing (MPS) of a large number of diverse genetic events at a large number of diversegenomic loci. In some instances, the disclosed methods and systems may comprise the use of one or more global alignment algorithms. In some instances, the disclosed methods and systems may comprise the use of one or more local alignment algorithms. Examples of alignment algorithms that may be used include, but are not limited to, the Burrows-Wheeler Alignment (BWA) software bundle (see, e.g., Li, et al. (2009), “Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform”, Bioinformatics 25:1754-60; Li, et al. (2010), Fast and Accurate Long-Read Alignment with Burrows-Wheeler Transform”, Bioinformatics epub. PMID: 20080505), the Smith-Waterman algorithm (see, e.g., Smith, et al. (1981), "Identification of Common Molecular Subsequences", J. Molecular Biology 147(1): 195-197), the Striped Smith-Waterman algorithm (see, e.g., Farrar (2007), “Striped Smith-Waterman Speeds Database Searches Six Times Over Other SIMD Implementations”, Bioinformatics 23(2): 156- 161), the Needleman- Wunsch algorithm (Needleman, et al. (1970) "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins", J. Molecular Biology 48(3):443-53), or any combination thereof.

[0167] In some instances, the methods and systems disclosed herein may also comprise the use of a sequence assembly algorithm, e.g., the Arachne sequence assembly algorithm (see, e.g., Batzoglou, et al. (2002), “ARACHNE: A Whole-Genome Shotgun Assembler”, Genome Res. 12:177-189).

[0168] In some instances, the alignment method used to analyze sequence reads is not individually customized or tuned for detection of different variants (e.g., point mutations, insertions, deletions, and the like) at different genomic loci. In some instances, different alignment methods are used to analyze reads that are individually customized or tuned for detection of at least a subset of the different variants detected at different genomic loci. In some instances, different alignment methods are used to analyze reads that are individually customized or tuned to detect each different variant at different genomic loci. In some instances, tuning can be a function of one or more of: (i) the genetic locus (e.g., gene loci, micro satellite locus, or other subject interval) being sequenced, (ii) the tumor type associated with the sample, (iii) the variant being sequenced, or (iv) a characteristic of the sample or the subject. The selection or use of alignment conditions that are individually tuned to a number of specific subject intervals to be sequenced allows optimization of speed, sensitivity, and specificity. The method is particularly effective when the alignment of reads for a relatively large number of diverse subject intervals are optimized.

[0169] In some instances, the method includes the use of an alignment method optimized for rearrangements in combination with other alignment methods optimized for subject intervals not associated with rearrangements.

[0170] In some instances, the methods disclosed herein further comprise selecting or using an alignment method for analyzing, e.g., aligning, a sequence read, wherein said alignment method is a function of, is selected responsive to, or is optimized for, one or more of: (i) tumor type, e.g., the tumor type in the sample; (ii) the location (e.g., a gene locus) of the subject interval being sequenced; (iii) the type of mutation or variant (e.g., a point mutation, insertion, deletion, substitution, copy number variation (CNV), rearrangement, or fusion) in the subject interval being sequenced; (iv) the site (e.g., nucleotide position) being analyzed; (v) the type of sample (e.g., a sample described herein); and / or (vi) adjacent sequence(s) in or near the subject interval being evaluated (e.g., according to the expected propensity thereof for misalignment of the subject interval due to, e.g., the presence of repeated sequences in or near the subject interval).

[0171] In some instances, the methods disclosed herein allow for the rapid and efficient alignment of troublesome reads, e.g., a read having a rearrangement. Thus, in some instances where a read for a subject interval comprises a nucleotide position with a rearrangement, e.g., a translocation, the method can comprise using an alignment method that is appropriately tuned and that includes: (i) selecting a rearrangement reference sequence for alignment with a read, wherein said rearrangement reference sequence aligns with a rearrangement (in some instances, the reference sequence is not identical to the genomic rearrangement); and (ii) comparing, e.g., aligning, a read with said rearrangement reference sequence.

[0172] In some instances, alternative methods may be used to align troublesome reads. These methods are particularly effective when the alignment of reads for a relatively large number of diverse subject intervals is optimized. By way of example, a method of analyzing a sample can comprise: (i) performing a comparison (e.g., an alignment comparison) of a read using a first set of parameters (e.g., using a first mapping algorithm, or by comparison with a first reference sequence), and determining if said read meets a first alignment criterion (e.g., the read can be aligned with said first reference sequence, e.g., with less than a specific number of mismatches); (ii) if said read fails to meet the first alignment criterion, performing a second alignment comparison using a second set of parameters, (e.g., using a second mapping algorithm, or by comparison with a second reference sequence); and (iii) optionally, determining if said read meets said second criterion (e.g., the read can be aligned with said second reference sequence, e.g., with less than a specific number of mismatches), whereinsaid second set of parameters comprises use of, e.g., said second reference sequence, which, compared with said first set of parameters, is more likely to result in an alignment with a read for a variant (e.g., a rearrangement, insertion, deletion, or translocation).

[0173] In some instances, the alignment of sequence reads in the disclosed methods may be combined with a mutation calling method as described elsewhere herein. As discussed herein, reduced sensitivity for detecting actual mutations may be addressed by evaluating the quality of alignments (manually or in an automated fashion) around expected mutation sites in the genes or genomic loci (e.g., gene loci) being analyzed. In some instances, the sites to be evaluated can be obtained from databases of the human genome (e.g., the HG19 human reference genome) or cancer mutations (e.g., COSMIC). Regions that are identified as problematic can be remedied with the use of an algorithm selected to give better performance in the relevant sequence context, e.g., by alignment optimization (or re-alignment) using slower, but more accurate alignment algorithms such as Smith-Waterman alignment. In cases where general alignment algorithms cannot remedy the problem, customized alignment approaches may be created by, e.g., adjustment of maximum difference mismatch penalty parameters for genes with a high likelihood of containing substitutions; adjusting specific mismatch penalty parameters based on specific mutation types that are common in certain tumor types (e.g. C~^T in melanoma); or adjusting specific mismatch penalty parameters based on specific mutation types that are common in certain sample types (e.g. substitutions that are common in FFPE).

[0174] Reduced specificity (increased false positive rate) in the evaluated subject intervals due to misalignment can be assessed by manual or automated examination of all mutation calls in the sequencing data. Those regions found to be prone to spurious mutation calls due to misalignment can be subjected to alignment remedies as discussed above. In cases where no algorithmic remedy is found possible, “mutations” from the problem regions can be classified or screened out from the panel of targeted loci.

[0175] Base calling refers to the raw output of a sequencing device, e.g., the determined sequence of nucleotides in an oligonucleotide molecule. Mutation calling refers to the process of selecting a nucleotide value, e.g., A, G, T, or C, for a given nucleotide position being sequenced. Typically, the sequence reads (or base calling) for a position will provide more than one value, e.g., some reads will indicate a T and some will indicate a G. Mutation calling is the process of assigning a correct nucleotide value, e.g., one of those values, to the sequence. Although it is referred to as “mutation” calling, it can be applied to assign a nucleotide value to any nucleotide position, e.g., positions corresponding to mutant alleles,wild-type alleles, alleles that have not been characterized as either mutant or wild-type, or to positions not characterized by variability.

[0176] In some instances, the disclosed methods may comprise the use of customized or tuned mutation calling algorithms or parameters thereof to optimize performance when applied to sequencing data, particularly in methods that rely on massively parallel sequencing (MPS) of a large number of diverse genetic events at a large number of diverse genomic loci (e.g., gene loci, microsatellite regions, etc.) in samples, e.g., samples from a subject having cancer. Optimization of mutation calling is described in the art, e.g., as set out in International Publication No. WO2012 / 092426.

[0177] Methods for mutation calling can include one or more of the following: making independent calls based on the information at each position or certain positions in the reference sequence (e.g., examining the sequence reads; examining the base calls and quality scores; calculating the probability of observed bases and quality scores given a potential genotype; and assigning genotypes (e.g., using Bayes’ rule)); removing false positives (e.g., using depth thresholds to reject SNPs with read depth much lower or higher than expected; local realignment to remove false positives due to small indels); and performing linkage disequilibrium (LD) / imputation-based analysis to refine the calls.

[0178] Equations used to calculate the genotype likelihood associated with a specific genotype and position are described in, e.g., Li, H. and Durbin, R. Bioinformatics, 2010; 26(5): 589-95. The prior expectation for a particular mutation in a certain cancer type can be used when evaluating samples from that cancer type. Such likelihood can be derived from public databases of cancer mutations, e.g., Catalogue of Somatic Mutation in Cancer (COSMIC), HGMD (Human Gene Mutation Database), The SNP Consortium, Breast Cancer Mutation Data Base (BIC), and Breast Cancer Gene Database (BCGD).

[0179] Examples of LD / imputation-based analysis are described in, e.g., Browning, B.L. and Yu, Z. Am. J. Hum. Genet. 2009, 85(6):847-61. Examples of low-coverage SNP calling methods are described in, e.g., Li, Y., et al., Annu. Rev. Genomics Hum. Genet. 2009, 10:387- 406.

[0180] After alignment, detection of substitutions can be performed using a mutation calling method (e.g., a Bayesian mutation calling method) which is applied to selected bases or at each base in each of the subject intervals, e.g., exons of a gene or other locus to be evaluated, where presence of alternate alleles is observed. This method will compare the probability of observing the read data in the presence of a mutation with the probability of observing theread data in the presence of base-calling error alone. Mutations can be called if this comparison is sufficiently strongly supportive of the presence of a mutation.

[0181] An advantage of a Bayesian mutation detection approach is that the comparison of the probability of the presence of a mutation with the probability of base-calling error alone can be weighted by a prior expectation of the presence of a mutation at the site. If some reads of an alternate allele are observed at a frequently mutated site for the given cancer type, then presence of a mutation may be confidently called even if the amount of evidence of mutation does not meet the usual thresholds. This flexibility can then be used to increase detection sensitivity for even rarer mutations / lower purity samples, or to make the test more robust to decreases in read coverage. The likelihood of a random base-pair in the genome being mutated in cancer is ~le-6. The likelihood of specific mutations occurring at many sites in, for example, a typical multigenic cancer genome panel can be orders of magnitude higher. These likelihoods can be derived from public databases of cancer mutations (e.g., COSMIC).

[0182] Indel calling is a process of finding bases in the sequencing data that differ from the reference sequence by insertion or deletion, typically including an associated confidence score or statistical evidence metric. Methods of indel calling can include the steps of identifying candidate indels, calculating genotype likelihood through local re-alignment, and performing LD-based genotype inference and calling. Typically, a Bayesian approach is used to obtain potential indel candidates, and then these candidates are tested together with the reference sequence in a Bayesian framework.

[0183] Algorithms to generate candidate indels are described in, e.g., McKenna, A., et al., Genome Res. 2010; 20(9): 1297-303; Ye, K., et al., Bioinformatics, 2009; 25(21):2865-71; Lunter, G., and Goodson, M., Genome Res. 2011; 21(6):936-9; and Li, H., et al. (2009), Bioinformatics 25(16):2078-9.

[0184] Methods for generating indel calls and individual-level genotype likelihoods include, e.g., the Dindel algorithm (Albers, C.A., et al., Genome Res. 2011;21(6):961-73). For example, the Bayesian EM algorithm can be used to analyze the reads, make initial indel calls, and generate genotype likelihoods for each candidate indel, followed by imputation of genotypes using, e.g., QCALL (Le S.Q. and Durbin R. Genome Res. 2011;21(6):952-60). Parameters, such as prior expectations of observing the indel can be adjusted e.g., increased or decreased), based on the size or location of the indels.

[0185] Methods have been developed that address limited deviations from allele frequencies of 50% or 100% for the analysis of cancer DNA. (See, e.g., SNVMix -Bioinformatics. 2010 March 15; 26(6): 730-736.) Methods disclosed herein, however, allow consideration of thepossibility of the presence of a mutant allele at frequencies (or allele fractions) ranging from 1% to 100% (z.e., allele fractions ranging from 0.01 to 1.0), and especially at levels lower than 50%.

[0186] In some instances, the mutation calling method used to analyze sequence reads is not individually customized or fine-tuned for detection of different mutations at different genomic loci. In some instances, different mutation calling methods are used that are individually customized or fine-tuned for at least a subset of the different mutations detected at different genomic loci. In some instances, different mutation calling methods are used that are individually customized or fine-tuned for each different mutant detected at each different genomic loci. The customization or tuning can be based on one or more of the factors described herein, e.g., the type of cancer in a sample, the gene or locus in which the subject interval to be sequenced is located, or the variant to be sequenced. This selection or use of mutation calling methods individually customized or fine-tuned for a number of subject intervals to be sequenced allows for optimization of speed, sensitivity, and specificity of mutation calling.

[0187] In some instances, a nucleotide value is assigned for a nucleotide position in each of X unique subject intervals using a unique mutation calling method, and X is at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, or greater. The calling methods can differ, and thereby be unique, e.g., by relying on different Bayesian prior values.

[0188] In some instances, assigning said nucleotide value is a function of a value which is or represents the prior (e.g., literature) expectation of observing a read showing a variant, e.g., a mutation, at said nucleotide position in a tumor of type.

[0189] In some instances, the method comprises assigning a nucleotide value (e.g., calling a mutation) for at least any of 10, 20, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 nucleotide positions, wherein each assignment is a function of a unique value (as opposed to the value for the other assignments) which is or represents the prior (e.g., literature) expectation of observing a read showing a variant, e.g., a mutation, at said nucleotide position in a tumor of type.

[0190] In some instances, assigning said nucleotide value is a function of a set of values which represent the probabilities of observing a read showing said variant at said nucleotide position if the variant is present in the sample at a specified frequency (e.g., 1%, 5%, 10%,etc.) and / or if the variant is absent (e.g., observed in the reads due to base-calling error alone).

[0191] In some instances, the mutation calling methods described herein can include the following: (a) acquiring, for a nucleotide position in each of said X subject intervals: (i) a first value which is or represents the prior (e.g., literature) expectation of observing a read showing a variant, e.g., a mutation, at said nucleotide position in a tumor of type X; and (ii) a second set of values which represent the probabilities of observing a read showing said variant at said nucleotide position if the variant is present in the sample at a frequency (e.g., 1%, 5%, 10%, etc.) and / or if the variant is absent (e.g., observed in the reads due to basecalling error alone); and (b) responsive to said values, assigning a nucleotide value (e.g., calling a mutation) from said reads for each of said nucleotide positions by weighing, e.g., by a Bayesian method described herein, the comparison among the values in the second set using the first value (e.g., computing the posterior probability of the presence of a mutation), thereby analyzing said sample.

[0192] Additional description of exemplary nucleic acid sequencing methods, mutation calling methods, and methods for analysis of genetic variants is provided in, e.g., U.S. Patent No. 9,340,830, U.S. Patent No. 9,792,403, U.S. Patent No. 11,136,619, U.S. Patent No. 11,118,213, and International Publication No. WO2020 / 236941.Methods of Use

[0193] Described below are methods of using the detection of clonal hematopoietic and / or germline mutation(s) for constructing a molecular profile and / or for diagnosing, treating, evaluating, or monitoring a subject (e.g., a patient) for a disease (e.g., cancer) or CHIP as described herein.

[0194] In some embodiments, the methods of the present disclosure further comprise generating a molecular profile for the individual, based, at least in part, on detecting the mutation in the one or more genes. In some embodiments, the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof. In some embodiments, the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test.

[0195] A comprehensive genomic profiling (CGP) is a next-generation sequencing (NGS) approach used to assess hundreds of genes (including relevant cancer biomarkers) in a singleassay. A gene expression profiling test provides information about all of the genes in a cell or tissue that are making messenger RNA and may be used to help diagnose a disease or condition, such as cancer; this test may also be used to help plan treatment, find out how well treatment is working, make a prognosis, or predict whether the cancer will relapse or metastasize. A cancer hotspot panel test assesses for the presence of specific common cancer mutations at specific gene loci. A DNA methylation test (for example, the Luminometric Methylation Assay) examines the methylation patterns (e.g., absolute or relative) of DNA isolated from the subject that may impact biochemical processes that are indicative of cancer development, progression, metastasis, etc. A DNA or RNA fragmentation test examines, for example, the fragmentation patterns of cell-free DNA or RNA or circulating tumor DNA or RNA (e.g., by a Fragment Analyzer), which may be altered in cancer. For example, DNA fragmentation is not evenly distributed in the genome such that the genomic location(s) and the types and / or features of DNA fragments may be indicative of specific diseases, e.g., cancers.

[0196] In some embodiments, the molecular profile for the individual further comprises results from a nucleic acid sequencing-based test. In some embodiments, the results from a nucleic acid sequencing-based test is compiled into a genomic profile. In some embodiments, the genomic profile may comprise information on the presence of genes (or variant sequences thereof), copy number variations, epigenetic traits, proteins (or modifications thereof), and / or other biomarkers in an individual’s genome and / or proteome, as well as information on the individual’s corresponding phenotypic traits and the interaction between genetic or genomic traits, phenotypic traits, and environmental factors.

[0197] In some embodiments, the disclosed methods for determining the presence of clonal hematopoietic and / or germline gene mutation(s) may be implemented as part of a genomic profiling process that comprises identification of the presence of one or more mutations at one or more gene loci in a nucleic acid library derived from a liquid sample from a subject (e.g., a human patient) as part of detecting, monitoring, predicting a risk factor, or selecting a treatment for a particular disease, e.g., cancer, or CHIP. In some instances, the variant panel selected for genomic profiling may comprise the detection of variant sequences at a selected set of gene loci. In some instances, the variant panel selected for genomic profiling may comprise detection of variant sequences at a number of gene loci through comprehensive genomic profiling (CGP), which is a next-generation sequencing (NGS) approach used to assess hundreds of genes (including relevant cancer biomarkers) in a single assay. Inclusion of the disclosed methods for determining the presence of clonal hematopoietic and / orgermline gene mutation(s) as part of a genomic profiling process (or inclusion of the output from the disclosed methods as part of the genomic profile of the subject) can improve the validity of, e.g., disease detection calls and treatment decisions, made on the basis of the genomic profile by, for example, independently confirming the presence of clonal hematopoietic and / or germline gene mutation(s) in a given patient sample and filtering out the mutation if the mutation is determined to be a CH or germline gene mutation.

[0198] In some embodiments, the method further comprises selecting a treatment, administering a treatment, or applying a treatment to the individual based at least in part on the generated molecular profile.

[0199] In some embodiments, the method further comprises generating a report indicating the presence or absence of the one or more mutations in a sample or mixture of nucleic acid molecules of the present disclosure.

[0200] In some embodiments, the method further comprises transmitting the report to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office. In some embodiments, the report is transmitted via a computer network or a peer-to-peer connection. In some instances, all or a portion of the report may be displayed in a graphical user interface of an online or web-based healthcare portal. In some embodiments, the report is provided or delivered to the individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from obtaining a sample from the individual. In some embodiments, the report is provided or delivered to an individual or entity within any of about 1 day or more, about 7 days or more, about 14 days or more, about 21 days or more, about 30 days or more, about 45 days or more, or about 60 days or more from detecting a CH or germline mutation.

[0201] In some embodiments, a report generated according to the methods provided herein comprises one or more of: information about the presence or absence of a mutation detected in the sample(s); an identifier for the individual from which the sample(s) was obtained; information on the role of the mutation in disease (e.g., such as in cancer); information on prognosis, resistance, or potential or suggested therapeutic options (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein); information on the likely effectiveness, acceptability, or the advisability of applying a therapeutic option (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein) to the individual; arecommendation or information on the administration of a treatment (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein); or a recommendation or information on the dosage or treatment regimen of a treatment (such as an anti-cancer therapy provided herein, or a treatment selected or identified according to the methods provided herein), e.g., in combination with other treatments (e.g., a second therapeutic agent). In some embodiments, the report generated is a personalized cancer report.

[0202] In another aspect is provided a method as described herein that further comprises diagnosing a subject with a disease (e.g., cancer) or CHIP, recommending a treatment, selecting a treatment, administering a treatment, and / or evaluating the efficacy of a treatment for a subject with a disease (e.g., cancer) or CHIP. In some embodiments, the determination and filtering out of clonal hematopoietic and / or germline mutation(s) using the disclosed methods may be used as a prognostic or diagnostic indicator associated with the sample. For example, in some instances, the prognostic or diagnostic indicator may comprise an indicator of the presence of a disease (e.g., cancer) in the sample, an indicator of the probability that a disease (e.g., cancer) is present in the sample, an indicator of the probability that the subject from which the sample was derived will develop a disease (e.g., cancer) (i.e., a risk factor), or an indicator of the likelihood that the subject from which the sample was derived will respond to a particular therapy or treatment.

[0203] In some embodiments, the method further comprises recommending an anti-cancer therapy for the individual based, at least in part, on detecting the mutation in the one or more genes. An anti-cancer agent or anti-cancer treatment may refer to a compound that is effective in the treatment of cancer cells. Examples of anti-cancer agents or anti-cancer therapies include, but not limited to, alkylating agents, antimetabolites, natural products, hormones, chemotherapy, radiation therapy, immunotherapy, surgery, or a therapy configured to target a defect in a specific cell signaling pathway, e.g., a defect in a DNA mismatch repair (MMR) pathway.

[0204] In some embodiments, the method further comprises selecting an anti-cancer therapy for the individual based, at least in part, on detecting and filtering out the one or more mutations in the one or more genes.

[0205] In some embodiments, the method further comprises generating a report comprising one or more anti-cancer therapies identified for the individual based, at least in part, on detecting and filtering out the one or more mutations in the one or more genes.

[0206] In some embodiments, the method further comprises administering an anti-cancer therapy to the individual based, at least in part, on detecting and filtering out the one or more mutations in the one or more genes.

[0207] In some embodiments, the method further comprises determining an effective amount of the anti-cancer therapy to administer to the individual based, at least in part, on detecting and filtering out the one or more mutations in the one or more genes.

[0208] In some embodiments, the anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti- angiogenic therapy, an anti- DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof. In some embodiments, the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)- based therapy. In some embodiments, the nucleic acid comprises a double- stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In some embodiments, the anti-cancer therapy comprises chemotherapy, radiation therapy, immunotherapy, a targeted therapy, or surgery. In some embodiments, the anti-cancer therapy comprises a targeted anti-cancer therapy.

[0209] In some embodiments, the anti-cancer therapy comprises abemaciclib (Verzenio), abiraterone acetate (Zytiga), acalabrutinib (Calquence), ado-trastuzumab emtansine (Kadcyla), afatinib dimaleate (Gilotrif), aldesleukin (Proleukin), alectinib (Alecensa), alemtuzumab (Campath), alitretinoin (Panretin), alpelisib (Piqray), amivantamab-vmjw (Rybrevant), anastrozole (Arimidex), apalutamide (Erleada), asciminib hydrochloride (Scemblix), atezolizumab (Tecentriq), avapritinib (Ayvakit), avelumab (Bavencio), axicabtagene ciloleucel (Yescarta), axitinib (Inlyta), belantamab mafodotin-blmf (Blenrep), belimumab (Benlysta), belinostat (Beleodaq), belzutifan (Welireg), bevacizumab (Avastin), bexarotene (Targretin), binimetinib (Mektovi), blinatumomab (Blincyto), bortezomib (Velcade), bosutinib (Bosulif), brentuximab vedotin (Adcetris), brexucabtagene autoleucel (Tecartus), brigatinib (Alunbrig), cabazitaxel (Jevtana), cabozantinib (Cabometyx), cabozantinib (Cabometyx, Cometriq), canakinumab (Haris), capmatinib hydrochloride(Tabrecta), carfilzomib (Kyprolis), cemiplimab-rwlc (Libtayo), ceritinib (LDK378 / Zykadia), cetuximab (Erbitux), cobimetinib (Cotellic), copanlisib hydrochloride (Aliqopa), crizotinib (Xalkori), dabrafenib (Tafinlar), dacomitinib (Vizimpro), daratumumab (Darzalex), daratumumab and hyaluronidase-fihj (Darzalex Faspro), darolutamide (Nubeqa), dasatinib (Sprycel), denileukin diftitox (Ontak), denosumab (Xgeva), dinutuximab (Unituxin), dostarlimab-gxly (Jemperli), durvalumab (Imfinzi), duvelisib (Copiktra), elotuzumab (Empliciti), enasidenib mesylate (Idhifa), encorafenib (Braftovi), enfortumab vedotin-ejfv (Padcev), entrectinib (Rozlytrek), enzalutamide (Xtandi), erdafitinib (Balversa), erlotinib (Tarceva), everolimus (Afinitor), exemestane (Aromasin), fam-trastuzumab deruxtecan-nxki (Enhertu), fedratinib hydrochloride (Inrebic), fulvestrant (Faslodex), gefitinib (Iressa), gemtuzumab ozogamicin (Mylotarg), gilteritinib (Xospata), glasdegib maleate (Daurismo), hyaluronidase-zzxf (Phesgo), ibrutinib (Imbruvica), ibritumomab tiuxetan (Zevalin), idecabtagene vicleucel (Abecma), idelalisib (Zydelig), imatinib mesylate (Gleevec), infigratinib phosphate (Truseltiq), inotuzumab ozogamicin (Besponsa), iobenguane 1131 (Azedra), ipilimumab (Yervoy), isatuximab-irfc (Sarclisa), ivosidenib (Tibsovo), ixazomib citrate (Ninlaro), lanreotide acetate (Somatuline Depot), lapatinib (Tykerb), larotrectinib sulfate (Vitrakvi), lenvatinib mesylate (Lenvima), letrozole (Femara), lisocabtagene maraleucel (Breyanzi), loncastuximab tesirine-lpyl (Zynlonta), lorlatinib (Lorbrena), lutetium Lu 177-dotatate (Lutathera), margetuximab-cmkb (Margenza), midostaurin (Rydapt), mobocertinib succinate (Exkivity), mogamulizumab-kpkc (Poteligeo), moxetumomab pasudotox-tdfk (Lumoxiti), naxitamab-gqgk (Danyelza), necitumumab (Portrazza), neratinib maleate (Nerlynx), nilotinib (Tasigna), niraparib tosylate monohydrate (Zejula), nivolumab (Opdivo), obinutuzumab (Gazyva), ofatumumab (Arzerra), olaparib (Lynparza), olaratumab (Lartruvo), osimertinib (Tagrisso), palbociclib (Ibrance), panitumumab (Vectibix), panobinostat (Farydak), pazopanib (Votrient), pembrolizumab (Keytruda), pemigatinib (Pemazyre), pertuzumab (Perjeta), pexidartinib hydrochloride (Turalio), polatuzumab vedotin-piiq (Polivy), ponatinib hydrochloride (Iclusig), pralatrexate (Folotyn), pralsetinib (Gavreto), radium 223 dichloride (Xofigo), ramucirumab (Cyramza), regorafenib (Stivarga), ribociclib (Kisqali), ripretinib (Qinlock), rituximab (Rituxan), rituximab and hyaluronidase human (Rituxan Hycela), romidepsin (Istodax), rucaparib camsylate (Rubraca), ruxolitinib phosphate (Jakafi), sacituzumab govitecan-hziy (Trodelvy), seliciclib, selinexor (Xpovio), selpercatinib (Retevmo), selumetinib sulfate (Koselugo), siltuximab (Sylvant), sipuleucel-T (Provenge), sirolimus protein-bound particles (Fyarro), sonidegib (Odomzo), sorafenib (Nexavar), sotorasib (Lumakras), sunitinib (Sutent), tafasitamab-cxix (Monjuvi),tagraxofusp-erzs (Elzonris), talazoparib tosylate (Talzenna), tamoxifen (Nolvadex), tazemetostat hydrobromide (Tazverik), tebentafusp-tebn (Kimmtrak), temsirolimus (Torisel), tepotinib hydrochloride (Tepmetko), tisagenlecleucel (Kymriah), tisotumab vedotin-tftv (Tivdak), tocilizumab (Actemra), tofacitinib (Xeljanz), tositumomab (Bexxar), trametinib (Mekinist), trastuzumab (Herceptin), tretinoin (Vesanoid), tivozanib hydrochloride (Fotivda), toremifene (Fareston), tucatinib (Tukysa), umbralisib tosylate (Ukoniq), vandetanib (Caprelsa), vemurafenib (Zelboraf), venetoclax (Venclexta), vismodegib (Erivedge), vorinostat (Zolinza), zanubrutinib (Brukinsa), ziv-aflibercept (Zaltrap), or any combination thereof.

[0210] In some embodiments, the method further comprises monitoring disease progression or recurrence (e.g., cancer or tumor progression or recurrence) in a subject. For example, in some instances, the method may be used to determine and filter out clonal hematopoietic and / or germline gene mutation(s) in a first liquid sample obtained from the subject (e.g., human patient) at a first time point to diagnose the subject with a disease (e.g., cancer) or CHIP, and then may be subsequently used to determine and filter out clonal hematopoietic and / or germline gene mutation(s) in a second liquid sample obtained from the subject at a second time point, where comparison of the first and the second determination and filtering out of clonal hematopoietic and / or germline gene mutation(s) allows one to monitor disease progression or recurrence. In some instances, the first time point is chosen before the subject has been administered a therapy or treatment, and the second time point is chosen after the subject has been administered the therapy or treatment.

[0211] In some embodiments, the disclosed methods may be used for adjusting a therapy or treatment (e.g., an anti-cancer treatment or anti-cancer therapy) for a subject, e.g., by adjusting a treatment dose and / or selecting a different treatment in response to a change in the determination and filtering out of clonal hematopoietic and / or germline gene mutation(s).

[0212] In some embodiments, the disclosed methods may be used to select a subject (e.g., a patient) for a clinical trial based on the presence of clonal hematopoietic and / or germline mutation(s) determined for one or more gene loci. In some instances, patient selection for clinical trials based on, e.g., identification of clonal hematopoietic and / or germline mutation(s) at one or more gene loci, may accelerate the development of targeted therapies and improve the healthcare outcomes for treatment decisions.Systems and Computer- Readable Storage Media

[0213] Further described herein are systems for detecting one or more clonal hematopoietic (CH) or germline gene mutations according to any one of the methods described herein.

[0214] An exemplary system includes, for example, one or more processors and a memory unit communicatively coupled to the one or more processors and configured to store instructions that, when executed by the one or more processors, cause the system to: obtain a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA); analyze the plurality of sequence reads for the presence of a mutation in one or more genes; and detect, based on the analyzing, the mutation in the one or more genes in the sample, thereby detecting a clonal hematopoietic (CH) or germline gene mutation.

[0215] In some embodiments, the disclosed systems may be used for detecting and filtering out clonal hematopoietic and / or germline gene mutations in any of a variety of samples as described herein (e.g., a hematological sample or liquid biopsy sample derived from the subject).

[0216] In some embodiments, the plurality of gene loci for which sequencing data is processed to determine clonal hematopoietic or germline mutations may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 gene loci.

[0217] In some embodiments, the nucleic acid sequence data is acquired using a next generation sequencing technique (also referred to as a massively parallel sequencing technique) having a read-length of less than about 400 bases, less than about 300 bases, less than about 200 bases, less than about 150 bases, less than about 100 bases, less than about 90 bases, less than about 80 bases, less than about 70 bases, less than about 60 bases, less than about 50 bases, less than about 40 bases, or less than about 30 bases.

[0218] In some embodiments, the disclosed systems may further comprise sample processing and library preparation workstations, microplate-handling robotics, fluid dispensing systems, temperature control modules, environmental control chambers, additional data storage modules, data communication modules (e.g., Bluetooth®, WiFi, intranet, or internet communication hardware and associated software), display modules, one or more local and / or cloud-based software packages (e.g., instrument / system control software packages,sequencing data analysis software packages), etc., or any combination thereof. In some embodiments, the systems may comprise, or be part of, a computer system or computer network as described elsewhere herein.

[0219] In another aspect, provided herein is a non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, e.g., a method according to any of the methods disclosed herein. For example, in some embodiments, the method comprises obtaining (e.g., using one or more processors) a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA); analyzing (e.g., using one or more processors) the plurality of sequence reads for the presence of a mutation in one or more genes; and detecting, based on the analyzing (e.g., using one or more processors), the mutation in the one or more genes, thereby detecting a clonal hematopoietic (CH) or germline gene mutation.

[0220] In some embodiments, the plurality of sequence reads is obtained by sequencing, e.g., using a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique. In some embodiments, the massively parallel sequencing technique comprises next generation sequencing (NGS).

[0221] In some embodiments, the disclosed systems may further comprise a sequencer, e.g., a next generation sequencer (also referred to as a massively parallel sequencer). Examples of next generation (or massively parallel) sequencing platforms include, but are not limited to, Roche / 454’s Genome Sequencer (GS) FLX system, Illumina / Solexa’ s Genome Analyzer (GA), Illumina’s HiSeq® 2500, HiSeq® 3000, HiSeq® 4000 and NovaSeq® 6000 sequencing systems, Life / APG’s Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator’s G.007 system, Helicos BioSciences’ HeliScope Gene Sequencing system, ThermoFisher Scientific’s Ion Torrent Genexus system, or Pacific Biosciences’ PacBio® RS system.

[0222] In some embodiments, the one or more program instructions when executed by the one or more processors are further configured to generate, based at least in part on thedetecting, a molecular profile for a sample from which the mixture of nucleic acid molecules was obtained. In some embodiments, the sample is a liquid biopsy sample.

[0223] In some embodiments, the sample is obtained from an individual. In some embodiments, the individual is administered a treatment based at least in part on the molecular profile. In some embodiments, the molecular profile further comprises (a) results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof; and / or (b) results from a nucleic acid sequencing-based test.

[0224] FIG. 5 illustrates an example of a computing device or system in accordance with one embodiment. Device 500 can be a host computer connected to a network. Device 500 can be a client computer or a server. As shown in FIG. 5, device 500 can be any suitable type of microprocessor-based device, such as a personal computer, workstation, server or handheld computing device (portable electronic device) such as a phone or tablet. The device can include, for example, one or more processor(s) 510, input devices 520, output devices 530, memory or storage devices 540, communication devices 560, and nucleic acid sequencers 570. Software module 550 residing in memory or storage device 540 may comprise, e.g., an operating system as well as software for executing the methods described herein. Input device 520 and output device 530 can generally correspond to those described herein and can either be connectable or integrated with the computer.

[0225] Input device 520 can be any suitable device that provides input, such as a touch screen, keyboard or keypad, mouse, or voice-recognition device. Output device 530 can be any suitable device that provides output, such as a touch screen, haptics device, or speaker.

[0226] Storage 540 can be any suitable device that provides storage (e.g., an electrical, magnetic, or optical memory including a RAM (volatile and non-volatile), cache, hard drive, or removable storage disk). Communication device 560 can include any suitable device capable of transmitting and receiving signals over a network, such as a network interface chip or device. The components of the computer can be connected in any suitable manner, such as via a wired media (e.g., a physical system bus 580, Ethernet connection, or any other wire transfer technology) or wirelessly (e.g., Bluetooth®, Wi-Fi®, or any other wireless technology).

[0227] Software module 550, which can be stored as executable instructions in storage 540 and executed by processor(s) 510, can include, for example, an operating system and / or theprocesses that embody the functionality of the methods of the present disclosure e.g., as embodied in the devices as described herein).

[0228] Software module 550 can also be stored and / or transported within any non-transitory computer-readable storage medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described herein, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a computer-readable storage medium can be any medium, such as storage 540, that can contain or store processes for use by or in connection with an instruction execution system, apparatus, or device. Examples of computer-readable storage media may include memory units like hard drives, flash drives and distribute modules that operate as a single functional unit. Also, various processes described herein may be embodied as modules configured to operate in accordance with the embodiments and techniques described above. Further, while processes may be shown and / or described separately, those skilled in the art will appreciate that the above processes may be routines or modules within other processes.

[0229] Software module 550 can also be propagated within any transport medium for use by or in connection with an instruction execution system, apparatus, or device, such as those described above, that can fetch instructions associated with the software from the instruction execution system, apparatus, or device and execute the instructions. In the context of this disclosure, a transport medium can be any medium that can communicate, propagate, or transport programming for use by or in connection with an instruction execution system, apparatus, or device. The transport readable medium can include, but is not limited to, an electronic, magnetic, optical, electromagnetic, or infrared wired or wireless propagation medium.

[0230] Device 500 may be connected to a network (e.g., network 604, as shown in FIG. 6 and / or described below), which can be any suitable type of interconnected communication system. The network can implement any suitable communications protocol and can be secured by any suitable security protocol. The network can comprise network links of any suitable arrangement that can implement the transmission and reception of network signals, such as wireless network connections, T1 or T3 lines, cable networks, DSL, or telephone lines.

[0231] Device 500 can be implemented using any operating system, e.g., an operating system suitable for operating on the network. Software module 550 can be written in any suitable programming language, such as C, C++, Java, or Python. In various embodiments,application software embodying the functionality of the present disclosure can be deployed in different configurations, such as in a client / server arrangement or through a Web browser as a Web-based application or Web service, for example. In some embodiments, the operating system is executed by one or more processors, e.g., processor(s) 510.

[0232] Device 500 can further include a sequencer 570, which can be any suitable nucleic acid sequencing instrument.

[0233] FIG. 6 illustrates an example of a computing system in accordance with one embodiment. In system 600, device 500 (e.g., as described above and illustrated in FIG. 5) is connected to network 604, which is also connected to device 606. In some embodiments, device 606 is a sequencer. Exemplary sequencers can include, without limitation, Roche / 454’s Genome Sequencer (GS) FLX System, Illumina / Solexa’s Genome Analyzer (GA), Illumina’s HiSeq® 2500, HiSeq® 3000, HiSeq® 4000 and NovaSeq® 6000 Sequencing Systems, Life / APG’s Support Oligonucleotide Ligation Detection (SOLiD) system, Polonator’s G.007 system, Helicos BioSciences’ HeliScope Gene Sequencing system, or Pacific Biosciences’ PacBio® RS system.

[0234] Devices 500 and 606 may communicate, e.g., using suitable communication interfaces via network 604, such as a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet. In some embodiments, network 604 can be, for example, the Internet, an intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Devices 500 and 606 may communicate, in part or in whole, via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. Additionally, devices 500 and 606 may communicate, e.g., using suitable communication interfaces, via a second network, such as a mobile / cellular network. Communication between devices 500 and 606 may further include or communicate with various servers such as a mail server, mobile server, media server, telephone server, and the like. In some embodiments, Devices 500 and 606 can communicate directly (instead of, or in addition to, communicating via network 604), e.g., via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. In some embodiments, devices 500 and 606 communicate via communications 608, which can be a direct connection or can occur via a network (e.g., network 604).

[0235] One or all of devices 500 and 606 generally include logic (e.g., http web server logic) or are programmed to format data, accessed from local or remote databases or other sources of data and content, for providing and / or receiving information via network 604 according to various examples described herein.

[0236] FIGS. 7A & 7B provide a non-limiting example of a process flowchart (process 700) for detecting one or more CH or germline mutations in accordance with some embodiments. As shown in FIG. 7A, process 700 begins at 702 with performing a size selection on a mixture of nucleic acid molecules of the present disclosure, e.g., comprising a first plurality of nucleic acid molecules (e.g., comprising nucleic acid molecules from nucleated cells such as bcDNA) and a second plurality of nucleic acid molecules (e.g., comprising nucleic acid molecules from cfDNA or ctDNA). As described herein, the size selection comprises separating the first plurality of nucleic acid molecules of the mixture (e.g., bcDNA) from the second plurality of nucleic acid molecules of the mixture (e.g., cfDNA or ctDNA) to generate a size-selected plurality of nucleic acid molecules of the present disclosure. Methods for size selection are disclosed herein. For example, in some embodiments, the size selection comprises contacting the mixture of nucleic acid molecules with SPRI beads, e.g., using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0 or between about 0.4 and about 0.6 to generate the size-selected plurality of nucleic acid molecules.

[0237] At 704, process 700 optionally comprises ligating one or more adapters onto nucleic acid molecules in the size- selected plurality of nucleic acid molecules, thereby generating ligated nucleic acids. In some embodiments, the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.

[0238] At 706, process 700 optionally comprises amplifying nucleic acids from the ligated nucleic acids.

[0239] At 708, process 700 optionally comprises capturing or enriching from the amplified nucleic acids a plurality of nucleic acids comprising nucleotide sequences corresponding to one or more genes. For example, the enriching can comprise combining one or more bait molecules with the amplified nucleic acids and hybridizing the one or more bait molecules to nucleic acids comprising nucleotide sequences corresponding to the one or more genes to produce nucleic acid hybrids.

[0240] At 710, process 700 comprises sequencing (e.g., by a sequencer) the plurality of nucleic acids to obtain a plurality of sequence reads that represent the captured nucleic acids. A variety of sequencing techniques are disclosed herein and contemplated for use in the methods of the present disclosure. As shown in FIG. 7B, at 712, process 700 comprises obtaining (e.g., by one or more processors) a plurality of sequence reads that represent thecaptured nucleic acids, wherein the plurality of sequence reads correspond to one or more genes.

[0241] At 714, process 700 comprises analyzing (e.g., by one or more processors) the plurality of sequence reads for the presence or absence of a mutation in the one or more genes.

[0242] At 716, process 700 comprises detecting (e.g., by one or more processors) a mutation in the one or more genes, e.g., based on the detecting at 714. For example, 716 can optionally comprise analyzing a mutation in one or more genes detected in the size-selected plurality of nucleic acid molecules, analyzing a mutation in one or more genes detected in cfDNA (e.g., separated from the bcDNA during size selection, from plasma, or from a different sample), and / or determining that a detected mutation is a CH or germline mutation when it is represented in the size-selected plurality of nucleic acid molecules.Exemplary Embodiments

[0243] The following exemplary embodiments are representative of some aspects of the invention:Embodiment 1. A method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising: detecting a mutation in one or more genes from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), thereby determining that the detected mutation is a CH or germline mutation.Embodiment 2. The method of embodiment 1, further comprising, prior to the detection, performing a size selection on the mixture of nucleic acid molecules, wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture.Embodiment 3. A method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising:performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules; and detecting, by one or more processors, a mutation in one or more genes in the size- selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.Embodiment 4. The method of embodiment 3, further comprising, after performing the size selection: detecting, by the one or more processors, a mutation in one or more genes in the second plurality of nucleic acid molecules.Embodiment 5. The method of embodiment 4, further comprising: analyzing a mutation in one or more genes detected in the size-selected plurality of nucleic acid molecules; and analyzing a mutation in one or more genes detected in the second plurality of nucleic acid molecules.Embodiment 6. The method of embodiment 5, further comprising, based on the analyzing: identifying a mutation in one or more genes as being from cfDNA when it is represented in the second plurality of nucleic acid molecules but not the size-selected plurality of nucleic acid molecules.Embodiment 7. The method of embodiment 3, further comprising detecting, by one or more processors, a mutation in one or more genes in a plurality of cfDNA molecules.Embodiment 8. The method of embodiment 7, further comprising: analyzing a mutation in one or more genes detected in the size-selected plurality of nucleic acid molecules; andanalyzing a mutation in one or more genes detected in the plurality of cfDNA molecules.Embodiment 9. The method of embodiment 5 or embodiment 8, further comprising, based on the analyzing: identifying a mutation in one or more genes as a CH or germline mutation when it is represented in the size-selected plurality of nucleic acid molecules.Embodiment 10. A method of detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising detecting a mutation in one or more genes by:(a) performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules;(b) optionally, ligating one or more adapters onto nucleic acid molecules in the size- selected plurality of nucleic acid molecules, thereby generating ligated nucleic acid molecules;(c) optionally, amplifying the ligated nucleic acid molecules;(d) optionally, capturing, from the amplified nucleic acid molecules, nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes;(e) sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the one or more genes;(f) analyzing, by one or more processors, the plurality of sequence reads; and(g) based on the analysis, detecting a mutation in the one or more genes, thereby determining that the detected mutation is a CH or germline mutation.Embodiment 11. The method of embodiment 10, further comprising receiving, at one or more processors, sequence read data for the plurality of sequence reads.Embodiment 12. The method of embodiment 11, wherein the analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes.Embodiment 13. The method of any one of embodiments 10-12, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules.Embodiment 14. The method of any one of embodiments 10-13, wherein the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.Embodiment 15. The method of any one of embodiments 10-14, wherein the sequencer comprises a next generation sequencer.Embodiment 16. The method of any one of embodiments 10-15, further comprising, after performing the size selection and prior to adapter ligation: subjecting the size-selected plurality of nucleic acid molecules to fragmentation.Embodiment 17. A method of detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising:(a) performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules;(b) preparing a nucleic acid sequencing library from the size- selected plurality of nucleic acid molecules;(c) amplifying said library;(d) selectively enriching for a plurality of nucleic acid molecules from the amplified library, said plurality of nucleic acid molecules comprising nucleotide sequence(s) corresponding to the one or more genes, thereby producing an enriched plurality of nucleic acid molecules;(e) sequencing, by a sequencer, the enriched plurality of nucleic acid molecules, thereby producing a plurality of sequence reads;(f) analyzing, by one or more processors, the plurality of sequence reads for presence or absence of a mutation in the one or more genes; and(g) detecting, based on the analysis, presence or absence of a mutation in the one or more genes,thereby determining that the detected mutation is a CH or germline mutation.Embodiment 18. The method of embodiment 17, wherein the selective enrichment comprises: combining one or more bait molecules with the amplified library, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules.Embodiment 19. The method of embodiment 17 or embodiment 18, further comprising receiving, at the one or more processors, sequence read data for the plurality of sequence reads.Embodiment 20. The method of embodiment 19, wherein analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes.Embodiment 21. The method of any one of embodiments 10-20, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non- PCR amplification technique, or an isothermal amplification technique.Embodiment 22. The method of any one of embodiments 10-21, wherein the sequencer implements a massively parallel sequencing (MPS) technique, a whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique.Embodiment 23. The method of embodiment 22, wherein the sequencer implements a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS).Embodiment 24. The method of any one of embodiments 1-23, wherein the size selection separates substantially all nucleic acid molecules of the second plurality of nucleic acid molecules from the first plurality of nucleic acid molecules.Embodiment 25. The method of any one of embodiments 1-24, wherein the size selection is performed using one or more of: gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), DNA chromatography, DNA precipitation followed by centrifugation, spin column-based DNA purification, ion exchange chromatography, or solid-phase reversible immobilization (SPRI).Embodiment 26. The method of embodiment 25, wherein the gel electrophoresis comprises use of agarose or polyacrylamide gel electrophoresis (PAGE).Embodiment 27. The method of embodiment 25, wherein the HPLC is ion-pair reversed-phase HPLC (IP RP HPLC).Embodiment 28. The method of embodiment 25, wherein the SPRI comprises use of SPRI beads.Embodiment 29. The method of embodiment 28, wherein the size selection comprises contacting the mixture of nucleic acid molecules with the SPRI beads.Embodiment 30. A method for detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising:(a) performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), wherein the size selection comprises contacting the mixture of nucleic acid molecules with solid-phase reversible immobilization (SPRI) beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0, thereby generating a size-selected plurality of nucleic acid molecules; and(b) detecting a mutation in one or more genes in the size- selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.Embodiment 31. The method of any one of embodiments 28-30, wherein the SPRI beads comprise paramagnetic SPRI beads.Embodiment 32. The method of embodiment 31, wherein the size selection comprises contacting the mixture of nucleic acid molecules with the SPRI beads using a ratio of volumeof beads to volume of nucleic acid molecules of between about 0.4 and about 0.6, or between about 0.45 and about 0.55.Embodiment 33. The method of embodiment 32, wherein the ratio of volume of beads to volume of nucleic acid molecules is any of about 0.4, about 0.45, about 0.5, about 0.55, or about 0.6.Embodiment 34. The method of embodiment 33, wherein the ratio of volume of beads to volume of nucleic acid molecules is about 0.5.Embodiment 35. The method of any one of embodiments 28-34, wherein the size selection comprises contacting a volume of about 55p L of the mixture of nucleic acid molecules with a volume of about 27.5 pL of SPRI beads.Embodiment 36. The method of any one of embodiments 29-35, further comprising, after contacting the mixture of nucleic acid molecules with the SPRI beads: washing the mixture of nucleic acid molecules under conditions in which the mixture of nucleic acid molecules is bound to the SPRI beads; and eluting the size- selected plurality of nucleic acid molecules from the SPRI beads.Embodiment 37. The method of any one of embodiments 1-36, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more, about 5000 base pairs or more, about 10,000 base pairs or more, about 15,000 base pairs or more, about 20,000 base pairs or more, about 25,000 base pairs or more, about 30,000 base pairs or more, about 35,000 base pairs or more, about 40,000 base pairs or more, about 45,000 base pairs or more, about 50,000 base pairs or more, about 55,000 base pairs or more, about 60,000 base pairs or more, about 65,000 base pairs or more, about 70,000 base pairs or more, about 75,000 base pairs or more, about 80,000 base pairs or more, about 85,000 base pairs or more, about 90,000 base pairs or more, about 95,000 base pairs or more, or about 100,000 base pairs or more.Embodiment 38. The method of embodiment 37, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of up to about 200,000 base pairs.Embodiment 39. The method of embodiment 37 or embodiment 38, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more.Embodiment 40. The method of embodiment 39, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 1000 base pairs and about 200,000 base pairs.Embodiment 41. The method of embodiment 37 or embodiment 38, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 50,000 base pairs or more.Embodiment 42. The method of embodiment 41, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs.Embodiment 43. The method of any one of embodiments 1-42, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than about 1000 base pairs.Embodiment 44. The method of embodiment 43, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 999 base pairs or less, about 900 base pairs or less, about 800 base pairs or less, about 700 base pairs or less, about 600 base pairs or less, about 500 base pairs or less, about 400 base pairs or less, about 300 base pairs or less, about 200 base pairs or less, or about 100 base pairs or less.Embodiment 45. The method of embodiment 43 or embodiment 44, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 50 base pairs or more, about 75 base pairs or more, about 100 base pairs or more, about 125 base pairs or more, about 150 base pairs or more, about 175 base pairs or more, about 200 base pairs or more, about 225 base pairs or more, about 250 base pairs or more, about 275 base pairs or more, about 300 base pairs or more, about 325 base pairs or more, about 350 base pairs or more, about 375 base pairs or more, about 400 base pairs or more, about 425 base pairs or more, about 450 base pairs or more, about 475 base pairs or more, or about 500 base pairs or more.Embodiment 46. The method of any one of embodiments 43-45, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and about 1000 base pairs.Embodiment 47. The method of embodiment 46, wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 100 base pairs and about 300 base pairs.Embodiment 48. The method of any one of embodiments 1-36, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between 1000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than 1000 base pairs.Embodiment 49. The method of any one of embodiments 1-36, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than 1000 base pairs.Embodiment 50. The method of any one of embodiments 1-36, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between 1000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and 1000 base pairs.Embodiment 51. The method of any one of embodiments 1-36, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and about 1000 base pairs.Embodiment 52. The method of any one of embodiments 1-51, wherein the mutation in the one or more genes is detected by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situhybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence- specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing.Embodiment 53. The method of embodiment 52, wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique, optionally, wherein the massively parallel sequencing (MPS) technique comprises nextgeneration sequencing (NGS).Embodiment 54. The method of any one of embodiments 1-15 and 18-53, further comprising selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size- selected plurality of nucleic acid molecules, thereby producing an enriched plurality of nucleic acid molecules.Embodiment 55. The method of embodiment 54, wherein the selective enrichment comprises: combining one or more bait molecules with the size- selected plurality of nucleic acid molecules, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules.Embodiment 56. The method of embodiment 54, wherein the selective enrichment comprises amplifying one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size- selected plurality of nucleic acid molecules, thereby producing the enriched plurality of nucleic acid molecules.Embodiment 57. The method of any one of embodiments 54-56, further comprising sequencing the enriched plurality of nucleic acid molecules to detect the mutation in the one or more genes.Embodiment 58. The method of any one of embodiments 1-8 and 17-57, further comprising:optionally, ligating one or more adapters onto nucleic acid molecules in the size- selected plurality of nucleic acid molecules, thereby generating ligated nucleic acids; optionally, amplifying nucleic acids from the ligated nucleic acids; optionally, capturing or enriching from the amplified nucleic acids a plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes; sequencing, by a sequencer, the plurality of nucleic acids to obtain a plurality of sequence reads corresponding to the one or more genes; analyzing the plurality of sequence reads; and based on the analysis, detecting the mutation in the one or more genes.Embodiment 59. The method of embodiment 58, wherein the plurality of nucleic acids comprising nucleotide sequences corresponding to the one or more genes is captured from the amplified nucleic acids by hybridization with one or more bait molecules.Embodiment 60. The method of embodiment 59, wherein the capturing comprises: combining the one or more bait molecules with the amplified nucleic acids, thereby hybridizing the one or more bait molecules to nucleic acids comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids; and isolating the nucleic acid hybrids.Embodiment 61. The method of any one of embodiments 58-60, wherein the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.Embodiment 62. The method of any one of embodiments 58-61, further comprising, prior to adapter ligation: subjecting the size-selected plurality of nucleic acid molecules to fragmentation.Embodiment 63. The method of any one of embodiments 58-62, wherein the sequencer comprises a next generation sequencer.Embodiment 64. The method of any one of embodiments 56-63, wherein the amplifying comprises performing a polymerase chain reaction (PCR) amplification technique, a non- PCR amplification technique, or an isothermal amplification technique.Embodiment 65. The method of any one of embodiments 57-64, wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique.Embodiment 66. The method of embodiment 65, wherein the sequencing comprises a massively parallel sequencing technique, and the massively parallel sequencing technique comprises next generation sequencing (NGS).Embodiment 67. The method of any one of embodiments 58-66, further comprising receiving, at one or more processors, sequence read data for the plurality of sequence reads.Embodiment 68. The method of embodiment 67, wherein the analyzing the plurality of sequence reads comprises identifying, using the one or more processors, presence or absence of sequence reads comprising a mutation in the one or more genes.Embodiment 69. The method of any one of embodiments 13, 18, 55, 59, and 60, wherein the one or more bait molecules comprise a capture nucleic acid molecule configured to hybridize to a nucleotide sequence corresponding to the one or more genes.Embodiment 70. The method of embodiment 69, wherein the capture nucleic acid molecule comprises between about 10 and about 30 nucleotides, between about 50 and about 1000 nucleotides, between about 100 and about 500 nucleotides, between about 100 and about 300 nucleotides, or between about 100 and about 200 nucleotides.Embodiment 71. The method of any one of embodiments 13, 18, 55, 59, 60, 69, and 70, wherein the one or more bait molecules are conjugated to an affinity reagent or to a detection reagent.Embodiment 72. The method of embodiment 71, wherein the affinity reagent is an antibody, an antibody fragment, or biotin, or wherein the detection reagent is a fluorescent marker.Embodiment 73. The method of any one of embodiments 69-72, wherein the capture nucleic acid molecule comprises a DNA, RNA, or mixed DNA / RNA molecule.Embodiment 74. The method of any one of embodiments 1-73, wherein the size- selected plurality of nucleic acid molecules comprises a ratio of nucleic acid molecules from nucleated cells to nucleic acid molecules from cfDNA of at least about 20:1.Embodiment 75. The method of any one of embodiments 1-74, wherein the size selection recovers at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of nucleic acid molecules from the first plurality of nucleic acid molecules.Embodiment 76. The method of embodiment 75, wherein the size selection recovers at least 50%, at least 60%, or at least 62% of nucleic acid molecules from the first plurality of nucleic acid molecules.Embodiment 77. The method of embodiment 75, wherein the size selection recovers at least about 60% of nucleic acid molecules from the first plurality of nucleic acid molecules.Embodiment 78. The method of any one of embodiments 1-77, wherein the size selection recovers less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% of nucleic acid molecules from the second plurality of nucleic acid molecules.Embodiment 79. The method of embodiment 78, wherein the size selection recovers less than about 3% of nucleic acid molecules from the second plurality of nucleic acid molecules.Embodiment 80. The method of any one of embodiments 1-79, wherein the first plurality of nucleic acid molecules comprises genomic DNA from the nucleated cells.Embodiment 81. The method of any one of embodiments 1-80, wherein the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells.Embodiment 82. The method of any one of embodiments 1-81, wherein the second plurality of nucleic acid molecules comprises circulating tumor DNA (ctDNA).Embodiment 83. The method of any one of embodiments 1-82, wherein the mixture of nucleic acid molecules is from a liquid biopsy sample obtained from an individual.Embodiment 84. The method of embodiment 83, further comprising obtaining the liquid biopsy sample from the individual.Embodiment 85. The method of embodiment 83 or embodiment 84, wherein the liquid biopsy is from whole blood, plasma, serum, urine, saliva, amniotic fluid, breast milk, pleural effusions or ascites, synovial fluid, or cerebrospinal fluid.Embodiment 86. The method of embodiment 85, wherein the liquid biopsy is from whole blood.Embodiment 87. The method of any one of embodiments 83-86, wherein the mixture of nucleic acid molecules is obtained from a buffy coat sample derived from the liquid biopsy.Embodiment 88. The method of embodiment 87, wherein the mixture of nucleic acid molecules is obtained by extracting buffy coat DNA (bcDNA) from the buffy coat sample.Embodiment 89. The method of embodiment 87 or embodiment 88, further comprising extracting buffy coat DNA (bcDNA) from the buffy coat sample in order to generate the mixture of nucleic acid molecules.Embodiment 90. The method of any one of embodiments 87-89, wherein the buffy coat sample is derived by centrifugation of a whole blood sample from the individual.Embodiment 91. The method of embodiment 90, further comprising centrifuging a whole blood sample from the individual to generate the buffy coat sample.Embodiment 92. The method of any one of embodiments 87-89, wherein the buffy coat sample is derived by microfluidic fractionation of a whole blood sample from the individual.Embodiment 93. The method of embodiment 92, further comprising subjecting a whole blood sample from the individual to microfluidic fractionation in order to generate the buffy coat sample.Embodiment 94. The method of any one of embodiments 87-93, wherein the buffy coat sample comprises one or more nucleated cells, optionally wherein the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells.Embodiment 95. The method of any one of embodiments 88-94, wherein the size selection is performed on bcDNA.Embodiment 96. The method of any one of embodiments 83-95, further comprising generating a molecular profile for the individual based at least in part on detecting the mutation in the one or more genes.Embodiment 97. The method of embodiment 96, wherein the molecular profile for the individual further comprises results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof.Embodiment 98. The method of embodiment 96 or embodiment 97, wherein the molecular profile for the individual further comprises results from a nucleic acid sequencingbased test.Embodiment 99. The method of any one of embodiments 96-98, further comprising selecting a treatment, administering a treatment, or applying a treatment to the individual based at least in part on the generated molecular profile.Embodiment 100. The method of any one of embodiments 1-99, further comprising generating a report indicating the presence or absence of the mutation in the one or more genes in the sample.Embodiment 101. The method of embodiment 100, further comprising transmitting the report to the individual, a caregiver, a healthcare provider, a physician, an oncologist, an electronic medical record system, a hospital, a clinic, a third-party payer, an insurance company, or a government office.Embodiment 102. The method of embodiment 101, wherein the report is transmitted via a computer network or a peer-to-peer connection.Embodiment 103. The method of any one of embodiments 1-102, wherein detection of the mutation in the one or more genes is used to diagnose or confirm a diagnosis of disease in an individual.Embodiment 104. The method of embodiment 103, wherein the mutation in the one or more genes is determined to be caused by clonal hematopoiesis of indeterminate potential (CHIP).Embodiment 105. The method of embodiment 104, wherein the disease is a cardiovascular or hematological disease.Embodiment 106. The method of any one of embodiments 83-105, wherein the individual has, is suspected of having, or is being tested for CHIP.Embodiment 107. The method of any one of embodiments 103-106, wherein the disease is a cancer.Embodiment 108. The method of any one of embodiments 83-107, wherein the individual has, is suspected of having, or is being tested for cancer.Embodiment 109. The method of embodiment 107 or embodiment 108, wherein the cancer is a carcinoma, a sarcoma, a lymphoma, a leukemia, a myeloma, a germ cell cancer, or a blastoma.Embodiment 110. The method of embodiment 109, wherein the cancer is a solid tumor.Embodiment 111. The method of embodiment 109, wherein the cancer is a B cell cancer(multiple myeloma), a melanoma, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of an oral cavity, cancer of a pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, a cancer of hematological tissue, an adenocarcinoma, an inflammatory myofibroblastic tumor, a gastrointestinal stromal tumor (GIST), colon cancer, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD), acute lymphocytic leukemia (ALL), acute myelocyticleukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia Vera, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), soft-tissue sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellular carcinoma, thyroid cancer, gastric cancer, head and neck cancer, small cell cancer, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia, chronic eosinophilic leukemia, neuroendocrine cancers, or a carcinoid tumor.Embodiment 112. The method of embodiment 109, wherein the cancer comprises acute lymphoblastic leukemia (Philadelphia chromosome positive), acute lymphoblastic leukemia (precursor B-cell), acute myeloid leukemia (FLT3+), acute myeloid leukemia (with an IDH2 mutation), anaplastic large cell lymphoma, basal cell carcinoma, B-cell chronic lymphocytic leukemia, bladder cancer, breast cancer (HER2 overexpressed / amplified), breast cancer (HER2+), breast cancer (HR+, HER2-), cervical cancer, cholangiocarcinoma, chronic lymphocytic leukemia, chronic lymphocytic leukemia (with 17p deletion), chronic myelogenous leukemia, chronic myelogenous leukemia (Philadelphia chromosome positive), classical Hodgkin lymphoma, colorectal cancer, colorectal cancer (dMMR / MSLH), colorectal cancer (KRAS wild type), cryopyrin-associated periodic syndrome, a cutaneous T- cell lymphoma, dermatofibrosarcoma protuberans, a diffuse large B-cell lymphoma, fallopian tube cancer, a follicular B-cell non-Hodgkin lymphoma, a follicular lymphoma, gastric cancer, gastric cancer (HER2+), gastroesophageal junction (GEJ) adenocarcinoma, a gastrointestinal stromal tumor, a gastrointestinal stromal tumor (KIT+), a giant cell tumor of the bone, a glioblastoma, granulomatosis with polyangiitis, a head and neck squamous cell carcinoma, a hepatocellular carcinoma, Hodgkin lymphoma, juvenile idiopathic arthritis, lupus erythematosus, a mantle cell lymphoma, medullary thyroid cancer, melanoma, amelanoma with a BRAF V600 mutation, a melanoma with a BRAF V600E or V600K mutation, Merkel cell carcinoma, multicentric Castleman's disease, multiple hematologic malignancies including Philadelphia chromosome-positive ALL and CML, multiple myeloma, myelofibrosis, a non-Hodgkin’s lymphoma, a nonresectable subependymal giant cell astrocytoma associated with tuberous sclerosis, a non-small cell lung cancer, a non-small cell lung cancer (ALK+), a non-small cell lung cancer (PD-L1+), a non-small cell lung cancer (with ALK fusion or ROS1 gene alteration), a non-small cell lung cancer (with BRAF V600E mutation), a non-small cell lung cancer (with an EGFR exon 19 deletion or exon 21 substitution (L858R) mutations), a non-small cell lung cancer (with an EGFR T790M mutation), ovarian cancer, ovarian cancer (with a BRCA mutation), pancreatic cancer, a pancreatic, gastrointestinal, or lung origin neuroendocrine tumor, a pediatric neuroblastoma, a peripheral T-cell lymphoma, peritoneal cancer, prostate cancer, a renal cell carcinoma, rheumatoid arthritis, a small lymphocytic lymphoma, a soft tissue sarcoma, a solid tumor (MSLH / dMMR), a squamous cell cancer of the head and neck, a squamous non-small cell lung cancer, thyroid cancer, a thyroid carcinoma, urothelial cancer, a urothelial carcinoma, or Waldenstrom's macroglobulinemia.Embodiment 113. The method of any one of embodiments 107-112, further comprising recommending an anti-cancer therapy for the individual based at least in part on detecting the mutation in the one or more genes.Embodiment 114. The method of any one of embodiments 107-113, further comprising selecting an anti-cancer therapy for the individual based, at least in part, on detecting the mutation in the one or more genes.Embodiment 115. The method of any one of embodiments 107-114, further comprising generating a report comprising one or more anti-cancer therapies identified for the individual based, at least in part, on detecting the mutation in the one or more genes.Embodiment 116. The method of any one of embodiments 107-115, further comprising administering an anti-cancer therapy to the individual based, at least in part, on detecting the mutation in the one or more genes.Embodiment 117. The method of any one of embodiments 113-116, wherein the anticancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, aradiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti-neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.Embodiment 118. The method of embodiment 117, wherein the cellular therapy is an adoptive therapy, a T cell-based therapy, a natural killer (NK) cell-based therapy, a chimeric antigen receptor (CAR)-T cell therapy, a recombinant T cell receptor (TCR) T cell therapy, a macrophage-based therapy, an induced pluripotent stem cell-based therapy, a B cell-based therapy, or a dendritic cell (DC)-based therapy.Embodiment 119. The method of embodiment 117, wherein the nucleic acid comprises a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), or a small hairpin RNA (shRNA).Embodiment 120. The method of any one of embodiments 113-116, wherein the anticancer therapy comprises chemotherapy, radiation therapy, immunotherapy, a targeted therapy, or surgery.Embodiment 121. The method of any one of embodiments 113-116, wherein the anticancer therapy comprises a targeted anti-cancer therapy.Embodiment 122. The method of any one of embodiments 113-116, wherein the anticancer therapy comprises abemaciclib (Verzenio), abiraterone acetate (Zytiga), acalabrutinib (Calquence), ado-trastuzumab emtansine (Kadcyla), afatinib dimaleate (Gilotrif), aldesleukin (Proleukin), alectinib (Alecensa), alemtuzumab (Campath), alitretinoin (Panretin), alpelisib (Piqray), amivantamab-vmjw (Rybrevant), anastrozole (Arimidex), apalutamide (Erleada), asciminib hydrochloride (Scemblix), atezolizumab (Tecentriq), avapritinib (Ayvakit), avelumab (Bavencio), axicabtagene ciloleucel (Yescarta), axitinib (Inlyta), belantamab mafodotin-blmf (Blenrep), belimumab (Benlysta), belinostat (Beleodaq), belzutifan (Welireg), bevacizumab (Avastin), bexarotene (Targretin), binimetinib (Mektovi), blinatumomab (Blincyto), bortezomib (Velcade), bosutinib (Bosulif), brentuximab vedotin (Adcetris), brexucabtagene autoleucel (Tecartus), brigatinib (Alunbrig), cabazitaxel (Jevtana), cabozantinib (Cabometyx), cabozantinib (Cabometyx, Cometriq), canakinumab (Haris), capmatinib hydrochloride (Tabrecta), carfilzomib (Kyprolis), cemiplimab-rwlc(Libtayo), ceritinib (LDK378 / Zykadia), cetuximab (Erbitux), cobimetinib (Cotellic), copanlisib hydrochloride (Aliqopa), crizotinib (Xalkori), dabrafenib (Tafinlar), dacomitinib (Vizimpro), daratumumab (Darzalex), daratumumab and hyaluronidase-fihj (Darzalex Faspro), darolutamide (Nubeqa), dasatinib (Sprycel), denileukin diftitox (Ontak), denosumab (Xgeva), dinutuximab (Unituxin), dostarlimab-gxly (Jemperli), durvalumab (Imfinzi), duvelisib (Copiktra), elotuzumab (Empliciti), enasidenib mesylate (Idhifa), encorafenib (Braftovi), enfortumab vedotin-ejfv (Padcev), entrectinib (Rozlytrek), enzalutamide (Xtandi), erdafitinib (Balversa), erlotinib (Tarceva), everolimus (Afinitor), exemestane (Aromasin), fam-trastuzumab deruxtecan-nxki (Enhertu), fedratinib hydrochloride (Inrebic), fulvestrant (Faslodex), gefitinib (Iressa), gemtuzumab ozogamicin (Mylotarg), gilteritinib (Xospata), glasdegib maleate (Daurismo), hyaluronidase-zzxf (Phesgo), ibrutinib (Imbruvica), ibritumomab tiuxetan (Zevalin), idecabtagene vicleucel (Abecma), idelalisib (Zydelig), imatinib mesylate (Gleevec), infigratinib phosphate (Truseltiq), inotuzumab ozogamicin (Besponsa), iobenguane 1131 (Azedra), ipilimumab (Yervoy), isatuximab-irfc (Sarclisa), ivosidenib (Tibsovo), ixazomib citrate (Ninlaro), lanreotide acetate (Somatuline Depot), lapatinib (Tykerb), larotrectinib sulfate (Vitrakvi), lenvatinib mesylate (Lenvima), letrozole (Femara), lisocabtagene maraleucel (Breyanzi), loncastuximab tesirine-lpyl (Zynlonta), lorlatinib (Lorbrena), lutetium Lu 177-dotatate (Lutathera), margetuximab-cmkb (Margenza), midostaurin (Rydapt), mobocertinib succinate (Exkivity), mogamulizumab-kpkc (Poteligeo), moxetumomab pasudotox-tdfk (Lumoxiti), naxitamab-gqgk (Danyelza), necitumumab (Portrazza), neratinib maleate (Nerlynx), nilotinib (Tasigna), niraparib tosylate monohydrate (Zejula), nivolumab (Opdivo), obinutuzumab (Gazyva), ofatumumab (Arzerra), olaparib (Lynparza), olaratumab (Lartruvo), osimertinib (Tagrisso), palbociclib (Ibrance), panitumumab (Vectibix), panobinostat (Farydak), pazopanib (Votrient), pembrolizumab (Keytruda), pemigatinib (Pemazyre), pertuzumab (Perjeta), pexidartinib hydrochloride (Turalio), polatuzumab vedotin-piiq (Polivy), ponatinib hydrochloride (Iclusig), pralatrexate (Folotyn), pralsetinib (Gavreto), radium 223 dichloride (Xofigo), ramucirumab (Cyramza), regorafenib (Stivarga), ribociclib (Kisqali), ripretinib (Qinlock), rituximab (Rituxan), rituximab and hyaluronidase human (Rituxan Hycela), romidepsin (Istodax), rucaparib camsylate (Rubraca), ruxolitinib phosphate (Jakafi), sacituzumab govitecan-hziy (Trodelvy), seliciclib, selinexor (Xpovio), selpercatinib (Retevmo), selumetinib sulfate (Koselugo), siltuximab (Sylvant), sipuleucel-T (Provenge), sirolimus protein-bound particles (Fyarro), sonidegib (Odomzo), sorafenib (Nexavar), sotorasib (Lumakras), sunitinib (Sutent), tafasitamab-cxix (Monjuvi), tagraxofusp-erzs (Elzonris), talazoparib tosylate (Talzenna),tamoxifen (Nolvadex), tazemetostat hydrobromide (Tazverik), tebentafusp-tebn (Kimmtrak), temsirolimus (Torisel), tepotinib hydrochloride (Tepmetko), tisagenlecleucel (Kymriah), tisotumab vedotin-tftv (Tivdak), tocilizumab (Actemra), tofacitinib (Xeljanz), tositumomab (Bexxar), trametinib (Mekinist), trastuzumab (Herceptin), tretinoin (Vesanoid), tivozanib hydrochloride (Fotivda), toremifene (Fareston), tucatinib (Tukysa), umbralisib tosylate (Ukoniq), vandetanib (Caprelsa), vemurafenib (Zelboraf), venetoclax (Venclexta), vismodegib (Erivedge), vorinostat (Zolinza), zanubrutinib (Brukinsa), ziv-aflibercept (Zaltrap), or any combination thereof.Embodiment 123. The method of any one of embodiments 107-122, further comprising determining an effective amount of the anti-cancer therapy to administer to the individual based, at least in part, on detecting the mutation in the one or more genes.Embodiment 124. The method of any one of embodiments 1-123, wherein the mutation in the one or more genes comprises one or more of: a point mutation; an insertion-deletion (indel); an in-frame deletion of one or more codons; an intragenic deletion; an intragenic insertion; a deletion of a full gene; an inversion; an interchromosomal or intrachromosomal translocation; a tandem duplication; a gene fusion; a genomic rearrangement that comprises an intron sequence; and / or a gene amplification or duplication.Embodiment 125. The method of any one of embodiments 1-124, wherein the one or more genes comprise ABL1, ACVR1B, AKT1, AKT2, AKT3, ALK, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID1A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BCOR, BCORL1, BCR, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BTG1, BTG2, BTK, CALR, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD22, CD274, CD70, CD74, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC, CREBBP, CRKL, CSF1R, CSF3R, CTCF, CTNNA1, CTNNB1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DNMT3A, DOT1L, EED, EGFR, EMSY (Cllorf30), EP300, EPHA3, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC4, ERG, ERRFI1, ESRI, ETV4, ETV5, ETV6, EWSR1, EZH2, EZR, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GATA3, GATA4, GATA6, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HDAC1,HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1, INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2D (MLL2), KRAS, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MERTK, MET, MITF, MKNK1, MLH1, MPL, MRE11A, MSH2, MSH3, MSH6, MST1R, MTAP, MTOR, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NT5C2, NTRK1, NTRK2, NTRK3, NUTM1, P2RY8, PALB2, PARK2, PARP1, PARP2, PARP3, PAX5, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CA, PIK3CB, PIK3R1, PIM1, PMS2, POLDI, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PRKAR1A, PRKCI, PTCHI, PTEN, PTPN11, PTPRO, QKI, RAC1, RAD21, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAFI, RARA, RBI, RBM10, REL, RET, RICTOR, RNF43, ROS1, RPTOR, RSPO2, SDC4, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SGK1, SLC34A2, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STK11, SUFU, SYK, TBX3, TEK, TERC, TERT, TET2, TGFBR2, TIPARP, TMPRSS2, TNFAIP3, TNFRSF14, TP53, TSC1, TSC2, TYRO3, U2AF1, VEGFA, VHL, WHSCI, WHSC1L1, WT1, XPO1, XRCC2, ZNF217, ZNF703, or any combination thereof.Embodiment 126. The method of any one of embodiments 1-124, wherein the one or more genes comprise ABL, ALK, ALL, B4GALNT1, BAFF, BCL2, BRAF, BRCA, BTK, CD19, CD20, CD3, CD30, CD319, CD38, CD52, CDK4, CDK6, CML, CRACC, CS1, CTLA-4, dMMR, EGFR, ERBB1, ERBB2, FGFR1-3, FLT3, GD2, HDAC, HER1, HR, IDH2, IL-ip, IL-6, IL-6R, JAK1, JAK2, JAK3, KIT, KRAS, MEK, MET, MSI-H, mTOR, PARP, PD-1, PDGFR, PDGFRa, PDGFRp, PD-L1, PI3K5, PIGF, PTCH, RAF, RANKL, RET, ROS1, SLAMF7, VEGF, VEGFA, VEGFB, or any combination thereof.Embodiment 127. A system for detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising: a memory configured to store one or more program instructions, and one or more processors configured to execute the one or more program instructions, the one or more program instructions when executed by the one or more processors are configured to:(a) obtain a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA);(b) analyze the plurality of sequence reads for the presence of a mutation in one or more genes; and(c) detect, based on the analyzing, the mutation in the one or more genes in the sample, thereby detecting a clonal hematopoietic (CH) or germline gene mutation.Embodiment 128. The system of embodiment 127, wherein the plurality of sequence reads is obtained by sequencing; optionally wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and further optionally wherein the massively parallel sequencing technique comprises next generation sequencing (NGS).Embodiment 129. The system of embodiment 127 or embodiment 128, wherein the one or more program instructions when executed by the one or more processors are further configured to generate, based at least in part on the detecting, a molecular profile for a sample from which the mixture of nucleic acid molecules was obtained.Embodiment 130. The system of embodiment 129, wherein the sample is obtained from an individual, and wherein the individual is administered a treatment based at least in part on the molecular profile.Embodiment 131. The system of embodiment 129 or embodiment 130, wherein the molecular profile further comprises (a) results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof; and / or (b) results from a nucleic acid sequencing-based test.Embodiment 132. The system of any one of embodiments 129-131, wherein the sample is a liquid biopsy sample.Embodiment 133. A non-transitory computer readable storage medium comprising one or more programs executable by one or more computer processors for performing a method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, the method comprising:(a) obtaining, using the one or more processors, a plurality of sequence reads of one or more nucleic acid molecules from a first plurality of nucleic acid molecules, wherein the first plurality of nucleic acid molecules is from nucleated cells, and wherein the first plurality of nucleic acid molecules is separated by size selection from a mixture of nucleic acid molecules that comprises the first plurality of nucleic acid molecules and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA);(b) analyzing, using the one or more processors, the plurality of sequence reads for the presence of a mutation in one or more genes; and(c) detecting, using the one or more processors and based on the analyzing, the mutation in the one or more genes, thereby detecting a clonal hematopoietic (CH) or germline gene mutation.Embodiment 134. The non-transitory computer readable storage medium of embodiment 133, wherein the plurality of sequence reads is obtained by sequencing; optionally wherein the sequencing comprises use of a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique; and further optionally wherein the massively parallel sequencing technique comprises next generation sequencing (NGS).Embodiment 135. The non-transitory computer readable storage medium of embodiment 133 or embodiment 134, wherein the method further comprises generating, based at least in part on the detecting, a molecular profile for a sample from which the mixture of nucleic acid molecules was obtained.Embodiment 136. The non-transitory computer readable storage medium of embodiment 135, wherein the sample is obtained from an individual, and wherein the individual is administered a treatment based at least in part on the molecular profile.Embodiment 137. The non-transitory computer readable storage medium of embodiment 135 or embodiment 136, wherein the molecular profile further comprises (a) results from a comprehensive genomic profiling (CGP) test, a gene expression profiling test, a cancer hotspot panel test, a DNA methylation test, a DNA fragmentation test, an RNA fragmentation test, or any combination thereof; and / or (b) results from a nucleic acid sequencing-based test.Embodiment 138. The non-transitory computer readable storage medium of any one of embodiments 135-137, wherein the sample is a liquid biopsy sample.EXAMPLESExample 1: Analysis of the purity of genomic DNA isolated from buffy coat samplesDesign

[0244] A bulk volume of sample containing an equivalent mass of cfDNA and bcDNA was generated. This sample was then used for downstream workflows and workflow analyses. Automated SPRI DNA clean-up workflows utilized the AMPure beads to bind to the DNA sample. The automated SPRI DNA clean-up workflow followed standard manufacturer’s protocols except for the ratio of AMPure beads-to-DNA sample; different ratios of AMPure beads-to-DNA sample were tested using bead ratios between 0.8x to 0.3x. In comparison, standard SPRI workflows typically use lx to 1.8x bead ratios. The goal of this reduction in ratio was to allow for the preferential selection of high molecular weight (HMW) DNA and the size exclusion of low molecular weight (LMW; z.e., contaminating) DNA (e.g., cfDNA) of less than 1,000 bp. DNA was then eluted off the AMPure beads using manufacturer’s protocol.

[0245] The eluted products, as well as the original material, then was characterized on the Fragment Analyzer. This analysis tool enabled the concentration of the cfDNA and the bcDNA to be measured within a single assay.Results

[0246] The Fragment Analyzer assessed the various DNA size fragments that were present in the sample after elution of the SPRI beads compared to non-purified DNA sample with 50% cfDNA and 50% bcDNA. As can be seen in FIG. 1, a progressive reduction in bead-to-DNAratio led to a significant reduction in the presence of LMW DNA species while still retaining bcDNA in the eluate.

[0247] FIG. 2 shows the average concentration and the percent recovery of LMW (50- l,000bp) and HMW (l,000bp-150kb) DNA species. Ratios at 0.5x and 0.6x were found to provide the highest HMW purity without compromised recovery of bcDNA species. Eluates from conditions using ratios below 0.5x demonstrated a dramatic decrease in overall bcDNA species recovery.

[0248] FIG. 3 graphically shows the concentrations of cfDNA and bcDNA species based on the AMPure bead ratio and the bulk volume DNA sample (“stock”) control. The concentration of cfDNA is progressively reduced in a ratio-dependent manner, whereas the retained and recovered bfDNA maintains a similar concentration for AMPure ratios of 0.5x- 0.8x before dropping off at 0.45x.

[0249] The data showed that DNA under 1000 bp was removed from DNA samples ranging in size from 50,000 to 200,000 bp using an automated SPRI clean-up workflow with a reduced AMPure bead-to-DNA sample ratio. It was surprising that the recovery of the HMW DNA was relatively high when performing the workflow described herein (-50%). A similar recovery rate of bcDNA was observed across a range of AMPure bead ratios, while the presence of the cfDNA was reduced as the AMPure bead ratio decreased.

[0250] It should be understood from the foregoing that, while particular implementations of the disclosed methods have been illustrated and described, various modifications can be made thereto and are contemplated herein. It is also not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the preferable embodiments herein are not meant to be construed in a limiting sense. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. Various modifications in form and detail of the embodiments of the invention will be apparent to a person skilled in the art. It is therefore contemplated that the invention shall also cover any such modifications, variations and equivalents.

Claims

CLAIMSWhat is claimed is:

1. A method for detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising: performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules; and detecting, by one or more processors, a mutation in one or more genes in the size- selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.

2. The method of claim 1, wherein the size selection is performed using one or more of: gel electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), DNA chromatography, DNA precipitation followed by centrifugation, spin columnbased DNA purification, ion exchange chromatography, or solid-phase reversible immobilization (SPRI).

3. A method detecting one or more clonal hematopoietic (CH) or germline gene mutations in a sample, comprising:(a) performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), wherein the size selection comprises contacting the mixture of nucleic acid molecules with solid-phase reversible immobilization (SPRI) beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.2 and about 1.0, thereby generating a size-selected plurality of nucleic acid molecules; and(b) detecting a mutation in one or more genes in the size- selected plurality of nucleic acid molecules, thereby determining that the detected mutation is a CH or germline mutation.

4. The method of claim 3, wherein the size selection comprises contacting the mixture of nucleic acid molecules with the SPRI beads using a ratio of volume of beads to volume of nucleic acid molecules of between about 0.4 and about 0.6, or between about 0.45 and about 0.55, optionally wherein the ratio of volume of beads to volume of nucleic acid molecules is any of about 0.4, about 0.45, about 0.5, about 0.55, or about 0.6.

5. The method of claim 3, further comprising, after contacting the mixture of nucleic acid molecules with the SPRI beads: washing the mixture of nucleic acid molecules under conditions in which the mixture of nucleic acid molecules is bound to the SPRI beads; and eluting the size- selected plurality of nucleic acid molecules from the SPRI beads; optionally wherein the SPRI beads comprise paramagnetic SPRI beads.

6. A method of detecting one or more clonal hematopoietic (CH) or germline gene mutations, comprising:(a) performing a size selection on a mixture of nucleic acid molecules, wherein the mixture comprises a first plurality of nucleic acid molecules from nucleated cells and a second plurality of nucleic acid molecules from cell-free DNA (cfDNA), and wherein the size selection comprises separating the first plurality of nucleic acid molecules of the mixture from the second plurality of nucleic acid molecules of the mixture, thereby generating a size- selected plurality of nucleic acid molecules;(b) preparing a nucleic acid sequencing library from the size- selected plurality of nucleic acid molecules;(c) amplifying said library;(d) selectively enriching for a plurality of nucleic acid molecules from the amplified library, said plurality of nucleic acid molecules comprising nucleotide sequence(s) corresponding to the one or more genes, thereby producing an enriched plurality of nucleic acid molecules;(e) sequencing, by a sequencer, the enriched plurality of nucleic acid molecules, thereby producing a plurality of sequence reads;(f) analyzing, by one or more processors, the plurality of sequence reads for presence or absence of a mutation in the one or more genes; and(g) detecting, based on the analysis, presence or absence of a mutation in the one or more genes, thereby determining that the detected mutation is a CH or germline mutation.

7. The method of claim 1, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 1000 base pairs or more, about 5000 base pairs or more, about 10,000 base pairs or more, about 15,000 base pairs or more, about 20,000 base pairs or more, about 25,000 base pairs or more, about 30,000 base pairs or more, about 35,000 base pairs or more, about 40,000 base pairs or more, about 45,000 base pairs or more, about 50,000 base pairs or more, about 55,000 base pairs or more, about 60,000 base pairs or more, about 65,000 base pairs or more, about 70,000 base pairs or more, about 75,000 base pairs or more, about 80,000 base pairs or more, about 85,000 base pairs or more, about 90,000 base pairs or more, about 95,000 base pairs or more, or about 100,000 base pairs or more, optionally wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of up to about 200,000 base pairs; and / or wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of less than about 1000 base pairs, optionally wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 999 base pairs or less, about 900 base pairs or less, about 800 base pairs or less, about 700 base pairs or less, about 600 base pairs or less, about 500 base pairs or less, about 400 base pairs or less, about 300 base pairs or less, about 200 base pairs or less, or about 100 base pairs or less, and optionally wherein the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of about 50 base pairs or more, about 75 base pairs or more, about 100 base pairs or more, about 125 base pairs or more, about 150 base pairs or more, about 175 base pairs or more, about 200 base pairs or more, about 225 base pairs or more, about 250 base pairs or more, about 275 base pairs or more, about 300 base pairs or more, about 325 base pairs or more, about 350 base pairs or more, about 375 base pairs or more, about 400 base pairs ormore, about 425 base pairs or more, about 450 base pairs or more, about 475 base pairs or more, or about 500 base pairs or more.

8. The method of claim 1, wherein the first plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50,000 base pairs and about 200,000 base pairs, and the second plurality of nucleic acid molecules comprises nucleic acid molecules or fragments thereof with a length of between about 50 base pairs and about 1000 base pairs.

9. The method of claim 1, further comprising: detecting a mutation in one or more genes in the second plurality of nucleic acid molecules; analyzing a mutation in one or more genes detected in the size-selected plurality of nucleic acid molecules; analyzing a mutation in one or more genes detected in the second plurality of nucleic acid molecules; and based on the analyzing: identifying a mutation in one or more genes as being from cfDNA when it is represented in the second plurality of nucleic acid molecules but not the size- selected plurality of nucleic acid molecules.

10. The method of claim 1, further comprising: ligating one or more adapters onto nucleic acid molecules in the size-selected plurality of nucleic acid molecules, thereby generating ligated nucleic acid molecules; optionally, amplifying the ligated nucleic acid molecules; optionally, capturing, from the amplified nucleic acid molecules, nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes; sequencing, by a sequencer, the captured nucleic acid molecules to obtain a plurality of sequence reads that represent the captured nucleic acid molecules, wherein one or more of the plurality of sequence reads correspond to the one or more genes; and analyzing, by one or more processors, the plurality of sequence reads;wherein the mutation in the one or more genes is detected in the size-selected plurality of nucleic acid molecules based on the analysis.

11. The method of claim 10, wherein the amplified nucleic acid molecules are captured by hybridization with one or more bait molecules; and / or wherein the one or more adapters comprise amplification primers, flow cell adapter sequences, substrate adapter sequences, sample index sequences, or unique molecular identifier (UMI) sequences.

12. The method of claim 1, wherein the mutation in the one or more genes is detected by one or more of: a nucleic acid hybridization assay, an amplification-based assay, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay, real-time PCR, a screening analysis, fluorescence in situ hybridization (FISH), spectral karyotyping, multicolor FISH (mFISH), comparative genomic hybridization, in situ hybridization, sequence- specific priming (SSP) PCR, high-performance liquid chromatography (HPLC), mass-spectrometric genotyping, or sequencing; optionally wherein the sequencing comprises a massively parallel sequencing (MPS) technique, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing, or a Sanger sequencing technique, optionally, wherein the massively parallel sequencing (MPS) technique comprises next-generation sequencing (NGS).

13. The method of claim 1, further comprising selectively enriching for one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size-selected plurality of nucleic acid molecules, thereby producing an enriched plurality of nucleic acid molecules; wherein optionally the selective enrichment comprises:(a) combining one or more bait molecules with the size- selected plurality of nucleic acid molecules, thereby hybridizing the one or more bait molecules to one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes and producing nucleic acid hybrids, and isolating the nucleic acid hybrids to produce the enriched plurality of nucleic acid molecules; or(b) amplifying one or more nucleic acid molecules comprising nucleotide sequences corresponding to the one or more genes in the size- selected plurality of nucleic acid molecules, thereby producing the enriched plurality of nucleic acid molecules.

14. The method of claim 1, wherein:(a) the size selection recovers at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of nucleic acid molecules from the first plurality of nucleic acid molecules, optionally wherein the size selection recovers at least 50%, at least 60%, or at least 62% of nucleic acid molecules from the first plurality of nucleic acid molecules; and / or(b) wherein the size selection recovers less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25%, or less than about 0.1% of nucleic acid molecules from the second plurality of nucleic acid molecules, optionally wherein the size selection recovers less than about 3% of nucleic acid molecules from the second plurality of nucleic acid molecules.

15. The method of claim 1, wherein:(a) the first plurality of nucleic acid molecules comprises genomic DNA from the nucleated cells, optionally wherein the nucleated cells comprise one or more of lymphocytes, monocytes, granulocytes, or nucleated blood cells; and / or(b) wherein the second plurality of nucleic acid molecules comprises circulating tumor DNA (ctDNA).

16. The method of claim 1, wherein the mixture of nucleic acid molecules is from a liquid biopsy sample obtained from an individual, optionally wherein the mixture of nucleic acid molecules is obtained from a buffy coat sample derived from the liquid biopsy.

17. The method of claim 1, wherein detection of the mutation in the one or more genes is used to diagnose or confirm a diagnosis of disease in an individual, optionally wherein the mutation in the one or more genes is determined to be caused by clonal hematopoiesis of indeterminate potential (CHIP), and optionally wherein the disease is a cardiovascular disease, hematological disease, or cancer.

18. The method of claim 17, wherein the disease is cancer, and the method further comprises administering an anti-cancer therapy to the individual based, at least in part, on detecting the mutation in the one or more genes; optionally wherein the anti-cancer therapy comprises one or more of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), or any combination thereof.

19. The method of claim 1, wherein the mutation in the one or more genes comprises one or more of: a point mutation; an insertion-deletion (indel); an in-frame deletion of one or more codons; an intragenic deletion; an intragenic insertion; a deletion of a full gene; an inversion; an interchromosomal or intrachromosomal translocation; a tandem duplication; a gene fusion; a genomic rearrangement that comprises an intron sequence; and / or a gene amplification or duplication.

20. The method of claim 1, wherein:(a) the one or more genes comprise ABL1, ACVR1B, AKT1, AKT2, AKT3, ALK, ALOX12B, AMER1, APC, AR, ARAF, ARFRP1, ARID1A, ASXL1, ATM, ATR, ATRX, AURKA, AURKB, AXIN1, AXL, BAP1, BARD1, BCL2, BCL2L1, BCL2L2, BCL6, BCOR, BCORL1, BCR, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BTG1, BTG2, BTK, CALR, CARD11, CASP8, CBFB, CBL, CCND1, CCND2, CCND3, CCNE1, CD22, CD274, CD70, CD74, CD79A, CD79B, CDC73, CDH1, CDK12, CDK4, CDK6, CDK8, CDKN1A, CDKN1B, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK1, CHEK2, CIC, CREBBP, CRKL, CSF1R, CSF3R, CTCF, CTNNA1, CTNNB1, CUL3, CUL4A, CXCR4, CYP17A1, DAXX, DDR1, DDR2, DIS3, DNMT3A, DOT1L, EED, EGFR, EMSY (Cllorf30), EP300, EPHA3, EPHB1, EPHB4, ERBB2, ERBB3, ERBB4, ERCC4, ERG, ERRFI1, ESRI, ETV4, ETV5, ETV6, EWSR1, EZH2, EZR, FAM46C, FANCA, FANCC, FANCG, FANCL, FAS, FBXW7, FGF10, FGF12, FGF14, FGF19, FGF23, FGF3, FGF4, FGF6, FGFR1, FGFR2, FGFR3, FGFR4, FH, FLCN, FLT1, FLT3, FOXL2, FUBP1, GABRA6, GATA3, GATA4, GATA6, GID4 (C17orf39), GNA11, GNA13, GNAQ, GNAS, GRM3, GSK3B, H3F3A, HDAC1, HGF, HNF1A, HRAS, HSD3B1, ID3, IDH1, IDH2, IGF1R, IKBKE, IKZF1,INPP4B, IRF2, IRF4, IRS2, JAK1, JAK2, JAK3, JUN, KDM5A, KDM5C, KDM6A, KDR, KEAP1, KEL, KIT, KLHL6, KMT2A (MLL), KMT2D (MLL2), KRAS, LTK, LYN, MAF, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K13, MAPK1, MCL1, MDM2, MDM4, MED12, MEF2B, MEN1, MERTK, MET, MITF, MKNK1, MLH1, MPL, MRE11A, MSH2, MSH3, MSH6, MST1R, MTAP, MTOR, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NFKBIA, NKX2-1, NOTCH1, NOTCH2, NOTCH3, NPM1, NRAS, NT5C2, NTRK1, NTRK2, NTRK3, NUTM1, P2RY8, PALB2, PARK2, PARP1, PARP2, PARP3, PAX5, PBRM1, PDCD1, PDCD1LG2, PDGFRA, PDGFRB, PDK1, PIK3C2B, PIK3C2G, PIK3CA, PIK3CB, PIK3R1, PIM1, PMS2, POLDI, POLE, PPARG, PPP2R1A, PPP2R2A, PRDM1, PRKAR1A, PRKCI, PTCHI, PTEN, PTPN11, PTPRO, QKI, RAC1, RAD21, RAD51, RAD51B, RAD51C, RAD51D, RAD52, RAD54L, RAFI, RARA, RBI, RBM10, REL, RET, RICTOR, RNF43, ROS1, RPTOR, RSPO2, SDC4, SDHA, SDHB, SDHC, SDHD, SETD2, SF3B1, SGK1, SLC34A2, SMAD2, SMAD4, SMARCA4, SMARCB1, SMO, SNCAIP, SOCS1, SOX2, SOX9, SPEN, SPOP, SRC, STAG2, STAT3, STK11, SUFU, SYK, TBX3, TEK, TERC, TERT, TET2, TGFBR2, TIPARP, TMPRSS2, TNFAIP3, TNFRSF14, TP53, TSC1, TSC2, TYRO3, U2AF1, VEGFA, VHL, WHSCI, WHSC1L1, WT1, XPO1, XRCC2, ZNF217, ZNF703, or any combination thereof; or(b) wherein the one or more genes comprise ABL, ALK, ALL, B4GALNT1, BAFF, BCL2, BRAF, BRCA, BTK, CD19, CD20, CD3, CD30, CD319, CD38, CD52, CDK4, CDK6, CML, CRACC, CS1, CTLA-4, dMMR, EGFR, ERBB1, ERBB2, FGFR1-3, FLT3, GD2, HD AC, HER1, HR, IDH2, IL-ip, IL-6, IL-6R, JAK1, JAK2, JAK3, KIT, KRAS, MEK, MET, MSI-H, mTOR, PARP, PD-1, PDGFR, PDGFRa, PDGFRp, PD-L1, PI3K5, PIGF, PTCH, RAF, RANKL, RET, ROS1, SLAMF7, VEGF, VEGFA, VEGFB, or any combination thereof.Ill

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