Unique molecule identification by jagged end indexing

Abstract

Methods for correcting consensus sequence reads are described. The disclosed methods can comprise: obtaining a sample of double stranded nucleic acid molecules having jagged ends; performing end repair on the jagged ends to introduce jagged end identifiers; preparing a sequencing library comprising the end repaired nucleic acid molecules; sequencing the library to generate sequence reads; mapping the sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, where the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read; grouping the mapped sequence reads into families based on the corresponding sequence information; determining a consensus sequence read for each family; and detecting a biological signal in the consensus sequence read for at least one family of a plurality of families.

Description

Docket No.: 197102019740UNIQUE MOLECULE IDENTIFICATION BY JAGGED END INDEXINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit to U.S. Provisional Application No. 63 / 736,412, filed December 19, 2024, the entire contents of which are incorporated herein by reference for all purposes.FIELD

[0002] The present disclosure relates generally to methods for analyzing genomic or multiomic profiling data, and more specifically to methods for improved sequencing error correction based on jagged end identifiers that, in turn, provide for improved sequence variant detection with or without molecular barcodes.BACKGROUND

[0003] The ability to identify and track unique DNA molecules through next generation sequencing workflows has several important implications for downstream data analysis. One example is that the detection of low-level variants using conventional sequencing library preparation techniques can be challenging due to sequence errors that arise from background PCR amplification error rates. Identification of unique DNA molecules allows one to distinguish between sequence differences arising from PCR amplification error rates and true sequence variants (z.e., biologically-relevant sequence variants). Standard practice has been to use unique molecular identifiers (UMIs). Conventionally, UMI’s have been understood to be comprised of three different types. Exogenous only (e.g., unique molecular barcodes), endogenous (e.g., using the molecule’s sequence information itself, and semi-exogenous (e.g., use of non-unique molecular barcodes in combination with other information (e.g., genomic / transcriptomic coordinates of the molecule).

[0004] When using molecular barcodes (unique or non-unique), the molecular barcodes are typically attached to each polynucleotide molecule during the library construction process prior to amplification. This can be done by ligation of a molecular barcode to one or both ends of either a double stranded or single stranded polynucleotide molecule. These barcodes are then used, alone (exogenously, unique barcodes) or in combination (exogenously, non- unique barcodes) to identify which molecules within an amplicon are progeny of a single unique DNA molecule, and thus to help to distinguish between true variants and1MF-365322596Docket No.: 197102019740 amplification artifacts once the amplicons have been sequenced. However, the use of molecular barcodes requires one to perform additional steps during the preparation of sequencing libraries, can be complicated to design effectively (especially when using an exogenously unique approach) and the addition of the molecular barcode sequences to each strand of a double- stranded DNA fragment can use up sequencing and computation capacity in a given sequencing run in order to determine the barcode sequences. This can be especially true in assays that require a larger number of nucleotides in each molecular barcode to achieve the desired uniqueness.

[0005] Additionally, endogenous methods have been found to be less effective given that sequencing information alone can be somewhat unreliable due to the number of errors that can occur during sequencing, and that in turn can cause the molecules / reads / fragments not to be identified appropriately during computational processing. This is mostly to do with the fact that the sequencing information is unknown prior to performing sequencing and, as a result, can be contaminated by any chemistry-based or sequencing-based errors that introduce inaccuracies in the sequencing information. As a result, different UMIs may be assigned to two different reads that both originated from the same molecule. For this reason, endogenous methods have conventionally been thought to have inferior performance in comparison to that of exogenous or semi-exogenous methods.BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed herein are methods for utilizing jagged end identifiers (e.g., markers) incorporated into end repaired DNA overhang sequences of of double stranded DNA fragments (e.g., cfDNA molecules) - more specifically, the 3’ and / or 5’ jagged ends of such fragments - in place of conventional types of UMIs to enable molecular tracking and facilitate sequencing error correction. The methods disclosed herein utilize end repair reactions for repairing jagged ends to introduce a jagged end identifier (or marker) into the 3’-end and / or 5’-end of DNA fragments comprising jagged ends (overhangs) that can then be used for molecular tracking and error correction of sequencing data. For example, replacing dCTP with 5hmC in double stranded library preparation (dsLP) end-repair reactions allows one to tag the 5’ jagged ends with methylated cytosines which are resistant to methyl conversion when performing methylation sequencing. This is one non-limiting example, with several other methods of implementation described herein.2MF-365322596Docket No.: 197102019740

[0007] The disclosed methods provide the advantages of a streamlined sequencing library workflow, can be used for both single-stranded library preparation (ssLP) and doublestranded library preparation (dsLP), and allow one to utilize sequencing capacity more efficiently than in conventional UMI approaches while still providing sequencing error correction capability. The disclosed methods can also provide greater accuracy and improved reliability compared to endogenous methods.

[0008] Disclosed herein are methods of detecting biological signals, the method comprising: obtaining a sample from a subject, the sample comprising a plurality of double stranded nucleic acid molecules having one or more jagged ends; performing end repair on the one or more jagged ends of the plurality of double stranded nucleic acid molecules, wherein the end repair introduces a jagged end identifier within a given double stranded molecule of the plurality of double stranded molecules as part of the end repair reaction; preparing a sequencing library comprising a plurality of end repaired nucleic acid molecules; sequencing the plurality of end repaired nucleic acid molecules to generate a plurality of sequence reads; mapping the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read; grouping the mapped sequence reads into a plurality of families based on the corresponding sequence information; based on the grouping, determining a consensus sequence read for each of the plurality of families; and detecting one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0009] In some embodiments, performing end repair further comprises inserting one or more methylated cytosines to fill in the one or more jagged ends of the plurality of double stranded molecules. In some embodiments, the one or more methylated cytosines are inserted via a polymerase reaction. In some embodiments, the method further comprises performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil. In some embodiments, mapping further comprises determining a position of a first methylated cytosine in the 5’ direction that is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position. In some embodiments, the conversion reaction is an enzymatic3MF-365322596Docket No.: 197102019740 conversion reaction. In some embodiments, the conversion reaction is a chemical conversion reaction. In some embodiments, the chemical conversion reaction is a bisulfite conversion reaction.

[0010] In some embodiments, performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules. In some embodiments, the one or more inosines are inserted via a polymerase reaction. In some embodiments, the one or more inosines are inserted by hydrolytic deamination of adenine. In some embodiments, mapping further comprises determining a position of a first inosine in the 5’ direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position.

[0011] In some embodiments, the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules. In some embodiments, the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprise circulating tumor DNA (ctDNA) molecules.

[0012] The method of any one of clauses 1 to 14, wherein the plurality of end repaired nucleic acid molecules are single stranded. In some embodiments, the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules. In some embodiments, the double-stranded nucleic acid molecules undergo double- stranded nucleic acid molecule library preparation. In some embodiments, the double-stranded nucleic acid molecule library preparation comprises denaturing the double-stranded nucleic acid molecules. In some embodiments, the double-stranded nucleic acid molecule library preparation comprises polyA-tailing the denatured double- stranded nucleic acid molecules. In some embodiments, the plurality of end repaired nucleic acid molecules undergo singlestranded nucleic acid molecule library preparation. In some embodiments, the single- stranded nucleic acid molecule library preparation comprises synthesizing a second nucleic acid strand.

[0013] In some embodiments, the method further comprises subjecting the plurality of double stranded nucleic acid molecules to an artificial jagged end process.4MF-365322596Docket No.: 197102019740

[0014] In some embodiments, the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

[0015] In some embodiments, the method further comprises partitioning the plurality of end repaired nucleic acid molecules based on a jagged end identifier into a first subsample and second subsample.

[0016] In some embodiments, the method further comprises amplifying the plurality of end repaired nucleic acid molecules. In some embodiments, the method further comprises ligating an adaptor to one or more ends of the plurality of end repaired nucleic acid molecules, wherein the adaptor includes at least a molecular barcode. In some embodiments, the corresponding sequence information includes information associated with the molecular barcode. In some embodiments, the method further comprises incorporating a nucleotide oligomer into the plurality of double stranded nucleic acid molecules or the plurality of end- repaired nucleic acid molecules. In some embodiments, incorporating the nucleotide oligomer comprises providing a ligase. In some embodiments, the ligase comprises a T4 DNA ligase. In some embodiments, the nucleotide oligomer comprises a barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules. In some embodiments, the nucleotide oligomer comprises a barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules. In some embodiments, the nucleotide oligomer comprises a first barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules, and a second barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules. In some embodiments, combined use of jagged end identifiers and incorporated nucleotide oligomers facilitates more accurate mapping, grouping, and determination of consensus sequence reads for each of the plurality of families.

[0017] In some embodiments, the method further comprises determining that at least a portion of the grouped sequence reads in a given family is due to a laboratory artifact, and where the laboratory artifact is not included in the consensus sequence read associated with the given family. In some embodiments, the laboratory artifact results from an amplification5MF-365322596Docket No.: 197102019740 error. In some embodiments, amplification error comprises a PCR error. In some embodiments, the laboratory artifact is a sequencing error.

[0018] Also disclosed herein are methods of correcting laboratory artifacts in sequence reads, the method comprising: receiving, at a processor, a plurality of sequence reads generated from a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules include a jagged end identifier; mapping, by a processor, the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information associated with the mapped sequence reads, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position and a stop position of a given mapped sequence read; grouping, by a processor, one or more of the plurality of mapped sequence reads into a plurality of families based the corresponding sequence information associated with a given mapped sequence read; based on the grouping, determining, by a processor, a consensus sequence for each of the plurality of families; and detecting, by a processor, one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0019] In some embodiments, the jagged end identifier has been inserted into a given nucleic acid molecule of the plurality of nucleic acid molecules by performing end repair on a plurality of double stranded nucleic acid molecules prior to preparing a sequencing library from a plurality of end repaired nucleic acid molecules. In some embodiments, the end repair comprises inserting one or more methylated cytosines to fill in one or more jagged end of a plurality of double stranded nucleic acid molecules. In some embodiments, the one or more methylated cytosines are inserted via a polymerase reaction. In some embodiments, the method further comprises performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil. In some embodiments, the plurality of double- stranded nucleic acid molecules comprise non-CpG-rich nucleic acid molecules. In some embodiments, the non-CpG-rich nucleic acid molecules map onto a non- CpG-rich region of the genome. In some embodiments, mapping further comprises determining a position of a first methylated cytosine in the 5’ direction that is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position.6MF-365322596Docket No.: 197102019740

[0020] In some embodiments, performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules. In some embodiments, the one or more inosines are inserted via a polymerase reaction. In some embodiments, the one or more inosines are inserted by hydrolytic deamination of adenine. In some embodiments, mapping further comprises determining a position of a first inosine in the 5’ direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position.

[0021] In some embodiments, the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules. In some embodiments, the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprises circulating tumor DNA (ctDNA) molecules. In some embodiments, the plurality of end repaired nucleic acid molecules are single stranded. In some embodiments, the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules.

[0022] In some embodiments, the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

[0023] Disclosed herein are methods comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules each have one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules. In some embodiments, the method further comprises attaching one or more adaptors to a plurality of nucleic acid molecules to create tagged molecules thereby generating a tagged nucleic acid molecule, wherein the plurality of adaptors each include one or more of a sample barcode and a molecular barcode.7MF-365322596Docket No.: 197102019740

[0024] Disclosed herein are methods for detecting a presence or an absence of cancer in a subject, comprising: (a) extracting a plurality of nucleic acid molecules from a first sample obtained from a subject, wherein the plurality of molecules have one or more jagged ends and the first sample is obtained at a first timepoint; (b) repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule; (d) amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; (f) selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and (g) sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data from a plurality of sequence reads; (h) repeating (a) to (g) with a second sample from the subject obtained at a second time point other than the first time point; and (i) detecting the presence or absence of cancer in the subject based on the sequence data changes between of the first sample and the second sample.

[0025] Disclosed herein are methods comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules have one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data associated with each of the plurality of enriched target nucleic acid molecules, wherein the sequence data includes a plurality of sequence reads; grouping the plurality of sequence reads based on at least the jagged end identifier associated with a particular sequence read to generate a plurality of families; determining, for each family, a consensus sequence for each family of the plurality of families; using the consensus sequence to detect one or more molecular alterations.

[0026] Disclosed herein are methods for marking a plurality of nucleic acid molecules, comprising: subjecting the plurality of nucleic acid molecules to a fragmentation process to8MF-365322596Docket No.: 197102019740 generate a plurality of jagged end nucleic acid molecules having one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; and selectively enriching the marked nucleic acid molecules for genomic regions of interest.

[0027] Disclosed herein are methods for identifying one or more molecular alterations in a sample obtained from a subject, comprising: repairing one or more jagged ends of a plurality of nucleic acid molecules extracted from the sample via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; determining a consensus sequence based on the jagged end identifier after the marked nucleic acid molecules are sequenced and identifying one or more molecular alterations in the sample based on the consensus sequence.

[0028] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.INCORPORATION BY REFERENCE

[0029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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:9MF-365322596Docket No.: 197102019740

[0031] FIG. 1 provides a non-limiting example of a process flowchart for detecting biological signals based on the use of jagged end identifiers, in accordance with one implementation of the disclosed methods.

[0032] FIG. 2 provides a non-limiting example of a process flowchart for detecting biological signals based on detection of jagged end identifiers in sequence read data, in accordance with another implementation of the disclosed methods.

[0033] FIG. 3 depicts an exemplary computing device or system in accordance with one embodiment of the present disclosure.

[0034] FIG. 4 depicts an exemplary computer system or computer network, in accordance with some instances of the systems described herein.

[0035] FIG. 5 provides a schematic illustration of a situation where a conventional start-stop deduplication approach fails to distinguish between two unique DNA molecules.

[0036] FIG. 6 provides a schematic illustration of a situation where a conventional start-stop deduplication approach succeeds, but the two unique DNA molecules can also be distinguished based on jagged end identifiers introduced during end repair using 5hmC in place of dCTP.

[0037] FIG. 7 provides a schematic illustration of a situation where two unique DNA molecules have the same start-stop locations but can still be distinguished based on jagged end identifiers introduced during end repair using 5hmC in place of dCTP.DETAILED DESCRIPTION

[0038] Methods for using jagged end identifiers (e.g., markers) incorporated into end repaired DNA overhang sequences of double stranded DNA fragments - more specifically, the 3’ and / or 5’ jagged ends of such fragments - in place of conventional UMIs to enable molecular tracking and facilitate sequencing error correction are described. These methods utilize end repair reactions for repairing jagged ends to introduce a jagged end identifier (or marker) into the 3’-end and / or 5’-end of DNA fragments comprising jagged ends (overhangs), that can then be used for molecular tracking and error correction of sequencing data. For example, in some implementations, replacing dCTP with 5hmC in double stranded library preparation (dsLP) end-repair reactions allows one to tag the 5’ jagged ends with methylated cytosines10MF-365322596Docket No.: 197102019740 which are resistant to methyl conversion when performing methylation sequencing. This is one non-limiting example, with several other methods of implementation described herein.

[0039] As noted above, the disclosed methods provide the advantages of a streamlined sequencing library workflow, can be used for both single-stranded library preparation (ssLP) and double- stranded library preparation (dsLP), and allow one to utilize sequencing capacity more efficiently than in UMI-based approaches while still providing sequencing error correction capability. The disclosed methods can also provide greater accuracy and improved reliability compared to endogenous methods for molecular tracking and error correction.

[0040] In some instances, for example, methods are described that comprise: obtaining a sample from a subject, the sample comprising a plurality of double stranded nucleic acid molecules having one or more jagged ends; performing end repair on the one or more double stranded nucleic acid molecules, where the end repair introduces a jagged end identifier within a given double stranded molecule of the plurality of double stranded molecules as part of the end repair; preparing a sequencing library comprising a plurality of end repaired nucleic acid molecules; sequencing the plurality of end repaired nucleic acid molecules to generate a plurality of sequence reads; mapping the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, where the sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read; grouping the mapped sequence reads into a plurality of families based on the sequence information; based on the grouping, determining a consensus sequence read for each of the plurality of families; and detecting one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0041] In some instances, methods (e.g., computer-implemented methods) are described that comprise: receiving, at a processor, a plurality of sequence reads generated from a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules include a jagged end identifier; mapping, by a processor, the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information associated with the mapped sequence reads, where the sequence information includes a jagged end position based on the jagged end identifier, a start position and a stop position of a given mapped sequence read; grouping, by a processor, one or more of the plurality of mapped sequence reads into a plurality of families based the sequence information associated11MF-365322596Docket No.: 197102019740 with a given mapped sequence read; based on the grouping, determining, by a processor, a consensus sequence for each of the plurality of families; and detecting, by a processor, one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0042] In some instances, performing end repair further comprises inserting one or more methylated cytosines (e.g., incorporating one or more 5hmC nucleotides, or other methyl- modified forms of deoxycytosine triphosphate (dCTP)) to fill in the one or more jagged end of the plurality of double stranded molecules. In some instances, performing end repair further comprises inserting one or more inosines (e.g., by incorporating one or more inosine nucleotides, or by performing hydrolytic deamination of one or more incorporated deoxyadenosine triphosphates (dATPs)) to fill in the one or more jagged ends of the plurality of double stranded molecules.

[0043] In some instances, the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) or cell free RNA (cfRNA). In some instances, the double stranded nucleic acid molecules are cell free DNA (cfDNA), and the cfDNA comprises circulating tumor DNA (ctDNA).

[0044] In some instances, the disclosed methods comprising the use of jagged end identifiers for correction of consensus sequence reads may be used as a stand-alone method for performing deduplication. In some instances, the disclosed methods comprising the use of jagged end identifiers may be used in combination with, e.g., start-stop deduplication and / or unique molecular identifiers (UMIs) to perform correction of consensus sequence reads.

[0045] In some instances, the one or more biological signals may comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.Definitions

[0046] 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.12MF-365322596Docket No.: 197102019740

[0047] 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.

[0048] “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.

[0049] 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.

[0050] 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.

[0051] As used herein, the term “disease” refers to any disorder of structure or function in a human, animal, or plant, especially one that has a known cause and / or a distinctive group of symptoms, signs, or anatomical changes. In some instances, the term “disease” as used herein refers to any disorder of structure or function in a human, animal, or plant that is related to a change (e.g., an alteration) in a nucleic acid sequence (e.g., a DNA and / or RNA sequence) that arises through a genetic, genomic, epigenomic, and / or proteomic process.

[0052] The terms “cancer” and “tumor” are used interchangeably herein. These terms refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a13MF-365322596Docket No.: 197102019740 metastatic lesion. As used herein, the term “cancer” includes premalignant, as well as malignant cancers.

[0053] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention (e.g., administration of an anti-cancer agent or anticancer therapy) 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.

[0054] As used herein, the term “subgenomic interval” (or “subgenomic sequence interval”) refers to a portion of a genomic sequence.

[0055] As used herein, the term "subject interval" refers to a subgenomic interval or an expressed subgenomic interval (e.g., the transcribed sequence of a subgenomic interval).

[0056] As used herein, the term “sequence read” is a computationally generated sequence generated by a sequencer to represent a sequence of bases of a single strand of a sequenced fragment. A sequence read can refer to a raw sequence read (e.g., a sequence read as obtained directly from a sequencing instrument), an aligned sequence read (e.g., a sequence read that has been aligned to a reference genome), a single-end sequence read, a paired-end sequence read, a merged sequence read (e.g., a sequence read based on merging a group of overlapping paired-end reads), a consensus sequence read (e.g., a sequence read based on performing error correction on a merged sequence read), a computationally reconstructed sequence read (e.g., a sequence read that has been computationally truncated at the 5’ and / or 3’ end), or any combination thereof.

[0057] As used herein, the terms “variant sequence”, “variant”, or “alteration” are used interchangeably and refer to a modified nucleic acid sequence relative to a corresponding “normal” or “wild-type” sequence. In some instances, a variant sequence may be a “short variant sequence” (or “short variant”), i.e., a variant sequence of less than about 50 base pairs in length. In some instances, a variant sequence may be a single nucleotide variant (SNV), an14MF-365322596Docket No.: 197102019740 insertion, a deletion, a copy number variation (CNV), a rearrangement, or a change in methylation status of a nucleotide residue (e.g., a change in cytosine methylation).

[0058] The terms “allele frequency” and “allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular allele relative to the total number of sequence reads for a genomic locus.

[0059] The terms “variant allele frequency” and “variant allele fraction” are used interchangeably herein and refer to the fraction of sequence reads corresponding to a particular variant allele relative to the total number of sequence reads for a genomic locus.

[0060] As used herein, the term “unique molecular identifier” refers to a marker used to enable molecular tracking and facilitate sequencing error correction. Unique molecular identifier (UMI) can comprise unique molecular barcodes (e.g., unique oligonucleotide barcode sequences) attached to an end of a nucleic acid fragment, non-unique molecular barcodes (e.g., non-unique oligonucleotide barcode sequences), endogenous sequence information (e.g., specific reference sequences, or start positions and / or stop positions of sequence reads that map to specific reference sequence region), jagged end identifiers, or any combination thereof.

[0061] As used herein, the term “molecular barcode” refers to an oligonucleotide barcode sequence that is attached (e.g., ligated) to an end of a nucleic acid fragment. In some instances, molecular barcodes may be unique molecular barcodes (e.g., the length of the barcode sequence can be selected such that the number of possible unique sequences represented by an oligonucleotide of the specified length is much larger than the number of unique nucleic acid fragment molecules to be labeled, thereby ensuring (to a high probability) that each unique molecular fragment is labeled with a different molecular barcode). In some instances, molecular barcodes may be non-unique molecular barcodes (e.g., more than one unique nucleic acid fragment molecule may be labeled with the same molecular barcode). In some instances, non-unique molecular barcodes may be combined with unique molecular barcodes, endogenous sequence information (as described above), and / or jagged end identifiers to enable molecular tracking and facilitate sequencing error correction.

[0062] As used herein, the term “jagged end identifier” refers to a marker introduced to the 3 ’-end and / or 5 ’-end of a unique nucleic acid fragment comprising an overhang that can subsequently be used to enable molecular tracking and facilitate sequencing error correction.15MF-365322596Docket No.: 197102019740Jagged end identifiers can be introduced through the end repair of DNA overhangs performed during sequencing library preparation and can comprise, e.g., a unique pattern of methylated cytosines or other modified nucleotide residues that occur at the 3 ’-end and / or 5 ’-end of a nucleic acid fragment. In some instances, jagged end identifiers may be used alone to enable molecular tracking and facilitate sequencing error correction. In some instances, jagged end identifiers may be used in combination with unique molecular barcodes and / or non-unique molecular barcodes to enable molecular tracking and facilitate sequencing error correction.

[0063] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Methods for correcting sequencing errors based on jagged end identifiers

[0064] The analysis of cell-free DNA (cfDNA) can be particularly complex when identifying low-frequency alterations or analyzing samples having relatively low DNA content. This is especially true when trying to identify signal in a sample for early stages of diseases like cancer or when looking for variants that have a very variant allele frequency.

[0065] One of the known problems with sequencing cfDNA is its error rate due to sequencing errors and / or other library contamination issues (e.g., errors introduced through amplification, methylation bias and / or errors during sequencing). In typical sequencing technologies this error rate can 1-3%. Generally this error rate is not an issue when you know the source of all of the molecules you are sequencing.

[0066] However, when you are collecting molecules from various sources and / or the frequency of, for example, a variant that you are trying to identify is at a very low variant allele frequency in the sample this noise reduce the accuracy of the assay. For example, cfDNA includes fragmented DNA from many sources including germline DNA, non-tumor cell free DNA, cell free fetal DNA (in pregnant women) and circulating tumor DNA (in subjects with oncogenesis). Therefore when the frequency of molecules with sequence variants at the same rate as the errors introduced during sequencing, then sequence variants may not be distinguishable from noise. This can lead to decreased sensitivity in the assay.

[0067] Standard practice has been to use unique molecular identifiers (UMIs) to identify or track molecules through the sequencing process to be able to more readily identify errors from true variants or alterations (e.g., single nucleotide variants (SNVs), insertions, deletions, copy number variations (CNVs), rearrangements, etc.). As mentioned above, conventional 16MF-365322596Docket No.: 197102019740UMI’s have been understood to be comprised of three different types. Exogenous only (e.g., unique molecular barcodes), endogenous (e.g., using the molecule’s sequence information itself, and semi-exogenous (e.g., use of non-unique molecular barcodes in combination with other information (e.g., genomic / transcriptomic coordinates of the molecule).

[0068] When using molecular barcodes (unique or non-unique), the molecular barcodes are typically attached into each polynucleotide during the library construction process prior to amplification. This can be done by ligation of a molecular barcode to one or both ends of either a double stranded or single stranded complements. These barcodes are then used, alone (exogenously unique) or in combination (exogenously non-unique) to identify which molecules within an amplicon are progeny of a single unique DNA molecule, and thus to help to distinguish between true variants and amplification artifacts once the amplicons have been sequenced. However, the use of molecular barcodes requires one to perform additional steps during the preparation of sequencing libraries, can be complicated to design effectively (especially when using an exogenously unique approach) and the addition of the molecular barcode sequences to each strand of a double-stranded DNA fragment can use up sequencing and computation capacity in a given sequencing run in order to determine the barcode sequences. This can be especially true in assays that require a larger number of nucleotides in each molecular barcode to achieve the desired uniqueness.

[0069] Additionally, endogenous methods have found to be less effective given that sequencing information alone can be somewhat unreliable due to the number of errors that can occur during sequencing that can cause the molecules / reads / fragments not to be identified appropriately during computational processing. This is mostly to do with the fact the sequencing information is unknown prior to sequencing and as a result can be contaminated by any chemistry or sequencing errors could create inaccuracies in the sequencing information. As a result, different UMIs may be assigned to two different reads that both originated from the same molecule. For this reason, endogenous methods have conventionally been thought to have inferior performance to exogenous or semi-exogenous methods.

[0070] The present embodiments, however, provide a new type of UMI that does not require and attachment or use molecular barcodes. It also does utilize only endogenous information about the nucleic acid molecule itself.17MF-365322596Docket No.: 197102019740

[0071] The methods, systems and assays described herein enable identification of unique, non-PCR duplicated nucleic acid molecules in either double stranded library preparation (dsLP) or single stranded library preparation (ssLP) workflows. In particular, the present invention leverages jagged ends (natural or synthetic) of nucleic acid fragments (e.g., cfDNA fragments) and their unique characteristics as unique molecular identifiers, rather than resorting to the conventional UMI approaches where a molecular barcode or tag is exogenously added to the molecule, e.g., the adaptor ligated to nucleic acid fragments as part of the sequencing library preparation process. In particular, in the present invention a marker is added during repair of jagged ends to identify the molecule (both the + and - strand) during computational processing. This solution poses a unique advantage over the conventional exogenous approaches (which add complexity to the assay) and solves a current pain point in library preparation workflows.

[0072] The disclosed methods allow one to bypass the use of traditional UMIs through utilizing the inherent characteristics of jagged ends on nucleic acid fragments. "Jagged ends" on nucleic acid fragments refer to when one strand of a double- stranded DNA molecule extends beyond the other at the end, thereby creating an overhang. Specifically, one strand has a single-stranded overhang while the other strand is blunt, resulting from DNA fragmentation processes often involving nuclease activity. This fragmentation can be done both naturally and synthetically through, e.g., sonification, enzymatic reactions, or other means.

[0073] To correct these jagged ends on nucleic acid fragments, end repair is a necessary step in double stranded library preparation (dsLP), and taking advantage of this required reaction to introduce jagged end identifiers is both advantageous and novel. In the context of single stranded library preparation (ssLP), the disclosed methods provide two benefits. First, jagged end information is lost in canonical ssLP but will be retained through the use of the methods described herein. Second, the disclosed methods provide an additional molecular tag (as with dsLP) to facilitate the identification of unique nucleic acid molecules present in the original sample.

[0074] FIG. 1 provides a non-limiting example of a flowchart for a process 100 for detecting biological signals based on the use of jagged end identifiers, in accordance with one implementation of the disclosed methods.18MF-365322596Docket No.: 197102019740

[0075] At step 102 in FIG. 1, a sample is obtained from a subject, the sample comprising a plurality of double stranded nucleic acid molecules having one or more jagged ends (e.g., 5’ overhanging ends and / or 3’ overhanging ends).

[0076] In some instances, the sample may be a tissue biopsy sample, a liquid biopsy sample, or a control sample (e.g., to verify that the workflow has been executed as expected). In some instances, the sample is a liquid biopsy sample and may comprise, e.g., blood, plasma, cerebrospinal fluid, sputum, stool, urine, bone aspirate, or saliva. In some instances, the sample is a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample is a liquid biopsy sample and may comprise cell-free DNA (cfDNA). In some instances, the cell-free DNA (cfDNA) or a portion thereof may comprise circulating tumor DNA (ctDNA).

[0077] Accordingly, in some instances, the plurality of double stranded nucleic acid molecules may be cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules. In some instances, the double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and may comprise circulating tumor DNA (ctDNA) molecules.

[0078] In some instances, the plurality of double stranded nucleic acid molecules may be naturally fragmented. In other instances, the method may further comprise subjecting the plurality of double stranded nucleic acid molecules to an artificial or synthetic jagged end process. For example, in some instances, the double stranded nucleic acid molecules may be treated with a restriction enzyme (e.g., a restriction endonuclease) to create jagged ends in fragments of nucleic acid molecules that formerly had blunt ends. Alternatively, the plurality of double stranded nucleic acid molecules may be subjected to a sonification process to create jagged end on the plurality of double stranded nucleic acid molecules.

[0079] At step 104 in FIG. 1, end repair is performed on the one or more double stranded nucleic acid molecules, where the end repair introduces a jagged end identifier within a given double stranded molecule of the plurality of double stranded molecules as part of performing the end repair reaction. A jagged end identifier is a “marker” that can be used, e.g., in lieu of or in combination with either conventional endogenous or exogenous UMIs described above to distinguish between unique nucleic acid molecules that were present in the sample, and to identify copies thereof that include amplification and / or sequencing errors.19MF-365322596Docket No.: 197102019740

[0080] In some instances, performing end repair may further comprise inserting one or more methylated cytosines (e.g., by incorporating one or more methyl-modified deoxycytosine triphosphates (dCTPs)) to fill in the one or more jagged ends of the plurality of double stranded molecules, particularly if the double stranded nucleic acid molecules are not from a CpG rich region. Examples of methyl-modified cytosine nucleotides that may be used include, but are not limited to, 5-methyl-cytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), pyrrolo-dC, and propynyl-dC (or any cytosine modification that is resistant to enzymatic or chemical conversion). The one or more methylated cytosines can be inserted (or incorporated), for example, via a polymerase reaction, e.g., a 3’ extension reaction to synthesize the complementary strand to a 5’ overhanging end.

[0081] In some instances, e.g., when the insertion of one or more methyl cytosines is used to introduce a jagged end identifier, the method can further comprise performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil residue. In some instances, the conversion reaction is an enzymatic conversion reaction. In some instances, the conversion reaction is a chemical conversion reaction, e.g., a bisulfite conversion reaction.

[0082] In some instances, performing end repair may further comprise inserting one or more inosines (e.g., by incorporating one or more inosine nucleotides; inosine is a nitrogenous base analog that can base pair with adenine, cytosine, or uracil) to fill in the one or more jagged ends of the plurality of double stranded molecules. In some instances, for example, one or more inosines (e.g., inosine nucleotides) may be incorporated via a polymerase reaction, e.g., a 3’ extension reaction to synthesize a complementary strand (or partially complementary strand) to a 5’ overhanging end and thereby generate a jagged end index. This would result in a marker comprising errors in the filled in portion of the double stranded molecule such that a region of the corresponding sequence read does not map effectively to the reference sequence, and would thus need to be soft-clipped (e.g., a process by which bases from the ends of sequence reads that do not align with the reference sequence are computationally removed / disregarded to improve alignment accuracy).

[0083] In some instances, the plurality of end repaired nucleic acid molecules produced by performing end repair may be single stranded, e.g., if the double stranded nucleic acid molecules have been denatured. In some instances, the plurality of end repaired nucleic acid molecules may be double stranded nucleic acid molecules.20MF-365322596Docket No.: 197102019740

[0084] At step 106 in FIG. 1, a sequencing library is prepared, the sequencing library comprising a plurality of end repaired nucleic acid molecules.

[0085] In some instances, the double- stranded nucleic acid molecules may undergo doublestranded nucleic acid molecule library preparation. In some instances, the double-stranded nucleic acid molecule library preparation may comprise denaturing the double- stranded nucleic acid molecules. In some instances, the double-stranded nucleic acid molecule library preparation may comprise performing polyA-tailing of the denatured double- stranded nucleic acid molecules.

[0086] In some instances, the plurality of end repaired nucleic acid molecules may undergo single- stranded nucleic acid molecule library preparation. In some instances, the singlestranded nucleic acid molecule library preparation may comprise synthesizing a second (complementary) nucleic acid strand.

[0087] In some instances, the method may further comprise partitioning the plurality of end repaired nucleic acid molecules based on a jagged end identifier into a first subsample and second subsample.

[0088] In some instance, the method may further comprise amplifying the plurality of end repaired nucleic acid molecules, e.g., using a PCR amplification technique.

[0089] In some instances, the method allows for use of both the jagged end identifier and molecular barcodes where increased certainty is required, the method may further comprise ligating an adaptor to one or more ends of the plurality of end repaired nucleic acid molecules, where the adaptor includes at least a molecular barcode. In some instances, the sequence information includes information associated with the molecular barcode.

[0090] In some instances, the method may further comprise incorporating a nucleotide oligomer into the plurality of double stranded nucleic acid molecules or the plurality of end- repaired nucleic acid molecules. In some instances, incorporating the nucleotide oligomer may comprise providing a ligase, e.g., a T4 DNA ligase. In some instances, the nucleotide oligomer may comprise a molecular barcode of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules. In some instances, the nucleotide oligomer may comprise a barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules. In some instances, the nucleotide oligomer may comprise a first barcode that21MF-365322596Docket No.: 197102019740 is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules, and a second barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

[0091] In some instances, the use of a nucleotide oligomer (e.g., a molecular barcode) in combination with the jagged end identifiers disclosed herein and / or start-stop deduplication may provide for more accurate mapping, grouping, and determination of consensus sequence reads for each of a plurality of families (e.g., by expanding the available coding space for distinguishing between unique nucleic acid molecules present in the original sample).

[0092] At step 108 in FIG. 1, the plurality end repaired nucleic acid molecules in the sequencing library is sequenced to generate a plurality of sequence reads.

[0093] In some instances, the sequencing may comprise, for example, whole genome sequencing (WGS), whole transcriptome sequencing (WTS) whole exome sequencing, targeted sequencing, direct sequencing, methylation sequencing or targeted RNA sequencing. In some instances, sequencing may be performed using, e.g., sequencing-by- synthesis (SBS) or Sanger sequencing. In some instances, the sequencing may comprise a paired-end sequencing technique that allows both ends of a nucleic acid fragment to be sequenced and generates high-quality sequence data for detection of, e.g., genomic rearrangements, repetitive sequence elements, gene fusions, and novel transcripts.

[0094] At step 110 in FIG. 1, the plurality of sequence reads is mapped to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, where the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read.

[0095] In some instances, the plurality of sequence reads may be mapped or aligned to, e.g., a human reference genome (e.g., human reference genome HG38, HG19, etc.).

[0096] In some instances, the mapping may further comprise determining or identifying a position of a jagged end identifier, e.g., a first methylated cytosine, in the 5’ direction that is not part of a CpG region in a given mapped sequence read, where a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position.22MF-365322596Docket No.: 197102019740

[0097] In some instances, the mapping my further comprise determining a position of a jagged end identifier, e.g., a first inosine, in the 5’ direction in the given mapped sequence read, where a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position. As noted above, incorporation of one or more inosines (e.g., through the use of diTP during end repair) would result in a marker comprising errors in the filled in portion of the double stranded molecule such that a region of the corresponding sequence read does not map effectively to the reference sequence, and would thus need to be soft-clipped.

[0098] At step 112 in FIG. 1, the mapped sequencing read are grouped into a plurality of families based on the sequence information.

[0099] At step 114 in FIG. 1, a consensus sequence read is determined for each of the plurality of families based on the grouping of sequence reads.

[0100] In some instances, the method may further comprise determining that at least a portion of the grouped sequence reads in a given family is due to a laboratory artifact, where the laboratory artifact is not included in the consensus sequence read or in an alteration detection analysis associated with the given family. In some instances, for example, the laboratory artifact may result from an amplification error (e.g., a PCR error) and / or a sequencing error.

[0101] At step 116 in FIG. 1, one or more biological signals are detected in the consensus sequence read for at least one family of the plurality of families. The one or more biological signals may comprise, for example, a genomic signal (e.g., a signal relating to a presence or absence of a short variant, a rearrangement (e.g., a fusion), a splice variant, a structural variant, an insertion, a deletion, copy number alterations etc.), an epigenetic signal (e.g., a signal relating to a methylation status, a methylation signature, etc.), a molecular characteristic (e.g., a length of a molecule, a jagged end characteristic, etc.), a transcriptomic signal (e.g., a signal relating to a gene expression level, or relating to a presence or absence of a short variant, a rearrangement (e.g., a fusion), a splice variant, a structural variant, an insertion, a deletion, a frameshift variant, etc.), a proteomic signal (e.g., a signal relating to a presence or absence of a peptide or protein as determined based on the consensus sequence read data), or any combination thereof.

[0102] FIG. 2 provides a non-limiting example of a flowchart for a process 200 for detecting biological signals based on detection of jagged end identifiers in sequence read data. Process23MF-365322596Docket No.: 197102019740200 can be performed, for example, using one or more electronic devices implementing a software platform. In some examples, process 200 is performed using a client-server system, and the blocks of process 200 are divided up in any manner between the server and a client device. In other examples, the blocks of process 200 are divided up between the server and multiple client devices. Thus, while portions of process 200 are described herein as being performed by particular devices of a client-server system, it will be appreciated that process 200 is not so limited. In other examples, process 200 is performed using only a client device or only multiple client devices. In process 200, some blocks are, optionally, combined, the order of some blocks is, optionally, changed, and some blocks are, optionally, omitted. In some examples, additional steps may be performed in combination with the process 200. Accordingly, the operations as illustrated (and described in greater detail below) are exemplary by nature and, as such, should not be viewed as limiting.

[0103] At step 202 in FIG. 2, sequence read data for a plurality of sequence reads generated from a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules include a jagged end identifier, is received, e.g., by one or more processors of a system configured to perform the process depicted in FIG. 2.

[0104] In some instances, the sequence read data may comprise data for sequence reads derived using, for example, whole genome sequencing (WGS), whole exome sequencing, targeted sequencing, direct sequencing methylation sequencing, or RNA sequencing. In some instances, sequencing may be performed using, e.g., sequencing-by-synthesis (SBS) or Sanger sequencing. In some instances, the sequencing may comprise a paired-end sequencing technique that allows both ends of a nucleic acid fragment to be sequenced and generates high-quality sequence data for detection of, e.g., genomic rearrangements, repetitive sequence elements, gene fusions, and novel transcripts.

[0105] In some instances, the jagged end identifier has been inserted into a given nucleic acid molecule of the plurality of nucleic acid molecules by performing end repair on a plurality of double stranded nucleic acid molecules prior to preparing a sequencing library from a plurality of end repaired nucleic acid molecules. The end repair can comprise, for example, filling in 5’ overhangs in double stranded nucleic acid molecules using a polymerase- catalyzed 3’ extension reaction. In some instances, the end repair reaction can comprise, e.g., inserting one or more methylated cytosines to fill in the one or more jagged ends of the plurality of double stranded molecules. In some instances, the end repair reaction can24MF-365322596Docket No.: 197102019740 comprise, e.g., inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

[0106] In some instances, the plurality of double stranded nucleic acid molecules may be cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules. In some instances, the plurality of double stranded nucleic acid molecules may be cell free DNA (cfDNA) molecules, and the cfDNA molecules may comprise circulating tumor DNA (ctDNA) molecules.

[0107] In some instances, the plurality of end repaired nucleic acid molecules may be single stranded. In some instances, the plurality of end repaired nucleic acid molecules may be double stranded nucleic acid molecules.

[0108] In some instances, the end repair may comprise inserting one or more methylated cytosines to fill in one or more jagged end of a plurality of double stranded nucleic acid molecules. In some instances, the one or more methylated cytosines may be inserted via a polymerase reaction. In some instances, the method may further comprise performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil.

[0109] In some instances, the plurality of double-stranded nucleic acid molecules may comprise non-CpG-rich nucleic acid molecules. In some instances, the non-CpG-rich nucleic acid molecules map onto a non-CpG-rich region of the genome.

[0110] In some instances, performing end repair may further comprise inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules. In some instances, the one or more inosines may be inserted via a polymerase reaction. In some instances, the one or more inosines may be inserted by hydrolytic deamination of adenine.

[0111] At step 204 in FIG. 2, the plurality of sequence reads is mapped to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, where the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read.25MF-365322596Docket No.: 197102019740

[0112] In some instances, the plurality of sequence reads may be mapped to, e.g., a human reference genome (e.g., human reference genome HG38, HG19, etc.).

[0113] In some instances, the mapping may further comprise determining a position of a first methylated cytosine in the 5’ direction that is not part of a CpG region in the given mapped sequence read, where a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position.

[0114] In some instances, the mapping may further comprise determining a position of a first inosine in the 5’ direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position. As noted above, incorporation of one or more inosines (e.g., through the use of diTP during end repair) would result in a marker comprising errors in the filled in portion of the double stranded molecule such that a region of the corresponding sequence read does not map effectively to the reference sequence, and would thus need to be soft-clipped.

[0115] At step 206 in FIG. 2, mapped sequencing read are grouped into a plurality of families based on the corresponding sequence information.

[0116] At step 208 in FIG. 2, a consensus sequence read is determined for each of the plurality of families based on the grouping of sequence reads.

[0117] In some instances, the method may further comprise determining that at least a portion of the grouped sequence reads in a given family is due to a laboratory artifact, where the laboratory artifact is not included in the consensus sequence read associated with the given family. In some instances, for example, the laboratory artifact may result from an amplification error (e.g., a PCR error) and / or a sequencing error.

[0118] In some instances, the use of a nucleotide oligomer (e.g., a molecular barcode) in combination with the jagged end identifiers disclosed herein and / or start-stop deduplication may provide for more accurate mapping, grouping, and determination of consensus sequence reads for each of a plurality of families (e.g., by expanding the available coding space for distinguishing between unique nucleic acid molecules present in the original sample).

[0119] At step 210 in FIG. 2, one or more biological signals are detected in the consensus sequence read for at least one family of the plurality of families. As noted above with respect26MF-365322596Docket No.: 197102019740 to FIG. 1, the one or more biological signals may comprise, for example, a genomic signal (e.g., a signal relating to a presence or absence of a short variant, a rearrangement (e.g., a fusion), a splice variant, a structural variant, an insertion, a deletion, etc.), an epigenetic signal (e.g., a signal relating to a methylation status, a methylation signature, etc.), a molecular characteristic (e.g., a length of a molecule, a jagged end characteristic, etc.), a transcriptomic signal (e.g., a signal relating to a gene expression level, or relating to a presence or absence of a short variant, a rearrangement (e.g., a fusion), a splice variant, a structural variant, an insertion, a deletion, a frameshift variant, etc.), a proteomic signal (e.g., a signal relating to a presence or absence of a peptide or protein as determined based on the consensus sequence read data), or any combination thereof.Methods of use

[0120] In some instances, the disclosed methods may further comprise one or more of the steps of: (i) obtaining the sample from the subject (e.g., a subject suspected of having or determined to have cancer), (ii) extracting nucleic acid molecules (e.g., a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules) from the sample, (iii) ligating or attaching one or more adapters and / or molecular barcodes to the nucleic acid molecules extracted from the sample (e.g., ligating or attaching one or more amplification primers, flow cell adaptor sequences, substrate adapter sequences, sample index sequences, unique molecular barcodes, and / or non-unique molecular barcodes), (iv) performing a methylation conversion reaction to convert, e.g., non-methylated cytosine to uracil, in the case that a methylation sequencing assay is being performed, (v) amplifying the nucleic acid molecules (e.g., using a polymerase chain reaction (PCR) amplification technique, a non-PCR amplification technique, or an isothermal amplification technique), (vi) optionally, capturing nucleic acid molecules from the amplified nucleic acid molecules (e.g., by hybridization to one or more bait molecules, where the bait molecules each comprise one or more nucleic acid molecules that each comprising a region that is complementary to a region of a captured nucleic acid molecule) depending on the sequencing method used, (vii) sequencing the nucleic acid molecules extracted from the sample (or library proxies derived therefrom) using, e.g., a next-generation (massively parallel) sequencing technique, a whole genome sequencing (WGS) technique, a whole exome sequencing technique, a targeted sequencing technique, a direct sequencing technique, or a Sanger sequencing technique) using, e.g., a next-generation (massively parallel) sequencer, (viii) performing error correction on sequence27MF-365322596Docket No.: 197102019740 reads using jagged end identifiers and / or other molecular barcoding methods to generate corrected sequence read data, (ix) determining whether one or more alterations are present based on the corrected sequence read data, (x) combining the nucleic acid sequence data (including, e.g., variant data, copy number data, methylation status data, etc., of the sequenced nucleic acid molecules) with other biomarker data modalities including, but not limited to, proteomics-based biomarker data (e.g., the detection of specific polypeptides, such as proteins) or fragmentomics-based biomarker data (e.g., the detection of certain attributes related to nucleic acid fragments, such as fragment size or the sequences of fragment ends), to determine, for example, the presence of ctDNA in the sample and / or to determine a diagnostic, prognostic, and / or treatment response prediction for the subject, and (xi) generating, displaying, transmitting, and / or delivering a report (e.g., an electronic, web-based, or paper report) to the subject (or patient), 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 instances, the report comprises output from the methods described herein. In some instances, all or a portion of the report may be displayed in the graphical user interface of an online or web-based healthcare portal. In some instances, the report is transmitted via a computer network or peer-to-peer connection.

[0121] The disclosed methods may be used with any of a variety of samples. For example, in some instances, the sample may comprise a tissue biopsy sample, a liquid biopsy sample, or a normal control. In some instances, the sample may be a liquid biopsy sample and may comprise blood, plasma, cerebrospinal fluid, sputum, stool, urine, bone aspirate or saliva. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA). In some instances, the cell-free DNA (cfDNA), or a portion thereof, may comprise circulating tumor DNA (ctDNA). In some instances, the liquid biopsy sample may comprise a combination of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA).

[0122] In some instances, the nucleic acid molecules extracted from a sample may comprise a mixture of tumor nucleic acid molecules and non-tumor nucleic acid molecules. In some instances, the tumor nucleic acid molecules may be derived from a tumor portion of a heterogeneous tissue biopsy sample, and the non-tumor nucleic acid molecules may be derived from a normal portion of the heterogeneous tissue biopsy sample. In some instances,28MF-365322596Docket No.: 197102019740 the sample may comprise a liquid biopsy sample, and the tumor nucleic acid molecules may be derived from a circulating tumor DNA (ctDNA) fraction of the liquid biopsy sample while the non-tumor nucleic acid molecules may be derived from a non-tumor, cell-free DNA (cfDNA) fraction of the liquid biopsy sample.

[0123] In some instances, the disclosed methods for correcting consensus sequence reads may be used as part of a method to diagnose (or as part of a diagnosis of) the presence of disease or other condition (e.g., cancer, genetic disorders (such as Down Syndrome and Fragile X), neurological disorders, or any other disease type where detection of variants, e.g., copy number alternations, are relevant to diagnosing, treating, or predicting said disease) in a subject e.g., a patient). In some instances, the disclosed methods may be applicable to any sequencing-based diagnosis of any of a variety of cancers as described elsewhere herein.

[0124] In some instances, the disclosed methods for correcting consensus sequence reads may be used as part of a method to predict genetic disorders in fetal DNA. (e.g., for invasive or non-invasive prenatal testing). For example, sequence read data obtained by sequencing fetal DNA extracted from samples obtained using invasive amniocentesis, chorionic villus sampling (cVS), or fetal umbilical cord sampling techniques, or obtained using non-invasive sampling of cell-free DNA (cfDNA) samples (which comprises a mix of maternal cfDNA and fetal cfDNA), may be processed according to the disclosed methods to identify variants, e.g., copy number alterations, associated with, e.g., Down Syndrome (trisomy 21), trisomy 18, trisomy 13, and extra or missing copies of the X and Y chromosomes.

[0125] In some instances, the disclosed methods for correcting consensus sequence reads or identifying errors in reads may be used as part of a method to select a subject (e.g., a patient) for a clinical trial based on, e.g., the detection of one or more variants at one or more gene loci. In some instances, patient selection for clinical trials based on, e.g., identification of one or more variants at one or more gene loci, may accelerate the development of targeted therapies and improve the healthcare outcomes for treatment decisions.

[0126] In some instances, the disclosed methods for correcting consensus sequence reads or identifying errors in reads may be used as part of a method to select an appropriate therapy or treatment (e.g., an anti-cancer therapy or anti-cancer treatment) for a subject. In some instances, for example, the anti-cancer therapy or treatment may comprise use of a poly (ADP-ribose) polymerase inhibitor (PARPi), a platinum compound, chemotherapy, radiation29MF-365322596Docket No.: 197102019740 therapy, a targeted therapy, an immunotherapy, a neoantigen-based therapy, surgery, or any combination thereof.

[0127] In some instances, the anti-cancer therapy or treatment may comprise a targeted anticancer therapy or treatment (e.g., a monoclonal antibody-based therapy, an enzyme inhibitorbased therapy, an antibody-drug conjugate therapy, a hormone therapy, and / or a targeted radiotherapy) that targets specific molecules required for cancer cell growth, division, and spreading. In some instances, the targeted anti-cancer therapy or treatment may comprise abemaciclib (Verzenio), abiraterone acetate (Zytiga), acalabrutinib (Calquence), ado- trastuzumab emtansine (Kadcyla), afatinib dimaleate (Gilotrif), 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), 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), ipilimumab (Yervoy), isatuximab-irfc30MF-365322596Docket No.: 197102019740(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), 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), 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.

[0128] In some instances, the anti-cancer therapy or treatment may comprise an immunotherapy (e.g., a cancer treatment that acts by stimulating the immune system to fight cancer). In some instances, the immunotherapy can be, for example, an immune system modulator (e.g., a cytokine, such as an interferon or interleukin), an immune checkpoint inhibitor (such as an anti-PD-1 or anti-PD-Ll antibody), a T-cell transfer therapy (e.g., a31MF-365322596Docket No.: 197102019740 tumor infiltrating lymphocyte (TIL) therapy in lymphocytes extracted from a patient’s tumor are selected for their ability to recognize tumor cells and propagated prior to reintroduction into the patient, or a CAR T-cell therapy in which a patient’s T-cells are modified to express the CAR protein prior to reintroduction into the patient), a monoclonal antibody-based therapy (e.g., a monoclonal antibody that binds to cell surface markers on cancer cells to facilitate recognition by the immune system), or a cancer treatment vaccine (e.g., a vaccine based on tumor cells, tumor-associated neoantigens, or dendritic cells, etc., that stimulates the immune system to fight cancer).

[0129] In some instances, the anti-cancer therapy or treatment may comprise a neoantigenbased therapy. Non-limiting examples of neoantigen-based therapies include T-cell receptor (TCR) engineered T-cell (TCR-T) therapies, chimeric antigen receptor T-cell (CAR-T) therapies, TCR bispecific antibody therapies, and cancer vaccines. TCR-T therapies are produced by genetically engineering a patient’s T-cells to express T-cell receptors that are specific to neoantigens of interest, and then infusing them back into the patient. CAR-T therapies are produced by genetically engineering a patient’s T-cells to express chimeric antigen receptor molecules which contain an intracellular signaling and co-signaling domain as well as an extracellular antigen-binding domain; CAR-T therapies don’t always rely on neoantigen presentation, but can be designed to be directed towards neoantigens. TCR bispecific antibody therapies are small, engineered antibody molecules that comprise a neoantigen-specific TCR on one end and a CD3-directed single-chain variable fragment on the other end. Cancer vaccines can include RNA molecules, DNA molecules, peptides, or a combination thereof that are designed to boost the immune system’s ability to find and destroy neoantigen-presenting cells.

[0130] In some instances, the disclosed methods for correcting consensus sequence reads or identifying errors in reads may be used as part of a method for treating a disease (e.g., a cancer) in a subject. For example, in response to identifying the presence of one or more variants in one or more gene loci using any sequencing-based method such as those disclosed herein, an effective amount of an anti-cancer therapy or anti-cancer treatment may be administered to the subject.

[0131] In some instances, the disclosed methods for correcting consensus sequence reads or identifying errors in reads may be used as part of a sequencing-based method for monitoring disease progression or recurrence (e.g., cancer or tumor progression or recurrence) in a 32MF-365322596Docket No.: 197102019740 subject. For example, in some instances, the methods may be used to determine a first biological signal (e.g., first mutational pattern) in a first sample obtained from the subject at a first time point, and used to determine a second biological signal (e.g., a second mutational pattern) in a second sample obtained from the subject at a second time point, where comparison of the first biological signal and the second biological signal 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.

[0132] In some instances, 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 of a biological signal.

[0133] In some instances, the value of a biological signal determined 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.

[0134] In some instances, the disclosed methods for correcting consensus sequence reads or identifying errors in reads may be implemented as part of a genomic profiling process that comprises identification of the presence of variant sequences at one or more gene loci in a sample derived from a subject as part of detecting, monitoring, predicting a risk factor, or selecting a treatment for a particular disease, e.g., cancer. 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 correcting consensus sequence reads as part of a genomic profiling process (or inclusion of the output from the disclosed methods for correcting33MF-365322596Docket No.: 197102019740 consensus sequence reads 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 genetic variant or other biological signal in a given patient sample.

[0135] In some instances, a 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.

[0136] In some instances, a genomic profile for the subject may comprise results from a comprehensive genomic profiling (CGP) test, a nucleic acid sequencing-based 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.

[0137] In some instances, the method can further include administering or applying a treatment or therapy e.g., an anti-cancer agent, anti-cancer treatment, or anti-cancer therapy) to the subject based on the generated genomic profile. 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.Samples

[0138] The disclosed methods and systems may be used with any of a variety of samples (also referred to herein as specimens) comprising nucleic acids (e.g., DNA or RNA) that are collected from a subject (e.g., a patient). Examples of a sample include, but are not limited to, a tumor sample, a tissue sample, a biopsy sample (e.g., a tissue biopsy, a liquid biopsy, or both), 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 cerebral spinal 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 (or34MF-365322596Docket No.: 197102019740 cell sample derived therefrom). In certain instances, the sample may be frozen sample or a formalin-fixed paraffin-embedded (FFPE) sample.

[0139] In some instances, the sample may be collected by tissue resection (e.g., surgical resection), needle biopsy, bone marrow biopsy, bone marrow aspiration, skin biopsy, endoscopic biopsy, fine needle aspiration, oral swab, nasal swab, vaginal swab or a cytology smear, scrapings, washings or lavages (such as a ductal lavage or bronchoalveolar lavage), etc.

[0140] In some instances, the sample is a liquid biopsy sample, and may comprise, e.g., whole blood, blood plasma, blood serum, urine, stool, sputum, saliva, or cerebrospinal fluid. In some instances, the sample may be a liquid biopsy sample and may comprise circulating tumor cells (CTCs). In some instances, the sample may be a liquid biopsy sample and may comprise cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), or any combination thereof.

[0141] In some instances, the sample may comprise one or more premalignant or malignant cells. Premalignant, as used herein, refers to a cell or tissue that is not yet malignant but is poised to become malignant. In certain instances, the sample may be acquired from a solid tumor, a soft tissue tumor, or a metastatic lesion. In certain instances, the sample may be acquired from a hematologic malignancy or pre-malignancy. In other instances, the sample may comprise a tissue or cells from a surgical margin. In certain instances, the sample may comprise tumor-infiltrating lymphocytes. In some instances, the sample may comprise one or more non-malignant cells. In some instances, the sample may be, or is part of, a primary tumor or a metastasis e.g., a metastasis biopsy sample). In some instances, the sample may be obtained from a site (e.g., a tumor site) with the highest percentage of tumor (e.g., tumor cells) as compared to adjacent sites (e.g., sites adjacent to the tumor). In some instances, the sample may be obtained from a site (e.g., a tumor site) with the largest tumor focus (e.g., the largest number of tumor cells as visualized under a microscope) as compared to adjacent sites (e.g., sites adjacent to the tumor).

[0142] In some instances, the disclosed methods may further comprise analyzing a primary control (e.g., a normal tissue sample). In some instances, the disclosed methods may further comprise determining if a primary control is available and, if so, isolating a control nucleic acid (e.g., DNA) from said primary control. In some instances, the sample may comprise any35MF-365322596Docket No.: 197102019740 normal control (e.g., a normal adjacent tissue (NAT)) if no primary control is available. In some instances, the sample may be or may comprise histologically normal tissue. In some instances, the method includes evaluating a sample, e.g., a histologically normal sample (e.g., from a surgical tissue margin) using the methods described herein. In some instances, the disclosed methods may further comprise acquiring a sub-sample enriched for non-tumor cells, e.g., by macro-dissecting non-tumor tissue from said NAT in a sample not accompanied by a primary control. In some instances, the disclosed methods may further comprise determining that no primary control and no NAT is available, and marking said sample for analysis without a matched control.

[0143] In some instances, samples obtained from histologically normal tissues (e.g., otherwise histologically normal surgical tissue margins) may still comprise a genetic alteration such as a variant sequence as described herein. The methods may thus further comprise re-classifying a sample based on the presence of the detected genetic alteration. In some instances, multiple samples (e.g., from different subjects) are processed simultaneously.

[0144] The disclosed methods and systems may be applied to the analysis of nucleic acids extracted from any of variety of tissue samples (or disease states thereof), e.g., solid tissue samples, soft tissue samples, metastatic lesions, or liquid biopsy samples. Examples of tissues include, but are not limited to, connective tissue, muscle tissue, nervous tissue, epithelial tissue, and blood. Tissue samples may be collected from any of the organs within an animal or human body. Examples of human organs include, but are not limited to, the brain, heart, lungs, liver, kidneys, pancreas, spleen, thyroid, mammary glands, uterus, prostate, large intestine, small intestine, bladder, bone, skin, etc.

[0145] In some instances, the nucleic acids extracted from the sample may comprise deoxyribonucleic acid (DNA) molecules. Examples of DNA that may be suitable for analysis by the disclosed methods include, but are not limited to, genomic DNA or fragments thereof, mitochondrial DNA or fragments thereof, cell-free DNA (cfDNA), and 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 (ctDNA) 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.36MF-365322596Docket No.: 197102019740

[0146] In some instances, DNA is extracted from nucleated cells from the sample. In some instances, a sample may have a low nucleated cellularity, e.g., when the sample is comprised mainly of erythrocytes, lesional cells that contain excessive cytoplasm, or tissue with fibrosis. In some instances, a sample with low nucleated cellularity may require more, e.g., greater, tissue volume for DNA extraction.

[0147] In some instances, the nucleic acids extracted from the sample may comprise ribonucleic acid (RNA) molecules. Examples of RNA that may be suitable for analysis by the disclosed methods include, but are not limited to, total cellular RNA, total cellular RNA after depletion of certain abundant RNA sequences (e.g., ribosomal RNAs), cell-free RNA (cfRNA), messenger RNA (mRNA) or fragments thereof, the poly(A)-tailed mRNA fraction of the total RNA, ribosomal RNA (rRNA) or fragments thereof, transfer RNA (tRNA) or fragments thereof, and mitochondrial RNA or fragments thereof. In some instances, RNA may be extracted from the sample and converted to complementary DNA (cDNA) using, e.g., a reverse transcription reaction. In some instances, the cDNA is produced by random-primed cDNA synthesis methods. In other instances, the cDNA synthesis is initiated at the poly(A) tail of mature mRNAs by priming with oligo(dT)-containing oligonucleotides. Methods for depletion, poly(A) enrichment, and cDNA synthesis are well known to those of skill in the art.

[0148] In some instances, the sample may comprise a tumor content (e.g., comprising tumor cells or tumor cell nuclei), or a non-tumor content (e.g., immune cells, fibroblasts, and other non-tumor cells). In some instances, the tumor content of the sample may constitute a sample metric. In some instances, the sample may comprise a tumor content of at least 5-50%, 10- 40%, 15-25%, or 20-30% tumor cell nuclei. In some instances, the sample may comprise a tumor content of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% tumor cell nuclei. In some instances, the percent tumor cell nuclei (e.g., sample fraction) is determined (e.g., calculated) by dividing the number of tumor cells in the sample by the total number of all cells within the sample that have nuclei. In some instances, for example when the sample is a liver sample comprising hepatocytes, a different tumor content calculation may be required due to the presence of hepatocytes having nuclei with twice, or more than twice, the DNA content of other, e.g., non-hepatocyte, somatic cell nuclei. In some instances, the sensitivity of detection of a genetic alteration, e.g., a variant sequence, or a determination of, e.g., microsatellite instability, may depend on the tumor content of the37MF-365322596Docket No.: 197102019740 sample. For example, a sample having a lower tumor content can result in lower sensitivity of detection for a given size sample.

[0149] In some instances, as noted above, the sample comprises nucleic acid (e.g., DNA, RNA (or a cDNA derived from the RNA), or both), e.g., from a tumor or from normal tissue. In certain instances, the sample may further comprise a non-nucleic acid component, e.g., cells, protein, carbohydrate, or lipid, e.g., from the tumor or normal tissue.Subjects

[0150] 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) or suspected of having the condition or disease. In some instances, the hyperproliferative disease is a cancer. In some instances, the cancer is a solid tumor or a metastatic form thereof. In some instances, the cancer is a hematological cancer, e.g., a leukemia or lymphoma.

[0151] In some instances, 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 a genetic mutation that increases his or her baseline risk for developing a cancer). 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. In some instances, the subject is in need of being monitored for development of a cancer. In some instances, the subject is in need of 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 is in need of being monitored for relapse of cancer. In some instances, the subject is in need of being monitored for minimum residual disease (MRD). In some instances, the subject has been, or is being treated, for cancer. In some instances, the subject has not been treated with an anticancer therapy (or anti-cancer treatment).

[0152] 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.

[0153] 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 38MF-365322596Docket No.: 197102019740 therapy, the sample comprises a resection, e.g., an original resection, or a resection following recurrence e.g., following a disease recurrence post- therapy).Cancers

[0154] In some instances, the sample is acquired from a subject having a cancer. Exemplary cancers include, but are not limited to, B cell cancer (e.g., multiple myeloma), melanomas, breast cancer, lung cancer (such as non-small cell lung carcinoma or NSCLC), bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, adenocarcinomas, inflammatory myofibroblastic tumors, 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 cancers, essential thrombocythemia, agnogenic myeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis, familiar hypereosinophilia, chronic eosinophilic leukemia, neuroendocrine cancers, carcinoid tumors, and the like.39MF-365322596Docket No.: 197102019740

[0155] 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 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+), 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.40MF-365322596Docket No.: 197102019740

[0156] In some instances, the cancer is a hematologic malignancy (or premaligancy). 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.Nucleic acid extraction and processing

[0157] DNA or RNA may be extracted from tissue samples, biopsy samples, blood samples, 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)). Protocols for RNA isolation are disclosed in, e.g., the Maxwell® 16 Total RNA Purification Kit Technical Bulletin (Promega Literature #TB351, August 2009, Promega Corporation, Madison, WI).

[0158] A typical DNA extraction procedure, for example, comprises (i) collection of the fluid sample, cell sample, or tissue sample from which DNA is to be extracted, (ii) disruption of cell membranes (i.e., cell lysis), if necessary, to release DNA and other cytoplasmic41MF-365322596Docket No.: 197102019740 components, (iii) treatment of the fluid sample or lysed 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 (iv) purification of DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during the cell membrane lysis step.

[0159] Disruption of cell membranes may be performed using a variety of mechanical shear (e.g., by passing through a French press or fine needle) or ultrasonic disruption techniques. The cell lysis step often comprises the use of detergents and surfactants to solubilize lipids the cellular and nuclear membranes. In some instances, the lysis step may further comprise use of proteases to break down protein, and / or the use of an RNase for digestion of RNA in the sample.

[0160] 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.

[0161] 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.

[0162] 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).

[0163] As noted above, in some instances the sample may comprise a formalin-fixed (also known as formaldehyde-fixed, or paraformaldehyde-fixed), paraffin-embedded (FFPE) tissue preparation. For example, the FFPE sample may be a tissue sample embedded in a matrix, e.g., an FFPE block. Methods to isolate nucleic acids (e.g., DNA) from formaldehyde- or42MF-365322596Docket No.: 197102019740 paraformaldehyde-fixed, paraffin-embedded (FFPE) tissues are disclosed in, e.g., Cronin, et al., (2004) Am J Pathol. 164(1):35— 42; Masuda, et al., (1999) Nucleic Acids Res . 27(22):4436-4443; Specht, et al., (2001) Am J Pathol. 158(2):419-429; the Ambion RecoverAll™ Total Nucleic Acid Isolation Protocol (Ambion, Cat. No. AM1975, September 2008); the Maxwell® 16 FFPE Plus LEV DNA Purification Kit Technical Manual (Promega Literature #TM349, February 2011); the E.Z.N.A.® FFPE DNA Kit Handbook (OMEGA bio- tek, Norcross, GA, product numbers D3399-00, D3399-01, and D3399-02, June 2009); and the QIAamp® DNA FFPE Tissue Handbook (Qiagen, Cat. No. 37625, October 2007). For example, the RecoverAll™ Total Nucleic Acid Isolation Kit uses xylene at elevated temperatures to solubilize paraffin-embedded samples and a glass-fiber filter to capture nucleic acids. The Maxwell® 16 FFPE Plus LEV DNA Purification Kit is used with the Maxwell® 16 Instrument for purification of genomic DNA from 1 to 10 pm sections of FFPE tissue. DNA is purified using silica-clad paramagnetic particles (PMPs), and eluted in low elution volume. The E.Z.N.A.® FFPE DNA Kit uses a spin column and buffer system for isolation of genomic DNA. QIAamp® DNA FFPE Tissue Kit uses QIAamp® DNA Micro technology for purification of genomic and mitochondrial DNA.

[0164] In some instances, the disclosed methods may further comprise determining or acquiring a yield value for the nucleic acid extracted from the sample and comparing the determined value to a reference value. For example, if the determined or acquired value is less than the reference value, the nucleic acids may be amplified prior to proceeding with library construction. In some instances, the disclosed methods may further comprise determining or acquiring a value for the size (or average size) of nucleic acid fragments in the sample, and comparing the determined or acquired value to a reference value, e.g., a size (or average size) of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 base pairs (bps). In some instances, one or more parameters described herein may be adjusted or selected in response to this determination.

[0165] After isolation, the nucleic acids are typically dissolved in a slightly alkaline buffer, e.g., Tris-EDTA (TE) buffer, or in ultra-pure water. In some instances, the isolated nucleic acids (e.g., genomic DNA) may be fragmented or sheared by using any of a variety of techniques known to those of skill in the art. For example, genomic DNA can be fragmented by physical shearing methods, enzymatic cleavage methods, chemical cleavage methods, and other methods known to those of skill in the art. Methods for DNA shearing are described in 43MF-365322596Docket No.: 197102019740Example 4 in International Patent Application Publication No. WO 2012 / 092426. In some instances, alternatives to DNA shearing methods can be used to avoid a ligation step during library preparation.Library preparation

[0166] In some instances, the nucleic acids isolated from the sample may be used to construct a library (e.g., a nucleic acid library as described herein). In some instances, the nucleic acids are fragmented using any of the methods described above, subjected to repair of chain end damage to introduce jagged end identifiers, and optionally ligated or attached to synthetic adapters, primers, and / or sample and / or optionally molecular barcodes (e.g., amplification primers, sequencing adapters, flow cell adapters, substrate adapters, sample barcodes or indexes, unique molecular barcodes (e.g., unique molecular identifier (UMI) sequences), and / or non-unique molecular barcodes), 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 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 strand-displacement 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.

[0167] In some instances, the resulting nucleic acid 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 instances, the library may include at least 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the genomic DNA. In some instances, the library may consist of cDNA copies of genomic DNA that includes copies of at least 44MF-365322596Docket No.: 19710201974095%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% 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.

[0168] In some instances, a library (e.g., a nucleic acid 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 acid molecules 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, a cancer or tumor.

[0169] 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 tumor phenotype. In some instances, a subgenomic interval comprises more than one nucleotide position. Such instances include sequences of at least 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 microsatellite 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 acid45MF-365322596Docket No.: 197102019740 molecule. In some instances, the subgenomic interval comprises a non-tumor nucleic acid molecule.Targeting gene loci for analysis

[0170] 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.

[0171] In some instances, 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 a cancer, e.g., a cancer described herein.

[0172] In some instances, 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.

[0173] In some instances, the selected gene loci (also referred to herein as target gene loci or target sequences), or fragments thereof, may include subject intervals comprising non-coding sequences, coding sequences, intragenic regions, or intergenic regions of the subject genome. For example, the subject intervals can include 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, an intron sequence or a fragment thereof.Target capture reagents

[0174] 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., gene sequences or fragments thereof) for analysis. In some instances, a target capture reagent (i.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., a46MF-365322596Docket No.: 197102019740 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 cDNA molecule derived from an RNA molecule, a microsatellite DNA sequence, and the like. 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 Patent Application Publication No. WO 2020 / 236941, the entire content of which is incorporated herein by reference.

[0175] 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., cancerous tissue specimens, 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.

[0176] 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, greater than 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.

[0177] In some instances, the overall length of the target capture reagent sequence can be between about 70 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 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,47MF-365322596Docket No.: 197102019740200, 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 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, or 230 bases can be used.

[0178] 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.

[0179] In some instances, the target-specific capture sequences in the target capture reagents are between about 40 nucleotides and 1000 nucleotides in length. In some instances, the target- specific capture sequence is between about 70 nucleotides and 300 nucleotides in length. In some instances, the target- specific sequence is between about 100 nucleotides and 200 nucleotides in length. In yet other instances, the target-specific sequence is between about 120 nucleotides and 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 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.

[0180] 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.

[0181] 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 for48MF-365322596Docket No.: 197102019740 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.

[0182] 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.

[0183] 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 from one or more subjects; contacting the one or a plurality 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).

[0184] 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.

[0185] In some instances, the target capture reagents can be part of a kit which can optionally comprise instructions, standards, buffers or enzymes or other reagents.49MF-365322596Docket No.: 197102019740Hybridization conditions

[0186] As noted above, in some instances (e.g., when using a targeted sequencing method rather than a whole genome sequencing (WGS) or whole transcriptome sequencing (WTS) method), 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.

[0187] 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, the contents of which are incorporated herein by reference in their entireties.

[0188] Hybridization methods that can be adapted for use in the methods herein are described in the art, e.g., as described in International Patent Application Publication No. WO 2012 / 092426. Methods for hybridizing target capture reagents to a plurality of target nucleic acids are described in more detail in, e.g., International Patent Application Publication No. WO 2020 / 236941, the entire content of which is incorporated herein by reference.Sequencing methods

[0189] 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 nucleic50MF-365322596Docket No.: 197102019740 acid molecules in a high throughput fashion (e.g., wherein greater than 103, 104, 105or more than 105molecules are sequenced simultaneously).

[0190] Next-generation sequencing methods are known in the art, and are described in, e.g., Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46, which is incorporated herein by reference. Other examples of sequencing methods suitable for use when implementing the methods and systems disclosed herein are described in, e.g., International Patent Application Publication No. WO 2012 / 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.

[0191] The disclosed methods and systems may be implemented using sequencing platforms such as the Roche / 454 Genome Sequencer (GS) FLX System, Illumina / Solexa 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 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 Patent Application Publication No. WO 2020 / 236941, the entire content of which is incorporated herein by reference.

[0192] 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 molecules from a sample; (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 51MF-365322596Docket No.: 197102019740 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 acquiring 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 somatic mutation or germline 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.

[0193] 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, microsatellite 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.

[0194] 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 provides a 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.52MF-365322596Docket No.: 197102019740

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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).

[0199] 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).53MF-365322596Docket No.: 197102019740Alignment

[0200] 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 Patent Application Publication No. WO 2012 / 092426. Additional description of sequence alignment methods is provided in, e.g., International Patent Application Publication No. WO 2020 / 236941, the entire content of which is incorporated herein by reference.

[0201] 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.

[0202] 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 diverse genomic 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-Wheeler54MF-365322596Docket No.: 197102019740Alignment (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.

[0203] 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).

[0204] 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.

[0205] 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.55MF-365322596Docket No.: 197102019740

[0206] 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 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).

[0207] 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.

[0208] 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), wherein said second set of parameters comprises use of, e.g., said second reference sequence, which, 56MF-365322596Docket No.: 197102019740 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).

[0209] 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).

[0210] 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.Alignment of Methyl-Seq Sequence Reads

[0211] In some instances, e.g., for epigenetic analysis, the methods may include the use of an alignment method optimized for aligning sequence reads for DNA that has been converted using, e.g., a bisulfite reaction, to convert unmethylated cytosine residues to uracil (which is interpreted as a thymine in sequencing results). In some instances, sequence reads may be aligned to two genomes in silico, e.g., converted and unconverted versions of the reference57MF-365322596Docket No.: 197102019740 genome, using such alignment tools. Methylation occurs primarily at CpG sites, but may also occur less frequently at non-CpG sites (e.g., CHG or CHH sites).

[0212] In some instances, the sequence read data may be obtained using a nucleic acid sequencing method comprising the use of a bisulfite- or enzymatic-conversion reaction (e.g., during library preparation) to convert non-methylated cytosine to uracil (see, e.g., Li, et al. (2011), “DNA Methylation Detection: Bisulfite Genomic Sequencing Analysis”, Methods Mol. Biol. 791:11-21).

[0213] In some instances, the sequence read data may be obtained using a nucleic acid sequencing method comprising the use of alternative chemical and / or enzymatic reactions (e.g., during library preparation) to convert non-methylated cytosine to uracil (or to convert methylated cytosine to dihydrouracil). For example, enzymatic deamination of non- methylated cytosine using APOB EC to form uracil can be performed using, e.g., the Enzymatic Methyl-seq Kit from New England BioLabs (Ipswich, MA) which uses prior treatment with ten-eleven translocation methylcytosine dioxygenase 2 (TET2) to oxidize 5- mC and 5-hmC, thereby providing greater protection of the methylated cytosine from deamination by APOB EC). Liu, et al. (2019) recently described a bisulfite-free and base- level-resolution sequencing-based method, TET-Assisted Pyridine borane Sequencing (TAPS), for detection of 5mC and 5hmC. The method combines ten-eleven translocation methylcytosine dioxygenase (TET) -mediated oxidation of 5mC and 5hmC to 5- carboxylcytosine (5caC) with pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent PCR amplification converts DHU to thymine, thereby enabling conversion of methylated cytosines to thymine (Liu, et al. (2019), “Bisulfite-Free Direct Detection of 5- Methylcytosine and 5-Hydroxymethylcytosine at Base Resolution”, Nature Biotechnology, vol. 37, pp. 424-429).

[0214] In some instances, the sequence read data may be obtained using a nucleic acid sequencing method comprising the use of Methylated DNA Immunoprecipitation (MeDIP).

[0215] Examples of alignment tools optimized for aligning sequence reads for converted DNA include, but are not limited to, NovoAlign (Novocraft Technologies, Selangor, Malaysia), and the Bismark tool (Krueger, et al. (2011), “Bismark: A Flexible Aligner and Methylation Caller for Bisulfite-Seq Applications”, Bioinformatics 27(11): 1571-1572).58MF-365322596Docket No.: 197102019740Alteration (mutation) calling

[0216] Base calling refers to the raw output of a sequencing device, e.g., the determined sequence of nucleotides in an oligonucleotide molecule. Alternation (or mutation) calling refers to the process of selecting a nucleotide value, e.g., A, G, T, or C, for one or more nucleotide positions being sequenced. Typically, the sequence reads (or base calling) for a given position will provide more than one value, e.g., some reads will indicate a T and some will indicate a G for a given position. Alteration calling is the process of assigning a correct nucleotide value, e.g., one of those values, to each position of the one or more nucleotide positions being sequenced. Although often referred to as “alteration” or “mutation” calling, the process 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.

[0217] In some instances, the disclosed methods may comprise the use of customized or tuned alteration / 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, micro satellite regions, etc.) in samples, e.g., samples from a subject having cancer. Optimization of alteration / mutation calling is described in the art, e.g., as set out in International Patent Application Publication No. WO 2012 / 092426.

[0218] Methods for alteration / mutation calling can include one or more of the following: making independent calls based on the information at each position 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.

[0219] 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 from59MF-365322596Docket No.: 197102019740 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).

[0220] 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.

[0221] After alignment, detection of substitutions can be performed using an alteration / mutation calling method (e.g., a Bayesian calling method) which is applied to 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 an alteration / mutation with the probability of observing the read data in the presence of base-calling error alone. Alterations / mutations can be called if this comparison is sufficiently strongly supportive of the presence of a mutation.

[0222] An advantage of a Bayesian alteration / mutation detection approach is that the comparison of the probability of the presence of, e.g., a mutation with the probability of basecalling 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 an alteration / mutation may be confidently called even if the amount of evidence of alteration / mutation does not meet the usual thresholds. This flexibility can then be used to increase detection sensitivity for even rarer alterations / mutations and / or for 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).

[0223] 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 used60MF-365322596Docket No.: 197102019740 to obtain potential indel candidates, and then these candidates are tested together with the reference sequence in a Bayesian framework.

[0224] 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.

[0225] Methods for generating indel calls and individual-level genotype likelihoods include, e.g., the Dindel algorithm (Albers, C.A., et al., Genome Res. 201 l;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.

[0226] 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 the possibility of the presence of a mutant allele at frequencies (or allele fractions) ranging from 1% to 100% (i.e., allele fractions ranging from 0.01 to 1.0), and especially at levels lower than 50%. This approach is particularly important for the detection of mutations in, for example, low-purity FFPE samples of natural (multi-clonal) tumor DNA.

[0227] In some instances, the alteration / mutation calling method used to analyze sequence reads is not individually customized or fine-tuned for detection of different alterations / mutations at different genomic loci. In some instances, different alteration / mutation calling methods are used that are individually customized or fine-tuned for at least a subset of the different alterations / mutations detected at different genomic loci. In some instances, different alteration / mutation calling methods are used that are individually customized or fine-tuned for each different alteration / mutant detected at each different genomic locus. 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 alteration / mutation calling methods individually customized or fine-tuned for a number of61MF-365322596Docket No.: 197102019740 subject intervals to be sequenced allows for optimization of speed, sensitivity and specificity of alteration / mutation calling.

[0228] In some instances, a nucleotide value is assigned for a nucleotide position in each of X unique subject intervals using a unique alteration / 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.

[0229] 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.

[0230] In some instances, the method comprises assigning a nucleotide value (e.g., calling a mutation) for at least 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., an alteration or mutation, at said nucleotide position in a tumor of type.

[0231] 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).

[0232] In some instances, the alteration / 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 base-calling error alone); and (b) responsive to said values, assigning a nucleotide62MF-365322596Docket No.: 197102019740 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.

[0233] Additional description of exemplary nucleic acid sequencing methods, alteration / 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 Patent Application Publication No. WO 2020 / 236941, the entire contents of each of which is incorporated herein by reference.Methylation Status Calling

[0234] In some instances, the methods described herein may comprise the use of a methylation status calling method, e.g., to call the methylation status of the CpG sites based on the sequence reads and fragments (complementary pairs of forward and reverse sequence reads) derived from DNA that has been subjected to a chemical or enzymatic conversion reaction, e.g., to convert unmethylated cytosine residues to uracil (which is interpreted as a thymine in sequencing results). Examples of such methylation status calling tools include, but are not limited to, the Bismark tool (Krueger, et al. (2011), “Bismark: A Flexible Aligner and Methylation Caller for Bisulfite-Seq Applications”, Bioinformatics 27(11): 1571-1572), TARGOMICS (Garinet, et al. (2017), “Calling Chromosome Alterations, DNA Methylation Statuses, and Mutations in Tumors by Simple Targeted Next-Generation Sequencing - A Solution for Transferring Integrated Pangenomic Studies into Routine Practice?”, J.Molecular Diagnostics 19(5):776-787), Bicycle (Grana, et al. (2018) “Bicycle: A Bioinformatics Pipeline to Analyze Bisulfite Sequencing Data”, Bioinformatics 34(8): 1414- 5), SMAP (Gao, et al. (2015), “SMAP: A Streamlined Methylation Analysis Pipeline for Bisulfite Sequencing”, Gigascience 4:29), and MeDUSA (Wilson, et al. (2016), “Computational Analysis and Integration of MeDIP-Seq Methylome Data”, in: Kulski JK, editor, Next Generation Sequencing: Advances, Applications and Challenges. Rijeka: InTech, p. 153-69). See also, Rauluseviciute, et al. (2019), “DNA Methylation Data by Sequencing: Experimental Approaches and Recommendations for Tools and Pipelines for Data Analysis”, Clinical Epigenetics 11 : 193.63MF-365322596Docket No.: 197102019740Systems

[0235] Also disclosed herein are systems designed to implement any of the disclosed methods for correcting consensus sequence reads derived from a sample from a subject. The systems may comprise, e.g., 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: receive a plurality of sequence reads generated from a plurality of nucleic acid molecules, where the plurality of nucleic acid molecules include a jagged end identifier; map the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information associated with the mapped sequence reads, where the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position and a stop position of a given mapped sequence read; group one or more of the plurality of mapped sequence reads into a plurality of families based the corresponding sequence information associated with a given mapped sequence read; based on the grouping, determine a consensus sequence for each of the plurality of families; and detect one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0236] In some instances, 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.

[0237] In some instances, the disclosed systems may be used for correcting sequence reads derived from any of a variety of samples as described herein (e.g., a tissue sample, biopsy sample, hematological sample, or liquid biopsy sample derived from the subject).

[0238] In some instances, the plurality of gene loci for which sequencing data is processed to determine corrected consensus sequence reads may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more64MF-365322596Docket No.: 197102019740 than 1000 gene loci (or any number of gene loci within the range of 1 to more than 1000 gene loci).

[0239] In some instance, 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 400 bases, less than 300 bases, less than 200 bases, less than 150 bases, less than 100 bases, less than 90 bases, less than 80 bases, less than 70 bases, less than 60 bases, less than 50 bases, less than 40 bases, or less than 30 bases.

[0240] In some instances, 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 instances, the systems may comprise, or be part of, a computer system or computer network as described elsewhere herein.Computer systems and networks

[0241] FIG. 3 illustrates an example of a computing device or system in accordance with one embodiment. Device 300 can be a host computer connected to a network. Device 300 can be a client computer or a server. As shown in FIG. 3, device 300 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) 310, input devices 320, output devices 330, memory or storage devices 340, communication devices 360, and nucleic acid sequencers 370. Software 350 residing in memory or storage device 340 may comprise, e.g., an operating system as well as software for executing the methods described herein. Input device 320 and output device 330 can generally correspond to those described herein, and can either be connectable or integrated with the computer.

[0242] Input device 320 can be any suitable device that provides input, such as a touch screen, keyboard or keypad, mouse, or voice-recognition device. Output device 330 can be any suitable device that provides output, such as a touch screen, haptics device, or speaker.65MF-365322596Docket No.: 197102019740

[0243] Storage 340 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 360 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 380, Ethernet connection, or any other wire transfer technology) or wirelessly (e.g., Bluetooth®, Wi-Fi®, or any other wireless technology).

[0244] Software module 350, which can be stored as executable instructions in storage 340 and executed by processor(s) 310, can include, for example, an operating system and / or the processes that embody the functionality of the methods of the present disclosure (e.g., as embodied in the devices as described herein).

[0245] Software module 350 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 340, 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.

[0246] Software module 350 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 66MF-365322596Docket No.: 197102019740 electronic, magnetic, optical, electromagnetic or infrared wired or wireless propagation medium.

[0247] Device 300 may be connected to a network (e.g., network 404, as shown in FIG. 4 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.

[0248] Device 300 can be implemented using any operating system, e.g., an operating system suitable for operating on the network. Software module 350 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 Webbased application or Web service, for example. In some embodiments, the operating system is executed by one or more processors, e.g., processor(s) 310.

[0249] Device 300 can further include a sequencer 370, which can be any suitable nucleic acid sequencing instrument.

[0250] FIG. 4 illustrates an example of a computing system in accordance with one embodiment. In system 400, device 300 (e.g., as described above and illustrated in FIG. 3) is connected to network 404, which is also connected to device 406. In some embodiments, device 406 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, Pacific Biosciences’ PacBio® RS system, Ultima Genomics UG 100™ platform, or the Illumina NovaSeq X series platform.

[0251] Devices 300 and 406 may communicate, e.g., using suitable communication interfaces via network 404, such as a Local Area Network (LAN), Virtual Private Network (VPN), or the Internet. In some embodiments, network 404 can be, for example, the Internet, an67MF-365322596Docket No.: 197102019740 intranet, a virtual private network, a cloud network, a wired network, or a wireless network. Devices 300 and 406 may communicate, in part or in whole, via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. Additionally, devices 300 and 406 may communicate, e.g., using suitable communication interfaces, via a second network, such as a mobile / cellular network. Communication between devices 300 and 406 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 300 and 406 can communicate directly (instead of, or in addition to, communicating via network 404), e.g., via wireless or hardwired communications, such as Ethernet, IEEE 802.11b wireless, or the like. In some embodiments, devices 300 and 406 communicate via communications 408, which can be a direct connection or can occur via a network (e.g., network 404).

[0252] One or all of devices 300 and 406 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 404 according to various examples described herein.EXAMPLES

[0253] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present disclosure.Example 1 - Use of Jagged End Identifiers in Combination with Start-Stop Deduplication

[0254] This example provides an illustration of the use of jagged end identifiers in combination with start-stop deduplication to correctly identify unique nucleic acid molecules (e.g., DNA fragments) present in a sample.

[0255] FIG. 5 provides a schematic illustration of a situation where a conventional start-stop deduplication approach to tracking progeny from a single unique DNA molecule fails to distinguish between the progeny from two different unique DNA molecules. Start-stop deduplication uses the 5’ and 3’ ends of each read as a surrogate for the use of traditional UMIs. While this approach can be helpful when preparing sequencing libraries using low input quantities of nucleic acid, it suffers at higher input quantities of nucleic acid where the likelihood of having two unique molecules with the same start and stop locations increases. As illustrated in FIG. 5, performing end repair to fill in 5’ overhangs and blunt 3’ overhangs68MF-365322596Docket No.: 197102019740 can result in sequence reads corresponding to the two unique molecules that have the same start and stop locations.

[0256] FIG. 6 provides a schematic illustration of a situation where a conventional start-stop deduplication approach succeeds, but the two unique DNA molecules can also be distinguished based on jagged end identifiers introduced during end repair using 5hmC in place of dCTP. Jagged end indexing ( JEi) introduces an additional identifier to distinguish between unique molecules. When used in conjunction with star -stop location, the likelihood of successfully identifying unique molecules increases. Following introduction of jagged end identifiers, the samples can proceed through either dsLP or ssLP. For dsLP, the method provides an additional tag for unique molecule identification. For ssLP, jagged information has been retained which is lost with canonical ssLP and provides a tag for unique molecule identification.

[0257] FIG. 7 provides a schematic illustration of a situation where two unique DNA molecules have the same start-stop locations but can still be distinguished based on jagged end identifiers 702 and 704 introduced during end repair using 5hmC in place of dCTP. 5hmC is a methylated analogue of dCTP. Methylated cytosines are resistant to conversion in bisulfite and enzymatic conversion workflows for methylation sequencing, i.e., methylated cytosine remains cytosine while unmethylated cytosine is converted to uracil, and in turn to thymine (T) during PCR amplification. The jagged end index (JEi) marks the location of the jagged end and can be used as a unique identifier in situations where start-stop deduplication fails. Note that 5hmC incorporation is only useful in CH contexts (i.e., non-CpG regions). To probe CpG regions, one can incorporate dCTP and mark the position of the last T from the 3’ end of the sequence read in CpG contexts.EXEMPLARY IMPLEMENTATIONS

[0258] Exemplary implementations of the methods and systems described herein include:

[0259] Embodiment 1. A method of detecting biological signals, the method comprising: obtaining a sample from a subject, the sample comprising a plurality of double stranded nucleic acid molecules having one or more jagged ends; performing end repair on the one or more jagged ends of the plurality of double stranded nucleic acid molecules, wherein the end repair introduces a jagged end identifier within a given double stranded molecule of the plurality of double stranded molecules as part of the end repair reaction; preparing a69MF-365322596Docket No.: 197102019740 sequencing library comprising a plurality of end repaired nucleic acid molecules; sequencing the plurality of end repaired nucleic acid molecules to generate a plurality of sequence reads; mapping the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read; grouping the mapped sequence reads into a plurality of families based on the corresponding sequence information; based on the grouping, determining a consensus sequence read for each of the plurality of families; and detecting one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0260] Embodiment 2. The method of embodiment 1, wherein performing end repair further comprises inserting one or more methylated cytosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

[0261] Embodiment 3. The method of embodiment 2, wherein the one or more methylated cytosines are inserted via a polymerase reaction.

[0262] Embodiment 4. The method of embodiment 2 or claim 3, further comprising performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil.

[0263] Embodiment 5. The method of any one of embodiments 2 to 4, wherein mapping further comprises determining a position of a first methylated cytosine in the 5' direction that is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5' direction indicates the jagged end position.

[0264] Embodiment 6. The method of embodiment 4 or claim 5, wherein the conversion reaction is an enzymatic conversion reaction.

[0265] Embodiment 7. The method of embodiment 4 or claim 5, wherein the conversion reaction is a chemical conversion reaction.

[0266] Embodiment 8. The method of embodiment 7, wherein the chemical conversion reaction is a bisulfite conversion reaction.70MF-365322596Docket No.: 197102019740

[0267] Embodiment 9. The method of embodiment 1, wherein performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

[0268] Embodiment 10. The method of embodiment 9, wherein the one or more inosines are inserted via a polymerase reaction.

[0269] Embodiment 11. The method of embodiment 9, wherein the one or more inosines are inserted by hydrolytic deamination of adenine.

[0270] Embodiment 12. The method of any one of embodiments 9 to 11, wherein mapping further comprises determining a position of a first inosine in the 5' direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5' direction indicates the jagged end position.

[0271] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules.

[0272] Embodiment 14. The method of embodiment 13, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprise circulating tumor DNA (ctDNA) molecules.

[0273] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the plurality of end repaired nucleic acid molecules are single stranded.

[0274] Embodiment 16. The method of any one of embodiments 1 to 14, wherein the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules.

[0275] Embodiment 17. The method of embodiment 16, wherein the double-stranded nucleic acid molecules undergo double- stranded nucleic acid molecule library preparation.

[0276] Embodiment 18. The method of embodiment 17, wherein the double-stranded nucleic acid molecule library preparation comprises denaturing the double- stranded nucleic acid molecules.

[0277] Embodiment 19. The method of embodiment 18, wherein the double-stranded nucleic acid molecule library preparation comprises polyA-tailing the denatured double-stranded nucleic acid molecules.71MF-365322596Docket No.: 197102019740

[0278] Embodiment 20. The method of any of any one of embodiments 1 to 16, wherein the plurality of end repaired nucleic acid molecules undergo single- stranded nucleic acid molecule library preparation.

[0279] Embodiment 21. The method of embodiment 20, wherein the single-stranded nucleic acid molecule library preparation comprises synthesizing a second nucleic acid strand.

[0280] Embodiment 22. The method of any one of embodiments 1 to 21, further comprising subjecting the plurality of double stranded nucleic acid molecules to an artificial jagged end process.

[0281] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

[0282] Embodiment 24. The method of any one of embodiments 1 to 23, further comprising partitioning the plurality of end repaired nucleic acid molecules based on a jagged end identifier into a first subsample and second subsample.

[0283] Embodiment 25. The method of any one of embodiments 1 to 24, further comprising amplifying the plurality of end repaired nucleic acid molecules.

[0284] Embodiment 26. The method of any one of embodiments 1 to 25, further comprising ligating an adaptor to one or more ends of the plurality of end repaired nucleic acid molecules, wherein the adaptor includes at least a molecular barcode.

[0285] Embodiment 27. The method of embodiment 26, wherein the corresponding sequence information includes information associated with the molecular barcode.

[0286] Embodiment 28. The method of any of embodiment 26 or claim 27, further comprising incorporating a nucleotide oligomer into the plurality of double stranded nucleic acid molecules or the plurality of end-repaired nucleic acid molecules.

[0287] Embodiment 29. The method of embodiment 28, wherein incorporating the nucleotide oligomer comprises providing a ligase.

[0288] Embodiment 30. The method of embodiment 29, wherein the ligase comprises a T4 DNA ligase.72MF-365322596Docket No.: 197102019740

[0289] Embodiment 31. The method of any one of embodiments 28 to 30, wherein the nucleotide oligomer comprises a barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

[0290] Embodiment 32. The method of any one of embodiments 28 to 30, wherein the nucleotide oligomer comprises a barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

[0291] Embodiment 33. The method of any one of embodiments 28 to 30, wherein the nucleotide oligomer comprises a first barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules, and a second barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

[0292] Embodiment 34. The method of any of embodiments 28 to 33, wherein combined use of jagged end identifiers and incorporated nucleotide oligomers facilitates more accurate mapping, grouping, and determination of consensus sequence reads for each of the plurality of families.

[0293] Embodiment 35. The method of any of embodiments 1 to 34, further comprising determining that at least a portion of the grouped sequence reads in a given family is due to a laboratory artifact, and wherein the laboratory artifact is not included in the consensus sequence read associated with the given family.

[0294] Embodiment 36. The method of embodiment 35, wherein the laboratory artifact results from an amplification error.

[0295] Embodiment 37. The method of embodiment 36, wherein the amplification error comprises a PCR error.

[0296] Embodiment 38. The method of 35, wherein the laboratory artifact is a sequencing error.

[0297] Embodiment 39. A method of correcting laboratory artifacts in sequence reads, the method comprising: receiving, at a processor, a plurality of sequence reads generated from a73MF-365322596Docket No.: 197102019740 plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules include a jagged end identifier; mapping, by a processor, the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information associated with the mapped sequence reads, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position and a stop position of a given mapped sequence read; grouping, by a processor, one or more of the plurality of mapped sequence reads into a plurality of families based the corresponding sequence information associated with a given mapped sequence read; based on the grouping, determining, by a processor, a consensus sequence for each of the plurality of families; and detecting, by a processor, one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

[0298] Embodiment 40. The method of embodiment 39, wherein the jagged end identifier has been inserted into a given nucleic acid molecule of the plurality of nucleic acid molecules by performing end repair on a plurality of double stranded nucleic acid molecules prior to preparing a sequencing library from a plurality of end repaired nucleic acid molecules.

[0299] Embodiment 41. The method of embodiment 40, wherein the end repair comprises inserting one or more methylated cytosines to fill in one or more jagged end of a plurality of double stranded nucleic acid molecules.

[0300] Embodiment 42. The method of embodiment 41, wherein the one or more methylated cytosines are inserted via a polymerase reaction.

[0301] Embodiment 43. The method of embodiment 41 or claim 42, further comprising performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil.

[0302] Embodiment 44. The method of any one of embodiments 41 to 43, wherein the plurality of double-stranded nucleic acid molecules comprise non-CpG-rich nucleic acid molecules.

[0303] Embodiment 45. The method of embodiment 44, wherein the non-CpG-rich nucleic acid molecules map onto a non-CpG-rich region of the genome.

[0304] Embodiment 46. The method of any one of embodiments 41 to 45, wherein mapping further comprises determining a position of a first methylated cytosine in the 5' direction that74MF-365322596Docket No.: 197102019740 is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5' direction indicates the jagged end position.

[0305] Embodiment 47. The method of embodiment 40, wherein performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

[0306] Embodiment 48. The method of embodiment 47, wherein the one or more inosines are inserted via a polymerase reaction.

[0307] Embodiment 49. The method of embodiment 47, wherein the one or more inosines are inserted by hydrolytic deamination of adenine.

[0308] Embodiment 50. The method of any one of embodiments 47 to 49, wherein mapping further comprises determining a position of a first inosine in the 5' direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5' direction indicates the jagged end position.

[0309] Embodiment 51. The method of any one of embodiments 40 to 50, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules.

[0310] Embodiment 52. The method of embodiment 51, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprises circulating tumor DNA (ctDNA) molecules.

[0311] Embodiment 53. The method of any one of embodiments 40 to 52, wherein the plurality of end repaired nucleic acid molecules are single stranded.

[0312] Embodiment 54. The method of any one of embodiments 40 to 52, wherein the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules.

[0313] Embodiment 55. The method of any one of embodiments 39 to 54, wherein the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

[0314] Embodiment 56. A method comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules each have one or more jagged ends; repairing the one or more jagged ends via one 75MF-365322596Docket No.: 197102019740 or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules.

[0315] Embodiment 57. The method of embodiments 56, further comprising attaching one or more adaptors to a plurality of nucleic acid molecules to create tagged molecules thereby generating a tagged nucleic acid molecule, wherein the plurality of adaptors each include one or more of a sample barcode and a molecular barcode.

[0316] Embodiment 58. A method for detecting a presence or an absence of cancer in a subject, comprising: (a) extracting a plurality of nucleic acid molecules from a first sample obtained from a subject, wherein the plurality of molecules have one or more jagged ends and the first sample is obtained at a first timepoint; (b) repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule; (d) amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; (f) selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and (g) sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data from a plurality of sequence reads; (h) repeating (a) to (g) with a second sample from the subject obtained at a second time point other than the first time point; and (i) detecting the presence or absence of cancer in the subject based on the sequence data changes between of the first sample and the second sample.

[0317] Embodiment 59. A method comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules have one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of76MF-365322596Docket No.: 197102019740 each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data associated with each of the plurality of enriched target nucleic acid molecules, wherein the sequence data includes a plurality of sequence reads; grouping the plurality of sequence reads based on at least the jagged end identifier associated with a particular sequence read to generate a plurality of families; determining, for each family, a consensus sequence for each family of the plurality of families; using the consensus sequence to detect one or more molecular alterations.

[0318] Embodiment 60. A method for marking a plurality of nucleic acid molecules, comprising: subjecting the plurality of nucleic acid molecules to a fragmentation process to generate a plurality of jagged end nucleic acid molecules having one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; and selectively enriching the marked nucleic acid molecules for genomic regions of interest.

[0319] Embodiment 61. A method for identifying one or more molecular alterations in a sample obtained from a subject, comprising: repairing one or more jagged ends of a plurality of nucleic acid molecules extracted from the sample via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; determining a consensus sequence based on the jagged end identifier after the marked nucleic acid molecules are sequenced and identifying one or more molecular alterations in the sample based on the consensus sequence.

[0320] It should be understood from the foregoing that, while particular implementations of the disclosed methods and systems 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 77MF-365322596Docket No.: 197102019740 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.78MF-365322596

Claims

Docket No.: 197102019740CLAIMSWhat is claimed is:

1. A method of detecting biological signals, the method comprising: obtaining a sample from a subject, the sample comprising a plurality of double stranded nucleic acid molecules having one or more jagged ends; performing end repair on the one or more jagged ends of the plurality of double stranded nucleic acid molecules, wherein the end repair introduces a jagged end identifier within a given double stranded molecule of the plurality of double stranded molecules as part of the end repair reaction; preparing a sequencing library comprising a plurality of end repaired nucleic acid molecules; sequencing the plurality of end repaired nucleic acid molecules to generate a plurality of sequence reads; mapping the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position, and a stop position of a given mapped sequence read; grouping the mapped sequence reads into a plurality of families based on the corresponding sequence information; based on the grouping, determining a consensus sequence read for each of the plurality of families; and detecting one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

2. The method of claim 1, wherein performing end repair further comprises inserting one or more methylated cytosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

3. The method of claim 2, wherein the one or more methylated cytosines are inserted via a polymerase reaction.79MF-365322596Docket No.: 1971020197404. The method of claim 2 or claim 3, further comprising performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil.

5. The method of any one of claims 2 to 4, wherein mapping further comprises determining a position of a first methylated cytosine in the 5’ direction that is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position.

6. The method of claim 4 or claim 5, wherein the conversion reaction is an enzymatic conversion reaction.

7. The method of claim 4 or claim 5, wherein the conversion reaction is a chemical conversion reaction.

8. The method of claim 7, wherein the chemical conversion reaction is a bisulfite conversion reaction.

9. The method of claim 1, wherein performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

10. The method of claim 9, wherein the one or more inosines are inserted via a polymerase reaction.

11. The method of claim 9, wherein the one or more inosines are inserted by hydrolytic deamination of adenine.

12. The method of any one of claims 9 to 11, wherein mapping further comprises determining a position of a first inosine in the 5’ direction in the given mapped sequence read, and80MF-365322596Docket No.: 197102019740 wherein a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position.

13. The method of any one of claims 1 to 12, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules.

14. The method of claim 13, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprise circulating tumor DNA (ctDNA) molecules.

15. The method of any one of claims 1 to 14, wherein the plurality of end repaired nucleic acid molecules are single stranded.

16. The method of any one of claims 1 to 14, wherein the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules.

17. The method of claim 16, wherein the double- stranded nucleic acid molecules undergo double-stranded nucleic acid molecule library preparation.

18. The method of claim 17, wherein the double- stranded nucleic acid molecule library preparation comprises denaturing the double-stranded nucleic acid molecules.

19. The method of claim 18, wherein the double- stranded nucleic acid molecule library preparation comprises polyA-tailing the denatured double- stranded nucleic acid molecules.

20. The method of any one of claims 1 to 16, wherein the plurality of end repaired nucleic acid molecules undergo single-stranded nucleic acid molecule library preparation.

21. The method of claim 20, wherein the single- stranded nucleic acid molecule library preparation comprises synthesizing a second nucleic acid strand.81MF-365322596Docket No.: 19710201974022. The method of any one of claims 1 to 21, further comprising subjecting the plurality of double stranded nucleic acid molecules to an artificial jagged end process.

23. The method of any one of claims 1 to 22, wherein the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

24. The method of any one of claims 1 to 23, further comprising partitioning the plurality of end repaired nucleic acid molecules based on a jagged end identifier into a first subsample and second subsample.

25. The method of any one of claims 1 to 24, further comprising amplifying the plurality of end repaired nucleic acid molecules.

26. The method of any one of claims 1 to 25, further comprising ligating an adaptor to one or more ends of the plurality of end repaired nucleic acid molecules, wherein the adaptor includes at least a molecular barcode.

27. The method of claim 26, wherein the corresponding sequence information includes information associated with the molecular barcode.

28. The method of claim 26 or claim 27, further comprising incorporating a nucleotide oligomer into the plurality of double stranded nucleic acid molecules or the plurality of end- repaired nucleic acid molecules.

29. The method of claim 28, wherein incorporating the nucleotide oligomer comprises providing a ligase.

30. The method of claim 29, wherein the ligase comprises a T4 DNA ligase.82MF-365322596Docket No.: 19710201974031. The method of any one of claims 28 to 30, wherein the nucleotide oligomer comprises a barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

32. The method of any one of claims 28 to 30, wherein the nucleotide oligomer comprises a barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

33. The method of any one of claims 28 to 30, wherein the nucleotide oligomer comprises a first barcode that is unique for each molecule of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules, and a second barcode that is common to all of the molecules of the plurality of double stranded nucleic acid molecules or the plurality of end repaired nucleic acid molecules.

34. The method of any of claims 28 to 33, wherein combined use of jagged end identifiers and incorporated nucleotide oligomers facilitates more accurate mapping, grouping, and determination of consensus sequence reads for each of the plurality of families.

35. The method of any of claims 1 to 34, further comprising determining that at least a portion of the grouped sequence reads in a given family is due to a laboratory artifact, and wherein the laboratory artifact is not included in the consensus sequence read associated with the given family.

36. The method of claim 35, wherein the laboratory artifact results from an amplification error.

37. The method of claim 36, wherein the amplification error comprises a PCR error.

38. The method of claim 35, wherein the laboratory artifact is a sequencing error.

39. A method of correcting laboratory artifacts in sequence reads, the method comprising:83MF-365322596Docket No.: 197102019740 receiving, at a processor, a plurality of sequence reads generated from a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules include a jagged end identifier; mapping, by a processor, the plurality of sequence reads to a reference genome to generate a plurality of mapped sequence reads and corresponding sequence information associated with the mapped sequence reads, wherein the corresponding sequence information includes a jagged end position based on the jagged end identifier, a start position and a stop position of a given mapped sequence read; grouping, by a processor, one or more of the plurality of mapped sequence reads into a plurality of families based the corresponding sequence information associated with a given mapped sequence read; based on the grouping, determining, by a processor, a consensus sequence for each of the plurality of families; and detecting, by a processor, one or more biological signals in the consensus sequence read for at least one family of the plurality of families.

40. The method of claim 39, wherein the jagged end identifier has been inserted into a given nucleic acid molecule of the plurality of nucleic acid molecules by performing end repair on a plurality of double stranded nucleic acid molecules prior to preparing a sequencing library from a plurality of end repaired nucleic acid molecules.

41. The method of claim 40, wherein the end repair comprises inserting one or more methylated cytosines to fill in one or more jagged end of a plurality of double stranded nucleic acid molecules.

42. The method of claim 41, wherein the one or more methylated cytosines are inserted via a polymerase reaction.

43. The method of claim 41 or claim 42, further comprising performing a conversion reaction on the plurality of end repaired nucleic acid molecules to convert each methylated cytosine to a uracil.84MF-365322596Docket No.: 19710201974044. The method of any one of claims 41 to 43, wherein the plurality of double- stranded nucleic acid molecules comprise non-CpG-rich nucleic acid molecules.

45. The method of claim 44, wherein the non-CpG-rich nucleic acid molecules map onto a non-CpG-rich region of the genome.

46. The method of any one of claims 41 to 45, wherein mapping further comprises determining a position of a first methylated cytosine in the 5’ direction that is not part of a CpG region in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first methylated cytosine in the 5’ direction indicates the jagged end position.

47. The method of claim 40, wherein performing end repair further comprises inserting one or more inosines to fill in the one or more jagged ends of the plurality of double stranded molecules.

48. The method of claim 47, wherein the one or more inosines are inserted via a polymerase reaction.

49. The method of claim 47, wherein the one or more inosines are inserted by hydrolytic deamination of adenine.

50. The method of any one of claims 47 to 49, wherein mapping further comprises determining a position of a first inosine in the 5’ direction in the given mapped sequence read, and wherein a loci in the reference genome corresponding to the position of the first inosine in the 5’ direction indicates the jagged end position.

51. The method of any one of claims 40 to 50, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules or cell free RNA (cfRNA) molecules.85MF-365322596Docket No.: 19710201974052. The method of claim 51, wherein the plurality of double stranded nucleic acid molecules are cell free DNA (cfDNA) molecules, and wherein the cfDNA molecules comprises circulating tumor DNA (ctDNA) molecules.

53. The method of any one of claims 40 to 52, wherein the plurality of end repaired nucleic acid molecules are single stranded.

54. The method of any one of claims 40 to 52, wherein the plurality of end repaired nucleic acid molecules are double stranded nucleic acid molecules.

55. The method of any one of claims 39 to 54, wherein the one or more biological signals comprise a genomic signal, an epigenetic signal, a molecular characteristic, a transcriptomic signal, a proteomic signal, or any combination thereof.

56. A method comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules each have one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules.

57. The method of claim 56, further comprising attaching one or more adaptors to a plurality of nucleic acid molecules to create tagged molecules thereby generating a tagged nucleic acid molecule, wherein the plurality of adaptors each include one or more of a sample barcode and a molecular barcode.

58. A method for detecting a presence or an absence of cancer in a subject, comprising:86MF-365322596Docket No.: 197102019740(a) extracting a plurality of nucleic acid molecules from a first sample obtained from a subject, wherein the plurality of molecules have one or more jagged ends and the first sample is obtained at a first timepoint;(b) repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions thereby generating a marked nucleic acid molecule;(d) amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules;(f) selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and(g) sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data from a plurality of sequence reads;(h) repeating (a) to (g) with a second sample from the subject obtained at a second time point other than the first time point; and(i) detecting the presence or absence of cancer in the subject based on the sequence data changes between of the first sample and the second sample.

59. A method comprising: extracting a plurality of nucleic acid molecules from a sample obtained from a subject, wherein the plurality of nucleic acid molecules have one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a marked nucleic acid molecule; amplifying the plurality of marked nucleic acid molecules to generate a plurality of copies of each of the plurality of marked nucleic acid molecules; selectively enriching the copies for genomic regions of interest to generate a plurality of selectively enriched target nucleic acid molecules; and sequencing the plurality of the selectively enriched target nucleic acid molecules to generate sequence data associated with each of the plurality of enriched target nucleic acid molecules, wherein the sequence data includes a plurality of sequence reads; grouping the plurality of sequence reads based on at least the jagged end identifier associated with a particular sequence read to generate a plurality of families;87MF-365322596Docket No.: 197102019740 determining, for each family, a consensus sequence for each family of the plurality of families; using the consensus sequence to detect one or more molecular alterations.

60. A method for marking a plurality of nucleic acid molecules, comprising: subjecting the plurality of nucleic acid molecules to a fragmentation process to generate a plurality of jagged end nucleic acid molecules having one or more jagged ends; repairing the one or more jagged ends via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; and selectively enriching the marked nucleic acid molecules for genomic regions of interest.

61. A method for identifying one or more molecular alterations in a sample obtained from a subject, comprising: repairing one or more jagged ends of a plurality of nucleic acid molecules extracted from the sample via one or more end repair reactions, wherein the end repair introduces a jagged end identifier during the one or more end repair reactions, thereby generating a plurality of marked nucleic acid molecules before amplification of the plurality of nucleic acid molecules; determining a consensus sequence based on the jagged end identifier after the marked nucleic acid molecules are sequenced and identifying one or more molecular alterations in the sample based on the consensus sequence.88MF-365322596

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