Methods of nucleic acid analysis involving size separation

Abstract

The disclosure provides methods for analyzing a nucleic acid population. The method involves partitioning nucleic acids into at least two groups based on the length of the nucleic acid molecules. A first partition, enriched with longer nucleic acids, is subjected to at least one assay that identifies active chromatin regions and / or fragmentation profiles, providing insights into gene expression and chromosomal organization. A second partition, enriched with shorter nucleic acids, is analyzed for modifications, such as methylation status, or for detecting somatic mutations using targeted or untargeted approaches.

Description

Attorney Docket No.: GH0253WOMETHODS OF NUCLEIC ACID ANALYSIS INVOLVING SIZE SEPARATION CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Provisional Patent Application No. 63 / 725,478, filed November 26, 2024, which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present disclosure provides methods for analyzing a nucleic acid population (e.g., DNA such as cell-free DNA (cfDNA)) of varying lengths using selective partitioning based on the length of the nucleic acid molecules (e.g., cfDNA) followed by identifying active chromatin regions and / or fragmentation profiles of the nucleic acid molecules in one partition and identifying modifications, such as methylation status and / or somatic mutations using targeted or untargeted approaches, in a second partition. A sequencing library of the nucleic acid population (e.g., cfDNA) can be prepared. Such methods can be useful for accurately detecting the methylation status and variants present in a nucleic acid (e.g., cfDNA) sample, which, in turn, can be used to infer information about the cells and subject from which the nucleic acid sample is derived. In some embodiments, the nucleic acid molecule (e.g., cfDNA) is from a subject having or suspected of having a disease or disorder, such as cancer.INTRODUCTION AND SUMMARY

[0003] Cancer is responsible for millions of deaths per year worldwide. Early cancer detection may result in improved outcomes because early-stage cancer tends to be more susceptible to treatment.

[0004] Improperly controlled cell growth is a hallmark of cancer. Cancer is usually caused by the accumulation of mutations within an individual's normal cells, at least some resulting in improperly regulated cell division. Such mutations commonly include single nucleotide variations (SNVs), gene fusions, insertions and deletions (indels), transversions, translocations, and inversions. Cancers may also exhibit an accumulation of epigenetic changes, including modification of cytosine (e.g., 5-methylcytosine, 5- hydroxymethylcytosine, and other more oxidized forms) and association of DNA with chromatin proteins and transcription factors. Thus, cancer can be indicated by nonsequence modifications, such as methylation. Examples of methylation changes inAttorney Docket No.: GH0253WO cancer include local gains of DNA methylation, e.g., in the CpG islands at the transcription start sites of genes involved in normal growth control, DNA repair, cell cycle regulation, and / or cell differentiation. Hypermethylation can be associated with an aberrant loss of transcriptional capacity of involved genes and occurs at least as frequently as point mutations and deletions as a cause of altered gene expression. Furthermore, without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. The DNA from such cells may differ epigenetically from shed DNA in a healthy subject. As such, the distribution of epigenetically modified (e.g., methylated) DNA in certain DNA samples, such as cell-free DNA (cfDNA), may change upon carcinogenesis. Thus, sufficiently sensitive epigenetic (e.g., DNA methylation) profiling can be used to detect aberrant methylation in DNA of a sample.

[0005] Biopsies represent a traditional approach for detecting or diagnosing cancer in which cells or tissue are extracted from a possible cancer site and analyzed for relevant phenotypic and / or genotypic features. Biopsies have the drawback of being invasive. Cancer detection based on analysis of body fluids (“liquid biopsies”), such as blood, is an intriguing alternative based on the observation that DNA from cancer cells is released into body fluids. A liquid biopsy is noninvasive (sometimes requiring only a blood draw). However, it has been challenging to develop accurate and sensitive methods for analyzing liquid biopsy material in part because the amount of nucleic acids released into body fluids is low and variable, as is recovery of nucleic acids from such fluids in analyzable form. Further, the contribution of DNA from cells in or around a cancer or neoplasm to a sample may be relatively small relative to the contribution from other cells, and the DNA contributed from other cells may be uninformative as to cancer status. Isolating and processing cell-free DNA useful for further analysis in liquid biopsy procedures can be a useful part of these methods.

[0006] Current methods of cancer diagnostic assays of cell-free nucleic acids (e.g., cell-free DNA or cell-free RNA) may focus on the detection of tumor-related somatic variants, including single nucleotide variants (SNVs), copy number variations (CNVs), fusions, and indels (i.e., insertions or deletions), which are all mainstream targets for liquid biopsy. There is growing evidence that non-sequence modifications like methylation status and fragmentomic signal in cell-free DNA can provide informationAttorney Docket No.: GH0253WO on the source of cell-free DNA and disease level. Detailed knowledge of the nonsequence modifications of the cell-free DNA (e.g., when combined with somatic mutation calling) can improve assessments of tumor status.

[0007] Accordingly, there is a continued need for improved methods and compositions for analyzing non-sequence modifications (such as methylation status) in DNA, including cell-free DNA, e.g., in liquid biopsies.

[0008] A wide range of applications in genomics, including gene expression analysis, mutation detection, and genomic mapping utilize cell-free DNA (cfDNA) for the detection of disease. Such methods can be biased towards nucleic acids of shorter lengths (approximately less than 180 bp), wherein the methods preferentially amplify shorter nucleic acids in a population of mixed nucleic acids lengths (greater than, equal to, and less than 180 bp). While it has been shown that such fragments can facilitate improved analysis for some assays, such as mutation detection, significant information can be lost due to the inherent bias towards shorter nucleic acids.

[0009] Longer cfDNA fragments (e.g., at least 180 bp) present an opportunity for further application in genomics. Longer cfDNA fragments often originate from different genomic regions compared to shorter cfDNA fragments and can therefore provide a unique insight during disease detection. Traditional methods do not allow for analysis methods that combine the unique insights provided by different length nucleic acids. These challenges are particularly pronounced when dealing with samples of low- abundance nucleic acids or limited sample sizes. There is a need for a method that facilitates the parallel analysis of shorter and longer nucleic acids fragments within the analysis workflow to maximize the information that can be extracted from nucleic acid populations.

[0010] The present invention addresses the need for an improved method for the analysis of a nucleic acid population, through the utilization of nucleic acids of different lengths within the sample, provide other benefits, or at least provide the public with a useful choice.

[0011] In some embodiments, the present disclosure provides a method that allows for the analysis of a nucleic acid population by separating the nucleic acid population into at least two partitions according to nucleic acid length. This provides at least a first partition and a second partition where the nucleic acids in the first partition have aAttorney Docket No.: GH0253WO higher average length relative to the nucleic acids in the second partition. A variety of assays can be performed on these partitions. As nucleic acids of different lengths are likely to originate from different genomic loci, the insights they can provide differ. The assays performed on the partitions can be optimized for the targeted fragment lengths. In some embodiments, further details of the genomic landscape of the nucleic acid sample can be obtained and compared to analyses that do not include size selection.

[0012] Accordingly, in the first aspect, the present disclosure provides a method of analyzing a nucleic acid population, wherein the method comprises: (a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition; (b) performing a first assay on the first partition, wherein: (i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or (ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and (c) performing a second assay on the second partition, wherein: (i) the second assay determines the modification status of nucleic acids in the second partition; and / or (ii) the second assay detects somatic mutations of nucleic acids in the second partition.

[0013] In some embodiments, the nucleic acids in the first partition comprise nucleic acids longer than mononucleosomal cfDNA. In some embodiments, the length of the nucleic acids in the first partition are greater than or at least the length of mononucleosomal cfDNA. In some embodiments, the nucleic acids in the second partition comprise monucleosomal cfDNA and / or nucleic acids shorter than mononucleosomal cfDNA. In some embodiments, the length of the nucleic acids in the second partition are less than and / or equal to the length of mononucleosomal cfDNA.

[0014] Accordingly, the embodiments described herein are provided, which include, but are not limited to, the following.

[0015] Embodiment 1 is a method of analyzing a nucleic acid population comprising nucleic acids of different lengths, wherein the method comprises:(a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition;Attorney Docket No.: GH0253WO(b) performing a first assay on the first partition, wherein:(i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or(ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and(c) performing a second assay on the second partition, wherein:(i) the second assay determines the modification status of nucleic acids in the second partition; and / or(ii) the second assay detects somatic mutations of nucleic acids in the second partition.

[0016] Embodiment 2 is the method of the immediately preceding embodiment, wherein the nucleic acid population is obtained using a sample preparation method optimized for extracting longer nucleic acids.

[0017] Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein the nucleic acid population is partitioned using in vitro size selection.

[0018] Embodiment 4 is the method of any one of the preceding embodiments, wherein the nucleic acids in the first partition are at least 180 bp in length.

[0019] Embodiment 5 is the method of any one of the preceding embodiments, wherein the nucleic acids in the second partition are less than 180 bp in length.

[0020] Embodiment 6 is the method of any one of the preceding embodiments, wherein the separating the nucleic acid population into at least two partitions comprises binding nucleic acids with a higher average length with one or more beads.

[0021] Embodiment 7 is the method of the immediately preceding embodiment, wherein the one or more beads comprise a coating of silica and / or carboxyl groups.

[0022] Embodiment 8 is the method of embodiment 6 or embodiment 7, wherein the one or more beads comprise one or more paramagnetic beads.

[0023] Embodiment 9 is the method of any one of embodiments 6-8, wherein the one or more beads are in a solution comprising polyethylene glycol and a salt.

[0024] Embodiment 10 is the method of any one of embodiments 6-9, wherein the separating the nucleic acid population into at least two partitions comprises solid-phase reversible immobilization (SPRI).Attorney Docket No.: GH0253WO

[0025] Embodiment 11 is the method of any one of embodiments 1-5, wherein the separating the nucleic acid population into at least two partitions comprises column separation.

[0026] Embodiment 12 is the method of the immediately preceding embodiment, wherein the column separation comprises using a silica column.

[0027] Embodiment 13 is the method of embodiment 11 or 12, wherein the column separation comprises mixing the nucleic acid population with a buffer.

[0028] Embodiment 14 is the method of any one of embodiments 1-5, wherein the separating the nucleic acid population into at least two partitions comprises gel electrophoresis.

[0029] Embodiment 15 is the method of any one of the preceding embodiments, wherein the first assay and / or the second assay are untargeted.

[0030] Embodiment 16 is the method of any one of embodiments 1-14, wherein the first assay and / or the second assay are targeted such that the first partition and / or the second partition are enriched for a plurality of target genomic regions.

[0031] Embodiment 17 is the method of the immediately preceding embodiment, wherein enrichment for target genomic regions is performed using hybrid capture or targeted amplification.

[0032] Embodiment 18 is the method of any one of the preceding embodiments, wherein the first assay determines active chromatin regions of nucleic acids in the first partition.

[0033] Embodiment 19 is the method of the immediately preceding embodiment, wherein the first assay comprises identifying genomic features within the nucleic acids of the first partition.

[0034] Embodiment 20 is the method of the immediately preceding embodiment, wherein the genomic features comprise promoters and / or exons.

[0035] Embodiment 21 is the method of embodiment 19 or embodiment 20, further comprising identifying regions of active transcription using the identified genomic features.

[0036] Embodiment 22 is the method of any one of embodiments 19-21, further comprising identifying patterns of gene expression using the identified genomic features.Attorney Docket No.: GH0253WO

[0037] Embodiment 23 is the method of any one of the preceding embodiments, wherein the first assay comprises sequencing, optionally next-generation sequencing (NGS).

[0038] Embodiment 24 is the method of any one of embodiments 1-22, wherein the first assay comprises quantitative PCR (qPCR).

[0039] Embodiment 25 is the method of any one of the preceding embodiments, wherein the first assay determines the fragmentation profile of the nucleic acids in the first partition.

[0040] Embodiment 26 is the method of the immediately preceding embodiment, wherein the fragmentation profile is generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a next generation sequencing assay and the fragmentation profile is generated using data from the next generation sequencing assay.

[0041] Embodiment 27 is the method of any one of the preceding embodiments, wherein the second assay determines the modification status of nucleic acids in the second partition.

[0042] Embodiment 28 is the method of embodiment 27, wherein the second assay comprises a base-conversion based sequencing method.

[0043] Embodiment 29 is the method of the immediately preceding embodiment, wherein the base-conversion based sequencing method comprises bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, enzymatic methyl-seq (EM-seq) conversion, Tet-assisted pyridine borane sequencing (TAPS), APOBEC-coupled epigenetic (ACE) conversion, and / or direct methylation sequencing (DM-seq).

[0044] Embodiment 30 is the method of the immediately preceding embodiment, wherein the Tet-assisted conversion further comprises a substituted borane reducing agent, optionally wherein the substituted borane reducing agent comprises 2-picoline borane, borane pyridine, tert-butylamine borane, and / or ammonia borane.

[0045] Embodiment 31 is the method of embodiment 29 or embodiment 30, wherein the base-conversion based sequencing method comprises contacting the nucleic acids with a CpG-specific DNA methyltransferase (MTase) or a CpG-specificAttorney Docket No.: GH0253WO carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a deaminase.

[0046] Embodiment 32 is the method of the immediately preceding embodimeents, wherein the deaminase is a dsDNA deaminase.

[0047] Embodiment 33 is the method of embodiment 31 or 32, wherein the deaminase is a methyl insensitive deaminase.

[0048] Embodiment 34 is the method of the immediately preceding embodiment, wherein the methyl insensitive deaminase is an APOB EC enzyme, optionally wherein the APOBEC enzyme is APOBEC3A.

[0049] Embodiment 35 is the method of any one of embodiments 31 or 32, wherein the deaminase is a methyl sensitive deaminase.

[0050] Embodiment 36 is the method of the immediately preceding embodiment, wherein the methyl sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modification-sensitive DNA deaminase A (MsddA)-like deaminase.

[0051] Embodiment 37 is the method of embodiment 27, wherein the second assay comprises generating sub-partitions of nucleic acids according to the modification status of the nucleic acids, wherein at least one of the sub-partitions is enriched for nucleic acids with a modification of interest, wherein generating sub-partitions comprises providing a binding agent that preferentially binds to nucleic acids within the second partition that contain the modification of interest.

[0052] Embodiment 38 is the method of the immediately preceding embodiment, further comprising separating the nucleic acids bound to the binding agent from the unbound nucleic acids to produce an unbound sub-partition.

[0053] Embodiment 39 is the method of embodiment 37 or embodiment 38, further comprising eluting nucleic acids bound to the binding agent to provide a sub-partition containing nucleic acids comprising the modification.

[0054] Embodiment 40 is the method of any one of embodiments 37-39, wherein bound nucleic acids are subjected to a plurality of conditions which increasingly favor elution of the nucleic acids bound to the binding agent to provide multiple sub-partitions wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the modification compared to the nucleic acids eluted in the last of the plurality of conditions.Attorney Docket No.: GH0253WO

[0055] Embodiment 41 is the method of any one of embodiments 37-40, wherein the binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody or an antigen-binding fragment thereof.

[0056] Embodiment 42 is the method of any one of embodiments 27-41, further comprises quantifying the level of modifications, optionally using quantitative PCR (qPCR).

[0057] Embodiment 43 is the method of the immediately preceding embodiment, wherein the quantification is used to provide a modification score.

[0058] Embodiment 44 is the method of any one of embodiments 27-43, wherein the modification is a methylated nucleotide.

[0059] Embodiment 45 is the method of any one of embodiments 27-44, wherein the modification is 5-methylcytosine (5mC).

[0060] Embodiment 46 is the method of any one of the preceding embodiments, wherein the second assay detects somatic mutations within the nucleic acid population.

[0061] Embodiment 47 is the method of the immediately preceding embodiment, wherein the second assay comprises hybrid capture of nucleic acids comprising genomic regions of interest.

[0062] Embodiment 48 is the method of embodiment 46, wherein the second assay comprises targeted amplification of amplicons containing genomic regions of interest.

[0063] Embodiment 49 is the method of any one of embodiments 46-48, wherein the second assay comprises sequencing, optionally next generation sequencing (NGS), or qPCR.

[0064] Embodiment 50 is the method of any one of the preceding embodiments, further comprising preparing one or more sequencing libraries from at least a portion of the nucleic acids in the first partition and / or the second partition, further comprising sequencing the one or more sequencing libraries.

[0065] Embodiment 51 is the method of any one of embodiments 23-50, wherein the NGS comprises pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by-ligation, or sequencing-by-hybridization.

[0066] Embodiment 52 is the method of any one of embodiments 23-50, wherein the sequencing comprises nanopore-based sequencing or single-molecule real time (SMRT) sequencing.Attorney Docket No.: GH0253WO

[0067] Embodiment 53 is the method of any one of embodiments 23-50, wherein the sequencing comprises long-read sequencing.

[0068] Embodiment 54 is the method of any one of embodiments 23-50, wherein the sequencing comprises nanopore-based sequencing.

[0069] Embodiment 55 is the method of any one of embodiments 23-50, wherein the sequencing comprises 5-letter or 6-letter sequencing.

[0070] Embodiment 56 is the method of any one of embodiments 23-50 and 54, wherein the sequencing comprises nanopore-based sequencing and the method further comprises subjecting the nucleic acids in the sample to end repair to generate end- repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0071] Embodiment 57 is the method of any one of embodiments 23-50 and 52, wherein the sequencing comprises single-molecule real time (SMRT) sequencing and the method further comprises subjecting the nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end- repaired DNA molecules at one or more locations.

[0072] Embodiment 58 is the method of embodiment 56 or embodiment 57, wherein the modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, dUTP, a dNTP comprising fluorodeoxyuridine (FldU), a dNTP comprising 5-iododeoxyuridine (IdU), a dNTP comprising 5-ethynyldeoxyuridine (EdU), and / or a dNTP comprising 8oxoG.

[0073] Embodiment 59 is the method of any one of embodiments 56-58, further comprising subjecting the nucleic acids in the sample to end repair to generate end- repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is other than 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.Attorney Docket No.: GH0253WO

[0074] Embodiment 60 is the method of any one of embodiments 56-58, further comprising subjecting nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end- repaired DNA molecules at one or more locations.

[0075] Embodiment 61 is the method of any one of embodiments 56-58, further comprising subjecting the nucleic acids in the sample to end repair to generate end- repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, wherein the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations, and the repaired region is defined as:(i) the sequence between two non-methylated cytosines which span one or more methylated CpH cytosines; and / or(ii) the sequence between a methylated CpH cytosine and an end of a sequence read, wherein the methylated CpH cytosine is the CpH cytosine most distant from the end of the sequence read, or a subsequence thereof comprising one or more methylated CpH cytosines.

[0076] Embodiment 62 is the method of any one of embodiments 56-61, further comprising analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the nucleic acids in the sample.

[0077] Embodiment 63 is the method of any one of embodiments 56-62, wherein the method further comprises detecting the methylation status of cytosines in the nucleic acids in the sample, and wherein the analyzing the sequence data further comprises filtering out the one or more repaired regions of the end-repaired DNA molecules such that the one or more repaired regions are not used to determine the methylation status of cytosines in the nucleic acids in the sample.Attorney Docket No.: GH0253WO

[0078] Embodiment 64 is the method of any one of embodiments 56-63, wherein the method is for detecting the single nucleotide variants (SNVs) in the nucleic acids in the sample, and wherein the analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support.

[0079] Embodiment 65 is the method of any one of embodiments 56-64, further comprising analyzing the sequence data to determine a level of measured artifacts in the nucleic acids.

[0080] Embodiment 66 is the method of any one of the preceding embodiments, further comprising amplifying at least a portion of the nucleic acids using a DNA polymerase.

[0081] Embodiment 67 is the method of any one of embodiments 1-55 and 65-66, further comprising subjecting the nucleic acids in the sample to end repair to generate end-repaired nucleic acid molecules, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs).

[0082] Embodiment 68 is the method of the immediately preceding embodiment, wherein at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

[0083] Embodiment 69 is the method of embodiment 67 or embodiment 68, wherein the end repair is performed using a DNA polymerase that does not have 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

[0084] Embodiment 70 is the method of embodiment 67 or embodiment 68, wherein the end repair is performed using a DNA polymerase that has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0085] Embodiment 71 is the method of any one of embodiments 67-70, wherein the at least one type of dNTP which comprises a modified base, wherein the modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5- methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).Attorney Docket No.: GH0253WO

[0086] Embodiment 72 is the method of any one of embodiments 67-71, wherein the end repair occurs prior to step a).

[0087] Embodiment 73 is the method of any one of embodiments 67-71, wherein the end repair occurs after step a).

[0088] Embodiment 74 is the method of any one of the preceding embodiments, further comprising performing an A-tailing reaction, optionally after a step of subjecting the nucleic acids in the sample to end repair.

[0089] Embodiment 75 is the method of the immediately preceding embodiment, wherein the end-repair and the A-tailing reaction are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reaction are performed a single tube and / or optionally wherein the end-repair and the A-tailing reaction are performed without an intervening clean-up step.

[0090] Embodiment 76 is the method of embodiment 74 or embodiment 75, wherein the A-tailing is performed using a DNA polymerase that does not possess 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

[0091] Embodiment 77 is the method of any one of embodiments 74-76, wherein the A-tailing is performed using a thermostable DNA polymerase.

[0092] Embodiment 78 is the method of any one of the preceding embodiments, wherein one or more adapters are ligated to the end-repaired DNA molecules or one or more adapters are ligated to the nucleic acids in the sample.

[0093] Embodiment 79 is the method of the immediately preceding embodiment, wherein the one or more adapters comprise molecular barcodes.

[0094] Embodiment 80 is the method of embodiment 78 or embodiment 79, wherein at least one cytosine in the one or more adapters is a modification resistant cytosine, optionally wherein each cytosine in the one or more adapters is a modification resistant cytosine.

[0095] Embodiment 81 is the method of the immediately preceding embodiment, wherein the modification resistant cytosine is a deaminase resistant cytosine.

[0096] Embodiment 82 is the method of the immediately preceding embodiment, wherein the deaminase resistant cytosine is 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC),Attorney Docket No.: GH0253WO5-hydroxymethylcytosine (5hmC), glucosylated5-hydroxymethylcytosine (5ghmC), cytosine 5-methylenesulfonate (CMS), or N4-modified cytosine.

[0097] Embodiment 83 is the method of any one of embodiments 78-82, wherein the one or more adapters are Y-shaped adapters.

[0098] Embodiment 84 is the method of any one of the preceding embodiments, wherein the method comprises ligating one or more adapters to the nucleic acids after the step of separating the nucleic acid population into at least two partitions.

[0099] Embodiment 85 is the method of any one of embodiments 1-83, wherein the method comprises ligating one or more adapters to the nucleic acids prior to the step of separating the nucleic acid population into at least two partitions.

[0100] Embodiment 86 is the method of any one of the preceding embodiments, wherein the nucleic acids in the sample comprises barcodes.

[0101] Embodiment 87 is the method of any one of the preceding embodiments, further comprising enriching the nucleic acids in the sample for a plurality of target regions.

[0102] Embodiment 88 is the method of the immediately preceding embodiment, wherein the enriching the nucleic for a plurality of target regions occurs: i) after partitioning the nucleic in the sample into a plurality of subsamples; ii) prior to a step of sequencing the nucleic acids in the sample; iii) after amplifying the nucleic acids; and / or iv) prior to amplifying the nucleic acids.

[0103] Embodiment 89 is the method of embodiment 87 or embodiment 88, wherein the plurality of target regions comprises epigenetic target regions.

[0104] Embodiment 90 is the method of the immediately preceding embodiment, wherein the epigenetic target regions comprise hypermethylation variable target regions.

[0105] Embodiment 91 is the method of the immediately preceding embodiment, wherein the hypermethylation variable region comprises regions having a higher degree of methylation in at least one type of tissue or in blood than the degree of methylation in nucleic acids from a healthy subject.

[0106] Embodiment 92 is the method of any one of embodiments 89-91, wherein the epigenetic target regions comprise hypomethylation variable target regions.Attorney Docket No.: GH0253WO

[0107] Embodiment 93 is the method of the immediately preceding embodiment, wherein the hypomethylation variable region comprises regions having a lower degree of methylation in at least one type of tissue or in blood than the degree of methylation in nucleic acids from a healthy subject.

[0108] Embodiment 94 is the method of any one of embodiments 89-93, wherein the epigenetic target regions comprise a methylation control target region set.

[0109] Embodiment 95 is the method of any one of embodiments 89-94, wherein the epigenetic target region set comprise a fragmentation variable target region set.

[0110] Embodiment 96 is the method of the immediately preceding embodiment, wherein the fragmentation variable target region set comprises transcription start site regions.

[0111] Embodiment 97 is the method of embodiment 95 or embodiment 96, wherein the fragmentation variable target region set comprises CTCF binding regions.

[0112] Embodiment 98 is the method of any one of embodiments 87-97, wherein the plurality of target regions comprise sequence-variable target regions.

[0113] Embodiment 99 is the method of any one of the preceding embodiments, further comprising performing a methylation-preserving amplification of the nucleic acids of the sample.

[0114] Embodiment 100 is the method of the immediately preceding embodiment, wherein the methylation-preserving amplification is a linear, methylation-preserving amplification.

[0115] Embodiment 101 is the method of embodiment 99 or embodiment 100, wherein the methylation-preserving amplification comprises contacting the nucleic acids with a methyltransferase.

[0116] Embodiment 102 is the method of any one of embodiments 99-101, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and selfsustained sequence-based replication.

[0117] Embodiment 103 is the method of any one of embodiments 99-101, wherein the methylation-preserving amplification comprises thermocycled amplification.Attorney Docket No.: GH0253WO

[0118] Embodiment 104 is the method of any one of embodiments 99-101, wherein the methylation-preserving amplification comprises isothermal amplification.

[0119] Embodiment 105 is the method of any one of embodiments 99-104, wherein the methylation-preserving amplification occurs prior to step a).

[0120] Embodiment 106 is the method of any one of the preceding embodiments, wherein the nucleic acid population comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

[0121] Embodiment 107 is the method of any one of the preceding embodiments, wherein the sample is a tissue sample.

[0122] Embodiment 108 is the method of any one of the preceding embodiments, wherein the sample is a blood sample.

[0123] Embodiment 109 is the method of the immediately preceding embodiment, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a peripheral blood mononuclear cell (PBMC) sample.

[0124] Embodiment 110 is the method of any one of the preceding embodiments, wherein the sample is from a subject.

[0125] Embodiment 111 is the method of any one of the preceding embodiments, wherein the sample is from a subject and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.

[0126] Embodiment 112 is the method of any one of embodiments 110 or 111, wherein the subject is an animal.

[0127] Embodiment 113 is the method of the immediately preceding embodiment, wherein the subject is a human.

[0128] Embodiment 114 is the method of any one of embodiments 110-113, wherein the subject has or is at risk of having a cancer.

[0129] Embodiment 115 is the method of any one of embodiments 110-114, further comprising determining the presence or status of a cancer in the subject.

[0130] Embodiment 116 is the method of any one of embodiments 110-115, further comprising determining a likelihood that the subject has cancer.Attorney Docket No.: GH0253WO

[0131] Embodiment 117 is the method of embodiments 23-116, wherein the sequencing comprises generating a plurality of sequencing reads; and the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer.

[0132] Embodiment 118 is the method of the immediately preceding embodiment, further comprising detecting a presence or absence of nucleic acids originating or derived from a tumor cell using the mapped sequence reads.

[0133] Embodiment 119 is the method of the immediately preceding embodiment, further comprising determining a cancer recurrence score that is indicative of the presence or absence of the nucleic acids originating or derived from the tumor cell for the test subject, optionally further comprising determining a cancer recurrence status based on the cancer recurrence score, wherein the cancer recurrence status of the test subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the test subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

[0134] Embodiment 120 is the method of the immediately preceding embodiment, further comprising comparing the cancer recurrence score of the test subject with a predetermined cancer recurrence threshold, wherein the test subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.

[0135] Embodiment 121 is the method of any of the preceding embodiments, wherein nucleic acids in the first partition comprise nucleic acids longer than mononucleosomal cfDNA. Embodiment 122 is the method of any of the preceding embodiments, wherein the length of the nucleic acids in the first partition are greater than or at least the length of mononucleosomal cfDNA. Embodiment 123 is the method of any of the preceding embodiments, wherein the nucleic acids in the second partition comprise monucleosomal cfDNA and / or nucleic acids shorter than mononucleosomal cfDNA. Embodiment 124 is the method of any of the preceding embodiments, wherein theAttorney Docket No.: GH0253WO length of the nucleic acids in the second partition are less than and / or equal to the length of mononucleosomal cfDNA.

[0136] The various steps of the methods disclosed herein may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and / or by the same or different people.

[0137] In some embodiments, the results of the methods disclosed herein are used as an input to generate a report. The report may be in a paper or electronic format. For example, the results of the assays performed on the partitions as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such a report. Alternatively, or additionally, diagnostic information or therapeutic recommendations which are at least in part based on the methods disclosed herein can be included in the report.

[0138] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0139] FIG. 1 is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0140] Reference will now be made in detail to certain embodiments of the present disclosure. While the present disclosure will be described in conjunction with such embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the present disclosure as defined by the appended claims.

[0141] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearlyAttorney Docket No.: GH0253WO dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids.

[0142] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0143] Unless specifically noted in the above specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components.

[0144] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way.

[0145] All patents, patent applications, websites, other publications or documents and the like cited herein whether supra or infra, are expressly incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant, unless otherwise indicated.I. Definitions

[0146] “Solid tissue” or “solid tissue cells” as used herein means tissue or cells, respectively, in or derived from a solid tissue. Solid tissue cells exclude circulating cell types, such as cells normally present in blood or lymph. Examples of solid tissue types include but are not limited to colon, lung, breast, skin, prostate, stomach, pancreas, bladder, kidney, and liver.

[0147] A “reaction cleanup” refers to the removal of contaminants such as salts, enzymes, unincorporated dNTPs, primers, ethidium bromide, and other impurities that can interfere with downstream analysis. For example, when a reaction cleanup is performed between end repair and an A-tailing reaction, it removes unincorporated dNTPs such that the A-tailing reaction can be performed solely in the presence of dATP ( / .< . not dCTP, dGTP and dTTP, as used in the end tailing reaction). Reaction cleanups can be performed using commercially available kits such as MinElute Reaction Cleanup Kit (Qiagen).Attorney Docket No.: GH0253WO

[0148] “Regions of the end-repaired DNA that were synthesized during the end repair reaction”, also referred to as “repaired regions” or “synthesized regions,” refer to regions of the DNA that were not present in the DNA prior to the end repair and A- tailing reactions. They are regions which have been synthesized by the polymerases used in the end repair and / or A tailing reactions, if present. In instances where the A- tailing is performed in the same tube as the end repair reaction, all four types of dNTPs will be present, and thus the polymerases used for A-tailing may generate synthesized regions, e.g. through nick translation. In instances where the A-tailing is performed separately to the end repair reaction, and these steps are separated by a reaction cleanup, only dATP will be present in the A-tailing reaction, and thus the polymerases used for A-tailing will not typically generate synthesized regions because the dNTP components are not all present in the A-tailing reaction mix.

[0149] A “type of dNTP” refers to a dNTP comprising a specific base, including A, T, G or C. Accordingly, wherein an end repair reaction is performed with dNTPs, wherein at least one type of dNTP comprises a modified base, the end repair reaction may be performed using dCTP comprising 5mC, and dATP, dTTP and dGTP all comprising non-modified bases.

[0150] “Capable of identifying the base modification in the at least one type of dNTP” refers to the ability of a modification-sensitive sequencing method to detect the presence or absence of the base modification in the at least one type of dNTP comprising a modified base used in the end repair. This detection of the base modification may be direct, such as in nanopore sequencing or single molecule real time sequencing, wherein the sequencing data itself indicates the presence or absence of a base modification. Alternatively, the detection of the base modification may be indirect, for example wherein the method involves a conversion procedure which alters the base pairing specificity dependent on the base modification status. It is these changes in base pairing specificity which can be detected by the sequencing method, e.g. through the comparison of the sequencing data to a reference sequence. Moreover, a modificationsensitive sequencing method is capable of identifying the base modification in the at least one type of dNTP regardless of whether it can distinguish one base modification from all other base modifications. For example, one form of modification-sensitive sequencing is sequencing after bisulfite conversion. This method is capable ofAttorney Docket No.: GH0253WO distinguishing 5hmC and 5mC from unmethylated cytosine but cannot distinguish 5hmC from 5mC.

[0151] Bases of the “same identity” refer to the same base, regardless of modification status of that base. For example, cytosine is considered to be the “same identity” as 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), despite them having different modification statuses.

[0152] “Cell-free DNA,” “cfDNA molecules,” or simply “cfDNA” include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) into a fluid found in the organism and may be obtained from a sample of the fluid without the need to perform an in vitro cell lysis step.

[0153] As used herein, “cellular nucleic acids” means nucleic acids that are located within one or more cells from which the nucleic acids have originated, at least at the point a sample is taken or collected from a subject, even if those nucleic acids are subsequently removed (e.g., via cell lysis) as part of a given analytical process.

[0154] A “target region set” or “set of target regions” or “target regions” or “target regions of interest” or “regions of interest” or “genomic regions of interest” refers to a plurality of genomic loci or a plurality of genomic regions targeted for capture and / or targeted by a set of probes (e.g., through sequence complementarity).

[0155] “Sequence-variable target region set” refers to a set of target regions that may exhibit changes in sequence such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or transpositions in neoplastic cells (e.g., tumor cells and cancer cells).

[0156] “Epigenetic target region set” refers to target regions that may show sequence-independent changes in neoplastic cells (e.g., tumor cells or cancer cells) or that may show sequence-independent changes in cfDNA from subjects having cancer relative to cfDNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, changes in methylation (increases or decreases), nucleosome distribution, CCCTC-binding factor (“CTCF”) binding, transcription start sites, and regulatory protein binding regions. For present purposes, loci susceptible toAttorney Docket No.: GH0253WO neoplasia-, tumor-, or cancer-associated focal amplifications and / or gene fusions may also be included in an epigenetic target region set because detection of a change in copy number by sequencing or a fused sequence that maps to more than one locus in a reference genome tends to be more similar to detection of exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, e.g., in that the focal amplifications and / or gene fusions can be detected at a relatively shallow depth of sequencing because their detection does not depend on the accuracy of base calls at one or a few individual positions.

[0157] As used herein, an “epigenetic feature” refers to any feature of DNA or chromatin other than primary sequence (i.e., the sequence of A, C, G, and T bases). Epigenetic features include covalent modifications of bases, such as methylation, and modifications and positioning of histones and other stably DNA-associated proteins.

[0158] As used herein, a “differentially methylated region” (DMR) refers to a region of DNA having a detectably different degree of methylation in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type; or having a detectably different degree of methylation in at least one cell or tissue type obtained from a subject having a disease or disorder relative to the degree of methylation in the same region of DNA in the same cell or tissue type obtained from a healthy subject . In some embodiments, a DMR has a detectably higher degree of methylation (e.g., hypermethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject. In some embodiments, a DMR has a detectably lower degree of methylation (e.g., hypomethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type from a healthy subject.

[0159] As used herein, “type-specific” in the context of an epigenetic variation means an epigenetic variation that is present at a detectably different degree in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. Similarly, a “type-specific epigenetic target region” is an epigenetic target region that has a detectably different epigenetic characteristic in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types.Attorney Docket No.: GH0253WOExemplary epigenetic characteristics are discussed in the definition of epigenetic target regions set forth above. For example, a “type-specific differentially methylated region” is a region of DNA that has a detectably different degree of methylation in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. Examples of a type-specific differentially methylated region include tissuespecific differentially methylated regions, including those associated with copy -number gain in early cancer. In some embodiments, capturing, identification, and / or detection of type-specific differentially methylated regions facilitates identification of the cell or tissue type from which the DNA originated. The cell or tissue from which a typespecific differentially methylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue. In another example, a “type-specific fragment” of DNA is a DNA fragment arising from a type-specific fragmentation pattern that is present at a detectably different degree in one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, a type-specific fragment is only present in the specific cell or tissue type(s). In some embodiments, a type-specific fragment is present to a detectably greater extent in the specific cell or tissue type(s).

[0160] As used herein, a “blood sample” refers to a sample comprising whole blood or a component thereof (e.g., plasma, serum, buffy coat, plasma pellet).

[0161] As used herein, “partitioning” refers to physically separating or fractionating a mixture of nucleic acid molecules in a sample based on a characteristic of the nucleic acid molecules. The partitioning can be physical partitioning of molecules. Partitioning can involve separating the nucleic acid molecules into groups or sets based on the level of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned based on the level of methylation of the nucleic acid molecules. In some embodiments, the methods and systems used for partitioning may be found in PCT Patent Application No. PCT / US2017 / 068329, which is hereby incorporated by reference in its entirety.

[0162] As used herein, “partitioned set” or “partition” refers to a set of nucleic acid molecules partitioned into a set or group based on the differential binding affinity of the nucleic acid molecules or proteins associated with the nucleic acid molecules to a binding agent. A partitioned set may also be referred to as a subsample. The bindingAttorney Docket No.: GH0253WO agent binds preferentially to the nucleic acid molecules comprising nucleotides with epigenetic modification. For example, if the epigenetic modification is methylation, the binding agent can be a methyl binding domain (MBD) protein. In some embodiments, a partitioned set can comprise nucleic acid molecules belonging to a particular level or degree of epigenetic feature (for e.g., methylation). For example, the nucleic acid molecules can be partitioned into three sets - one set for highly methylated nucleic acid molecules (first subsample, hyper partition, hyper partitioned set or hypermethylated partitioned set), a second set for low methylated nucleic acid molecules (second subsample, hypo partition, hypo partitioned set or hypomethylated partitioned set), and a third set for intermediate methylated nucleic acid molecules (third subsample, intermediate partitioned set, intermediately methylated partitioned set, residual partitioned set, or residual partition). In another example, the nucleic acid molecules can be partitioned based on the number of methylated nucleotides - one partitioned set can have nucleic acid molecules with nine methylated nucleotides, and another partitioned set can have unmethylated nucleic acid molecules (zero methylated nucleotides).

[0163] As used herein, the form of the “originally isolated” sample refers to the composition or chemical structure of a sample at the time it was isolated and before undergoing any procedure that changes the chemical structure of the isolated sample. Similarly, a feature that is “originally present” in a molecule refers to a feature present in an “original molecule” or in molecules “originally comprising” the feature before the molecule undergoes any procedure that changes the chemical structure of the molecule.

[0164] As used herein, “base pairing specificity” refers to the standard DNA base (A, C, G, or T) for which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G) whereas uracil and cytosine have different base pairing specificity because uracil has base pairing specificity for A while cytosine has base pairing specificity for G. The ability of uracil to form a wobble pair with G, for example, is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0165] “Capturing” one or more target molecules refers to preferentially isolating or separating the one or more target molecules from non-target molecules.Attorney Docket No.: GH0253WO

[0166] A “captured set” of nucleic acids refers to nucleic acids that have undergone capture.

[0167] “Corresponding to a target region set” means that a nucleic acid, such as cfDNA, originated from a locus in the target region set or specifically binds one or more probes for the target-region set.

[0168] As used herein, a “label” is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of that to which it is attached.

[0169] As used herein, a “capture moiety” is a molecule that allows affinity separation of molecules linked to the capture moiety from molecules lacking the capture moiety. Exemplary capture moieties include biotin, which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.

[0170] As used herein, a “target-specific probe” means a probe that specifically binds to a target region, such as an epigenetic target region or a sequence-variable target region. In some embodiments, target-specific probes comprise a capture moiety to facilitate capture of the target region to which it specifically binds.

[0171] As used herein, a “tag” is a molecule, such as a nucleic acid, label, fluorophore, or peptide, containing information that indicates a feature of the molecule to which the tag is associated. For example, molecules can bear a sample tag (which distinguishes molecules in one sample from those in a different sample), a molecular tag / molecular barcode / barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios), a purification tag, and / or a detectable tag or label.

[0172] As used herein, a “target molecule” is a molecule, such as a protein, carbohydrate, nucleic acid, or lipid, that is targeted for capture, identification, and / or detection. In some embodiments, a target molecule is a nucleic acid comprising an epigenetic target region and / or a sequence-variable target region.

[0173] “Specifically binds” in the context of a probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or replicates thereof, to formAttorney Docket No.: GH0253WO a stable probe:target hybrid, while at the same time formation of stable probemon-target hybrids is minimized. Thus, a probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non-target sequence, to enable capture or detection of the target sequence. Appropriate hybridization conditions are well-known in the art, may be predicted based on sequence composition, or can be determined by using routine testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50- 9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated by reference herein).

[0174] DNA is “derived from cancerous cells” if it originated from a tumor cell. Cell free DNA derived from cancerous cells includes ctDNA or circulating tumor DNA Tumor cells are neoplastic cells that originated from a tumor, regardless of whether they remain in the tumor or become separated from the tumor (as in the cases, e.g., of metastatic cancer cells and circulating tumor cells).

[0175] The “capture yield” of a collection of probes for a given target region set refers to the amount (e.g., amount relative to another target region set or an absolute amount) of nucleic acid corresponding to the target set that the collection of probes captures under typical conditions. Exemplary typical capture conditions are an incubation of the sample nucleic acid and probes at 65°C for 10-18 hours in a small reaction volume (about 20 pL) containing stringent hybridization buffer. The capture yield may be expressed in absolute terms or, for a plurality of collections of probes, relative terms. When capture yields for a plurality of sets of target regions are compared, they are normalized for the footprint size of the target region set (e.g., on a per-kilobase basis). Thus, for example, if the footprint sizes of first and second target regions are 50 kb and 500 kb, respectively (giving a normalization factor of 0.1), then the DNA corresponding to the first target region set is captured with a higher yield than DNA corresponding to the second target region set when the mass per volume concentration of the captured DNA corresponding to the first target region set is more than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set. As a further example, using the same footprint sizes, if the captured DNA corresponding to the first target region set has a mass per volume concentration of 0.2 times the mass per volume concentration of the captured DNAAttorney Docket No.: GH0253WO corresponding to the second target region set, then the DNA corresponding to the first target region set was captured with a two-fold greater capture yield than the DNA corresponding to the second target region set.

[0176] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to addition of a methyl group to a cytosine at a CpG site (cytosine- phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ -> 3’ direction of the nucleic acid sequence)). In some embodiments, DNA methylation refers to addition of a methyl group to adenine, such as in N6-methyladenine (6mA). In some embodiments, DNA methylation is 5-methylation (modification of the carbon in the 5thposition of the cytosine ring). In some embodiments, 5-methylation refers to addition of a methyl group to the 5C position of the cytosine to create 5-methylcytosine (5mC). In some embodiments, methylation comprises a derivative of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5- fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon in the 3rdposition of the cytosine ring). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine to generate 3 -methylcytosine (3mC). Methylation can also occur at non- CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0177] The “modified nucleoside profile of DNA” means the position and identity of the nucleoside and the modification status of the nucleoside, such as methylations, within a DNA sequence. As described above, different modification sensitive sequencing methods can be used to detect such modifications. This includes methods which involve conversion followed by sequencing detect one or more different types of modified or unmodified nucleoside. For example, the TAPS method detects, but does not distinguish between, 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosineAttorney Docket No.: GH0253WO(5hmC). Hence, a method for analyzing the modified nucleoside profile of DNA in a sample typically means identifying particular modifications or groups of modification, such as 5mC and / or 5hmC. Modified nucleosides are identified according to the specific method / conversion procedure being used as described above. This generally involves comparing sequence data obtained from DNA that has been subjected to a conversion procedure to a reference sequence. Typically, the method involves (i) comparing the sequence data with (A) one or more pre-determined reference sequence; or (B) sequence data obtained by sequencing a sub-sample of the DNA that was not subjected to the conversion procedure, for example a subsample that was separated before subjecting a separate subsample to the conversion procedure, for example as described herein; and (ii) identifying point differences between the converted DNA sequences and the reference sequence(s) (A) or non-converted DNA sequences (B) as nucleosides (in the initial sample) having a modification status that permits a change in base pairing specificity on exposure to the conversion procedure.

[0178] As used herein, a modification or other feature is present in “a greater proportion” in a first sample or population of nucleic acid than in a second sample or population when the fraction of nucleotides with the modification or other feature is higher in the first sample or population than in the second population. For example, if in a first sample, one tenth of the nucleotides are mC, and in a second sample, one twentieth of the nucleotides are mC, then the first sample comprises the cytosine modification of 5-methylation in a greater proportion than the second sample.

[0179] As used herein, “without substantially altering base-pairing specificity” of a given nucleobase means that a majority of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample. In some embodiments, 75%, 90%, 95%, or 99% of molecules comprising that nucleobase that can be sequenced do not have alterations of the base pairing specificity of the second nucleobase relative to its base pairing specificity as it was in the originally isolated sample.

[0180] As used herein, “modified cytosine” refers to a cytosine in which at least one position of the cytosine has been substituted with a chemical moiety, such as a methyl or hydroxymethyl, that is different from the substituent at that position inAttorney Docket No.: GH0253WO unmodified cytosine. For the avoidance of doubt, “modified cytosine” does not include unmodified cytosine.

[0181] As used herein, a “combination” comprising a plurality of members refers to either of a single composition comprising the members or a set of compositions in proximity, e.g., in separate containers or compartments within a larger container, such as a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine, or other form of storage.

[0182] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypermethylated DNA can include DNA molecules comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, or at least 10 methylated residues.

[0183] As used herein, “type-specific hypermethylation” means an increased level or degree of methylation of nucleic acid molecules in at one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, capturing, identification, and / or detection of type-specific hypermethylated regions facilitates identification of the cell or tissue type from which the nucleic acid molecules originated. The cell or tissue from which a type-specific hypermethylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue.

[0184] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules within a population (e.g., sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules comprising 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0185] As used herein, “type-specific hypomethylation” means a decreased level or degree of methylation of nucleic acid molecules in at one cell or tissue type, or in a plurality of related cell or tissue types, relative to other cell or tissue types. In some embodiments, capturing, identification, and / or detection of type-specificAttorney Docket No.: GH0253WO hypomethylated regions facilitates identification of the cell or tissue type from which the nucleic acid molecules originated. The cell or tissue from which a type-specific hypomethylated region originated may be a wild type cell or tissue or a neoplastic cell or tissue.

[0186] As used herein, “methylation status” can refer to the presence or absence of methyl group on a DNA base (e.g. cytosine) at a particular genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., highly methylated, low methylated, intermediately methylated or unmethylated nucleic acid molecules). The methylation status can also refer to the number of nucleotides methylated in a particular nucleic acid molecule.

[0187] As used herein, “mutation” refers to a variation from a known reference sequence and includes mutations such as, for example, single nucleotide variants (SNVs), and insertions or deletions (indels). A mutation can be a germline or somatic mutation. In some embodiments, a reference sequence for purposes of comparison is a wildtype genomic sequence of the species of the subject providing a test sample, typically the human genome.

[0188] As used herein, the terms “neoplasm” and “tumor” are used interchangeably. They refer to abnormal growth of cells in a subject. A neoplasm or tumor can be benign, potentially malignant, or malignant. A malignant tumor is referred to as a cancer or a cancerous tumor.

[0189] As used herein, “next-generation sequencing” or “NGS” refers to sequencing technologies having increased throughput as compared to traditional Sanger- and capillary electrophoresis-based approaches, for example, with the ability to generate hundreds of thousands of relatively small sequence reads at a time. Some examples of next-generation sequencing techniques include, but are not limited to, sequencing by synthesis, sequencing by ligation, and sequencing by hybridization. In some embodiments, next-generation sequencing includes the use of instruments capable of sequencing single molecules. Examples of commercially available instruments for performing next-generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM and Ion GeneStudio S5.

[0190] As used herein, “nucleic acid tag” refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10Attorney Docket No.: GH0253WO nucleotides in length), used to distinguish nucleic acids from different samples (e.g., representing a sample index), distinguish nucleic acids from different partitions (e.g., representing a partition tag) or different nucleic acid molecules in the same sample (e.g., representing a molecular barcode), of different types, or which have undergone different processing. The nucleic acid tag comprises a predetermined, fixed, non-random, random or semi-random oligonucleotide sequence. Such nucleic acid tags may be used to label different nucleic acid molecules or different nucleic acid samples or sub-samples. Nucleic acid tags can be single-stranded, double-stranded, or at least partially doublestranded. Nucleic acid tags optionally have the same length or varied lengths. Nucleic acid tags can also include double-stranded molecules having one or more blunt-ends, include 5’ or 3’ single-stranded regions (e.g., an overhang), and / or include one or more other single-stranded regions at other locations within a given molecule. Nucleic acid tags can be attached to one end or to both ends of the other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information such as the sample of origin, form, or processing of a given nucleic acid. For example, nucleic acid tags can also be used to enable pooling and / or parallel processing of multiple samples comprising nucleic acids bearing different molecular barcodes and / or sample indexes in which the nucleic acids are subsequently being deconvolved by detecting (e.g., reading) the nucleic acid tags. Nucleic acid tags can also be referred to as identifiers (e.g. molecular identifier, sample identifier). Additionally, or alternatively, nucleic acid tags can be used as molecular identifiers (e.g., to distinguish between different molecules or amplicons of different parent molecules in the same sample or sub-sample). This includes, for example, uniquely tagging different nucleic acid molecules in a given sample, or non-uniquely tagging such molecules. In the case of non-unique tagging applications, a limited number of tags (i.e., molecular barcodes) may be used to tag each nucleic acid molecule such that different molecules can be distinguished based on their endogenous sequence information (for example, start and / or stop positions where they map to a selected reference genome, a sub-sequence of one or both ends of a sequence, and / or length of a sequence) in combination with at least one molecular barcode. Typically, a sufficient number of different molecular barcodes are used such that there is a low probability (e.g., less than about a 10%, less than about a 5%, less than about a 1%, or less than about a 0.1% chance) that any twoAttorney Docket No.: GH0253WO molecules may have the same endogenous sequence information (e.g., start and / or stop positions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode. Terms such as “library adapters having distinct molecular barcodes” encompass library adapters for uniquely or non-uniquely tagging molecules, in that regardless of whether the adapters are for unique or non-unique tagging, distinct barcodes will be present in the population of adapters.

[0191] As used herein, DNA that is “not immobilized” or that is “free in solution” refers to DNA that is not bound covalently or non-covalently to a solid support, such as a bead. Such DNA may be free in solution during any step (such as all steps) of the disclosed methods.

[0192] The terms “agent that recognizes a modified nucleobase in DNA,” such as an “agent that recognizes a modified cytosine in DNA” refers to a molecule or reagent that binds to or detects one or more modified nucleobases in DNA, such as methyl cytosine. A “modified nucleobase” is a nucleobase that comprises a difference in chemical structure from an unmodified nucleobase. In the case of DNA, an unmodified nucleobase is adenine, cytosine, guanine, or thymine. In some embodiments, a modified nucleobase is a modified cytosine. In some embodiments, a modified nucleobase is a methylated nucleobase. In some embodiments, a modified cytosine is a methyl cytosine, e.g., a 5-methyl cytosine. In such embodiments, the cytosine modification is a methyl. Agents that recognize a methyl cytosine in DNA include but are not limited to “methyl binding reagents,” which refer herein to reagents that bind to a methyl cytosine. Methyl binding reagents include but are not limited to methyl binding domains (MBDs) and methyl binding proteins (MBPs). In some such embodiments, the DNA may be singlestranded or double-stranded. Suitable agents include agents that recognize modified nucleotides in double-stranded DNA, single-stranded DNA, and both double-stranded and single-stranded DNA.

[0193] As used herein, “CpG-dense DNA” refers to a first subpopulation of DNA having an increased frequency of CpG dinucleotides relative to a second subpopulation of DNA. The first and second subpopulations may be present in the same sample or separated from each other, and in methods according to this disclosure, a sample can initially contain both subpopulations and separation of CpG-dense DNA from other DNA (such as unbound DNA, corresponding to the second subpopulation) can beAttorney Docket No.: GH0253WO effected by the methods. In some embodiments, CpG-dense nucleic acid molecules can include nucleic acid molecules comprising at least 1 CpG dinucleotide, at least 2 CpG dinucleotides, at least 3 CpG dinucleotides, at least 5 CpG dinucleotides, at least 10 CpG dinucleotides, at least 20 CpG dinucleotides, at least 30 CpG dinucleotides, at least 40 CpG dinucleotides, at least 50 CpG dinucleotides, at least 60 CpG dinucleotides, at least 70 CpG dinucleotides, at least 80 CpG dinucleotides, at least 90 CpG dinucleotides, or at least 100 CpG dinucleotides.

[0194] As used herein, “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides.Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG”, the nucleotides are in 5’ - 3’ order from left to right, and in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxy cytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases.

[0195] As used herein, “processing” refers to a set of steps used to generate a library of nucleic acids that is suitable for sequencing. The set of steps can include, but are not limited to, partitioning, end repairing, addition of sequencing adapters, tagging, and / or PCR amplification of nucleic acids.

[0196] As used herein, “quantitative measure” refers to an absolute or relative measure. A quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low). A quantitative measure can be a ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure.

[0197] As used herein, “reference sequence” refers to a known sequence used for purposes of comparison with experimentally determined sequences. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. AAttorney Docket No.: GH0253WO reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include non-contiguous segments that align with different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as, hgl9 and hg38.

[0198] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0199] As used herein, an “original sample” is a sample (e.g., of blood, plasma, or serum) as originally obtained from a source, such as a subject, tissue, or cell.

[0200] As used herein, a sample is a “proportionately methylated sample relative to an original sample” when it is an aliquot of the original sample or otherwise has not undergone manipulations that selectively enrich, capture, or deplete DNA molecules based on methylation or lack thereof. Proportionately methylated samples include samples or aliquots that were purified or isolated in a manner that does not substantially discriminate on the basis of methylation, such as ion exchange chromatography, size fractionation, hydrophobic interaction chromatography, or other techniques that do not rely on specific binding to methylated DNA or specific binding to unmethylated DNA. “Proportionately methylated samples,” as used herein, may have undergone processing steps such as end repair and adapter ligation (including end repair and adapter ligation using nucleotides and / or adapters comprising methylation or other modifications). In contrast, subsamples resulting from partitioning on the basis of methylation, e.g., using MBD or an antibody specific for methylated cytosine, are not proportionately methylated samples relative to their original sample.

[0201] As used herein, “sequencing” refers to any of a number of technologies used to determine the sequence (e.g., the identity and order of monomer units) of a biomolecule, e.g., a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole-genome sequencing, sequencing by hybridization, pyrosequencing, duplex sequencing, cycle sequencing, single-base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing, emulsion PCR, co-amplification atAttorney Docket No.: GH0253WO lower denaturation temperature-PCR (COLD-PCR), multiplex PCR, sequencing by reversible dye terminator, paired-end sequencing, near-term sequencing, exonuclease sequencing, sequencing by ligation, short-read sequencing, single-molecule sequencing, sequencing-by-synthesis, real-time sequencing, reverse-terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and a combination thereof. In some embodiments, sequencing can be performed by a gene analyzer such as, for example, gene analyzers commercially available from Illumina, Inc., Pacific Biosciences, Inc., or Applied Biosystems / Thermo Fisher Scientific, among many others.

[0202] As used herein, “sequence information” in the context of a nucleic acid polymer means the order and identity of monomer units (e.g., nucleotides, etc.) in that polymer.

[0203] As used herein, the terms “somatic mutation” or “somatic variation” are used interchangeably. They refer to a mutation in the genome that occurs after conception. Somatic mutations can occur in any cell of the body except germ cells and accordingly, are not passed on to progeny.

[0204] As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject”. For example, a subject can be an individual who has been diagnosed with having a cancer, is going to receive a cancer therapy, and / or has received at least one cancer therapy. The subject can be in remission of a cancer. As another example, the subject can be an individual who is diagnosed of having an autoimmune disease. As another example, the subject can be a female individual who is pregnant or who is planning on getting pregnant, who may have been diagnosed of or suspected of having a disease, e.g., a cancer, an auto-immune disease.Attorney Docket No.: GH0253WO

[0205] As used herein, “tumor fraction” refers to the proportion of cfDNA molecules that originated from tumor cells for a given sample, or sample-region pair.

[0206] As used herein, an “asymmetric adapter” is a double stranded adapter in which the two strands are not completely complementary or are otherwise distinguishable such that synthesis of a complementary sequence of one strand of the adapter results in a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y-shaped adapters and bubble adapters.

[0207] As used herein, a “Y-shaped adapter” refers to an adapter comprising two DNA strands comprising complementary and non-complementary parts, wherein the non-complementary parts form single-stranded arms. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that the complementary (doublestranded) part of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded arms may or may not be of identical length.

[0208] As used herein, a “bubble adapter” refers to an adapter comprising two DNA strands comprising a non-complementary part flanked by complementary parts, such that the adapter has a single stranded region located between double-stranded regions. The adapter can be attached to a sample or insert DNA molecule, e.g., by ligation, such that one of the complementary (double-stranded) parts of the adapter is proximal to the sample or insert DNA molecule. Prior to attachment, the double stranded portion of the Y-shaped adapter that would be attached to the insert or sample molecule may have a blunt end or an overhang, e.g., of one to three nucleotides. The single stranded portions of the two strands may or may not be of identical length.

[0209] The terms “or a combination thereof’ and “or combinations thereof’ as used herein refers to any and all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. TheAttorney Docket No.: GH0253WO skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0210] “Buffy coat” refers to the portion of a blood (such as whole blood) or bone marrow sample that contains all or most of the white blood cells and platelets of the sample. The buffy coat fraction of a sample can be prepared from the sample using centrifugation, which separates sample components by density. For example, following centrifugation of a whole blood sample, the buffy coat fraction is situated between the plasma and erythrocyte (red blood cell) layers. The buffy coat can contain both mononuclear (e.g, T cells, B cells, NK cells, dendritic cells, and monocytes) and polymorphonuclear (e.g, granulocytes such as neutrophils and eosinophils) white blood cells.

[0211] As used herein, “leukapheresis” refers to a procedure in which white blood cells (leukocytes) are isolated from a sample of blood collected from a subject. Leukapheresis may be performed, e.g., obtain cells for research, diagnostic, prognostic, or monitoring purposes, such as those described herein. Thus, as used herein, a “leukapheresis sample” refers to a sample comprising leukocytes collected from a subject using leukapheresis.

[0212] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and are found in the peripheral circulation. Such cells include, e.g., lymphocytes (T cells, B cells, and NK cells) as well as monocytes, and are isolated from blood samples (such as from a whole blood sample collected from a subject) using density gradient centrifugation.

[0213] As used herein, “amplify,” “amplifying,” or “amplification” refers to a process by which extra or multiple copies of a particular polynucleotide are formed. Amplification methods can include any suitable methods known in the art. As used herein, a nucleic acid molecule amplified using “methylation-preserving amplification” substantially maintains its methylation status post-amplification.

[0214] A “X1 / 7 / 7 / 7X2 mutation” in a specified polypeptide as used herein, where Xi and X2 are amino acids and nnn is a position in an amino acid sequence, refers to a substitution in the polypeptide of amino acid Xi present at position nnn of the full- length wild-type polypeptide with amino acid X2. The polypeptide is the humanAttorney Docket No.: GH0253WO polypeptide unless indicated otherwise. The polypeptide comprising the XmnnX mutation may, but does not necessarily, comprise additional differences from the wildtype sequence, including but not limited to truncations and deletions as well as other substitutions. For example, a “T1372S mutation” in TET2 refers to a substitution in a TET2 enzyme of the threonine present at position 1372 of the full-length wild-type human TET2 enzyme with a serine. Position 1372 of wild-type human TET2 aligns to position 258 and 248, respectively, of the truncated TET2 sequences disclosed as SEQ ID NOs: 23 and 24 of US Patent 10,961,525. Similarly, a “V1900X2 mutation” where X2 is A, C, G, I, or P in TET2 refers to a substitution in a TET2 enzyme of the valine present at position 1900 of the full-length wild-type human TET2 enzyme with an alanine, cysteine, glycine, isoleucine, or proline.

[0215] “Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.

[0216] The term “methylation-dependent nuclease” refers to a nuclease that preferentially cuts methylated DNA relative to unmethylated DNA. For example, a methylation-dependent nuclease may cut at or near a recognition sequence such as a restriction site in a manner dependent on methylation of at least one of the nucleobases in the recognition sequence, such as a cytosine. In some embodiments, the nucleolytic activity of the methylation-dependent nuclease is at least 10, 20, 50, or 100-fold higher on a methylated recognition site relative to an unmethylated control in a standard nucleolysis assay. Methylation-dependent nucleases include methylation-dependent restriction enzymes.

[0217] As used herein, “methylation-dependent restriction enzyme” or “MDRE” refers to a restriction enzyme that is dependent on methylation of the DNA (e.g. cytosine methylation) i.e., the presence or absence of methyl group in a nucleotide base alters the rate at which the enzyme cleaves the target DNA. In some embodiments, the methylation dependent restriction enzymes do not cleave the DNA if a particular nucleotide base is unmethylated at the recognition sequence. For example, MspJI is a methylation dependent restriction enzyme with a recognition sequence “mCNNR(N9)” and it does not cleave DNA if the absence of the methylated cytosine (mC) in the recognition sequence.Attorney Docket No.: GH0253WO

[0218] The term “methylation-sensitive nuclease” refers to a nuclease that preferentially cuts unmethylated DNA relative to methylated DNA. For example, a methylation-sensitive nuclease may cut at or near a recognition sequence such as a restriction site in a manner dependent on lack of methylation of at least one of the nucleobases in the recognition sequence, such as a cytosine. In some embodiments, the nucleolytic activity of the methylation-sensitive nuclease is at least 10, 20, 50, or 100- fold higher on an unmethylated recognition site relative to a methylated control in a standard nucleolysis assay. Methylation-sensitive nucleases include methylationsensitive restriction enzymes.

[0219] As used herein, “methylation sensitive restriction enzyme” or “MSRE” refers to a restriction enzyme that is sensitive to the methylation status of the DNA (e.g. cytosine methylation) i.e., the presence or absence of methyl group in a nucleotide base alters the rate at which the enzyme cleaves the target DNA. In some embodiments, the methylation sensitive restriction enzymes do not cleave the DNA if a particular nucleotide base is methylated at the recognition sequence. For example, Hpall is a methylation sensitive restriction enzyme with a recognition sequence “CCGG” and it does not cleave DNA if the second cytosine in the recognition sequence is methylated.

[0220] As used herein, “restriction enzyme” is an enzyme that recognizes and cleaves the DNA at or near a specific recognition site.

[0221] A “recognition sequence” refers to a sequence of nucleotides to which a binding agent binds. The binding agent can target a specific recognition sequence in which the sequence is invariable; or the binding agent can target a variable recognition sequence, e.g., that depends on a component of the binding agent, such as a guide RNA in the case of a Cas protein. The binding agent can be a nuclease (e.g., a restriction enzyme, meganuclease, or Cas nuclease, e.g., a Cas9 nuclease); a transcription activator-like effector (TALE); a zinc finger protein; an Argonaute protein; or a strand invading probe. The binding agent can be a catalytically dead protein, e.g., a dCas, such as a dCas9. The binding agent can use guide sequences (e.g., guide RNA) that directs the binding agent to the recognition sequence.Attorney Docket No.: GH0253WOII. Exemplary methodsA. Overview

[0222] Cancer formation and progression may arise from both genetic modification and epigenetic features of nucleic acids (e.g., deoxyribonucleic acid (DNA)). The present disclosure provides methods and systems for analyzing nucleic acids, such as cell-free DNA (cfDNA), by partitioning nucleic acids into at least two groups based on the length of each nucleic acid molecule in the sample. The present disclosure provides methods for processing nucleic acids (e.g., DNA such as cfDNA) and analyzing epigenetic and / or sequence-variable target regions by performing at least one distinct assay on each of the at least two partitions of nucleic acids.

[0223] Without wishing to be bound by any particular theory, cells in or around a cancer or neoplasm may shed more DNA than cells of the same tissue type in a healthy subject. As such, the distribution of tissue of origin of certain DNA samples, such as cfDNA, may change upon carcinogenesis. Thus, for example, a change in one or more of the modification status (such as the level of hypermethylation variable target regions that show lower methylation in healthy cfDNA than in at least one other tissue type or the level of hypomethylation variable target regions in the cfDNA), the fragmentation profile of and / or active chromatin regions present in cfDNA, and / or the somatic mutations present in cfDNA can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0224] Thus, methods of analyzing a nucleic acid population provided herein can be used to detect the fragmentation profile of, active chromatin regions, methylation status, and / or somatic mutations present in nucleic acid population (e.g., cfDNA), including for example, aberrant methylation in DNA of a sample. The nucleic acid population (e.g., cfDNA) can correspond to certain genomic regions (“differentially methylated regions” or “DMRs”) that are normally hypermethylated or hypomethylated in a given sample type (e.g., cfDNA from the bloodstream) but which may show an abnormal degree of methylation that correlates to a neoplasm or cancer, e.g., because of unusually increased contributions of tissues to the type of sample (e.g., due to increased shedding of DNA in or around the neoplasm or cancer) and / or from extents of methylation of the genome that are altered during development or that are perturbed by disease, for example, cancer or any cancer-associated disease.Attorney Docket No.: GH0253WO

[0225] In some embodiments, DNA methylation comprises addition of a methyl group to a cytosine residue at a CpG site (cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in a 5’ -> 3’ direction of the nucleic acid sequence). In some embodiments, DNA methylation comprises addition of a methyl group to an adenine residue, such as in N6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the carbon in the 5th position of the cytosine ring). In some embodiments, 5-methylation comprises addition of a methyl group to the 5C position of the cytosine residue to create 5-methylcytosine (m5c or 5-mC or 5mC). In some embodiments, methylation comprises a derivative of m5c. Derivatives of m5c include, but are not limited to, 5-hydroxymethylcytosine (5-hmC or 5hmC), 5- formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon in the 3rdposition of the cytosine ring). In some embodiments, 3C methylation comprises addition of a methyl group to the 3C position of the cytosine residue to generate 3 -methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at a CpA, CpT, or CpC site. DNA methylation can change the activity of methylated DNA region. For example, when DNA in a promoter region is methylated, transcription of the gene may be repressed. DNA methylation is critical for normal development and abnormality in methylation may disrupt epigenetic regulation. The disruption, e.g., repression, in epigenetic regulation may cause diseases, such as cancer. Promoter methylation in DNA may be indicative of cancer.

[0226] In some embodiments, the fragmentation profile comprises identifying the distribution of fragment lengths of the nucleic acids. In some embodiments, genome structure can affect fragmentation patterns. In some embodiments, some genomic regions may be more susceptible to fragmentation than other regions. The fragmentation patterns of a genomic region can be indicative of disease, such as cancer, wherein the genomic structure has been altered in the disease mechanism. In some embodiments, fragmentation profile, particularly the fragmentation profile of a targeted region, can provide insight into genomic structure and be indicative of disease.

[0227] In some embodiments, determining active chromatin regions of nucleic acids comprise determining nucleic acids that map to regions of the genome where active transcription is occurring. Active transcription can be a result of histone modificationsAttorney Docket No.: GH0253WO that facilitate the exposure of the DNA sequence, allowing transcription factors to bind within the region of the genome where active transcription is occurring. Regions of active chromatin can be associated with nucleosome-free regions. In some embodiments, identified active chromatin regions can also be used to infer gene expression patterns.

[0228] In some embodiments, determining somatic mutations comprises determining single nucleotide variants (SNVs), insertions / deletions (indels), copy number variations (CNVs), and / or structural rearrangements, such as gene fusions. In some embodiments, determining somatic mutations, can provide disease insight, including cancer. Cancer is usually caused by the accumulation of mutations within an individual's normal cells.

[0229] Many commercialized methods and methods undergoing development target specific cancer changes that occur in early stage cancers and pre-cancers. However, the accuracy of fragmentation profiling, detecting active chromatin regions, somatic mutation detection, methylation detection, molecular recovery, coverage uniformity, and methylation-specific enrichment efficacy can be improved in these methods (e.g., determining active chromatin regions fragmentation profiles of the nucleic acid molecules, and / or identifying modifications, such as methylation status), which would lead to improved clinical assay performance and / or assay cost reduction.

[0230] One exemplary embodiment is a method analyzing a nucleic acid population comprising nucleic acids of different lengths, comprising: (a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition; (b) performing a first assay on the first partition, wherein: (i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or (ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and (c) performing a second assay on the second partition, wherein: (i) the second assay determines the modification status of nucleic acids in the second partition; and / or (ii) the second assay detects somatic mutations of nucleic acids in the second partition.

[0231] Another exemplary embodiment is a method of analyzing a nucleic acid population comprising nucleic acids of different lengths. In some embodiments, theAttorney Docket No.: GH0253WO nucleic acid population is partitioned into two partitions according to nucleic acid length, wherein the first partition comprises nucleic acids which are equal to or longer than 180 bp, and the second partition comprises nucleic acids which shorter than 180 bp. In some embodiments, the first partition is subjected to fragmentation analysis. In some embodiments, prior to analysis, the first partition undergoes targeted amplification of amplicons containing at least one genomic region of interest. In some embodiments, the resulting sequence reads are mapped to a reference genome. In some embodiments, these sequence reads are utilized to create a fragmentation profile for the targeted genomic regions of interest. In some embodiments, the second partition is assayed for somatic mutations. The second partition can be assayed for somatic mutations at about the same time as the first partition is subjected to fragmentation analysis. In some embodiments, the second partition is amplified using primers that contain molecular barcodes and then sequenced. In some embodiments, the resulting sequence reads are grouped according to the molecular barcode they contain to produce a family of sequence reads. In some embodiments, each family correlates to a single parent nucleic acid in the original sample. For each family, a consensus sequence of the parent nucleic acid can be generated, identifying any somatic mutations in the original sample.

[0232] A further exemplary embodiment is a method of analyzing a cfDNA population comprising nucleic acids of different lengths. In some embodiments, prior to partitioning, cfDNA comprising genomic regions of interest can be separated from the nucleic acid population. In some embodiments, the separating cfDNA comprising genomic region of interest from the nucleic acid population uses hybrid capture. In some embodiments, baits, comprising probes that comprise a sequence complementary to the genomic region of interest in the cfDNA and are labelled with a capture group (e.g., biotin), are combined with the cfDNA sample. In some embodiments, the target cfDNAs hybridize with the baits. In some embodiments, cfDNA molecules are isolated using streptavidin-based beads that target the capture group (e.g., biotin capture group). In some embodiments, the baits are washed to release the cfDNAs of interest from the capture group. In some embodiments, the resulting cfDNA population is partitioned into two partitions according to nucleic acid length, wherein the second partition comprises mono-nucleosomal cfDNA, wherein the cfDNA is shorter than 180 bp, and the first partition comprises cfDNA longer than mono-nucleosomal cfDNA, wherein the cfDNAAttorney Docket No.: GH0253WO in the first partition comprises cfDNA equal to or longer than 180 bp. In some embodiments, the first partition is assayed to identify active chromatin. In some embodiments, cfDNA in the partition is sequenced using next generation sequencing and the resulting sequence reads are mapped to a reference genome. In some embodiments, known genomic features, such as promoters and exons, are identified within the mapped sequence reads and are used to infer or identify regions of active transcription. In parallel, a methylation detection assay can be performed on the second partition, wherein a methylation-based sub-partitioning assay is used to form a hypermethylated and hypomethylated sub-partition. In some embodiments, methyl-CpG binding domain protein 1 (MBD1) is used as a binding agent, wherein the binding agent targets the cfDNA in the second partition that comprises a 5mC modification. In some embodiments, the binding agents are eluted with solutions of increasing salt concentration. As the salt concentration increases, cfDNA with greater methylation levels can be eluted, resulting in a plurality of sub-partitions with increasing methylation levels. In some embodiments, each sub-partition is sequenced and mapped to a reference genome to identify methylation levels in a genomic region of interest.

[0233] Accordingly, in some embodiments, the present disclosure provides a method of analyzing a nucleic acid population. In some embodiments, the present disclosure provides a method of analyzing a nucleic acid population, wherein the method comprises: (a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition; (b) performing a first assay on the first partition, wherein: (i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or (ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and (c) performing a second assay on the second partition, wherein: (i) the second assay determines the modification status of nucleic acids in the second partition; and / or (ii) the second assay detects somatic mutations of nucleic acids in the second partition.

[0234] In some embodiments, the nucleic acid population is obtained using a sample preparation method optimized for extracting longer nucleic acids. In some embodiments, the nucleic acid population is partitioned using in vitro size selection.Attorney Docket No.: GH0253WO

[0235] In some embodiments, the nucleic acids in the first partition are at least 180 bp in length. In some embodiments the nucleic acids in the second partition are less than 180 bp in length.

[0236] In some embodiments, the separating the nucleic acid population into at least two partitions comprises binding nucleic acids with a higher average length with one or more beads. In some embodiments, the one or more beads comprise a coating of silica and / or carboxyl groups. In some embodiments, the one or more beads comprise one or more paramagnetic beads. In some embodiments, the one or more beads are in a solution comprising polyethylene glycol and a salt. In some embodiments, the separating the nucleic acid population into at least two partitions comprises solid-phase reversible immobilization (SPRI).

[0237] In some embodiments, the separating the nucleic acid population into at least two partitions comprises column separation. In some embodiments, the column separation comprises using a silica column. In some embodiments, the column separation comprises mixing the nucleic acid population with a buffer.

[0238] In some embodiments, the separating the nucleic acid population into at least two partitions comprises gel electrophoresis.

[0239] In some embodiments, the first assay and / or the second assay are untargeted. In some embodiments, the first assay and / or the second assay are targeted such that the first partition and / or the second partition are enriched for a plurality of target genomic regions.

[0240] In some embodiments, enrichment for target genomic regions is performed using hybrid capture or targeted amplification.

[0241] In some embodiments, the first assay determines active chromatin regions of nucleic acids in the first partition. In some embodiments, the first assay comprises identifying genomic features within the nucleic acids of the first partition. In some embodiments, the genomic features comprise promoters and / or exons. In some embodiments, the method further comprises identifying regions of active transcription using the identified genomic features. In some embodiments, the method further comprises identifying patterns of gene expression using the identified genomic features.

[0242] In some embodiments, the first assay comprises a next generation sequencing assay. In some embodiments, the first assay comprises quantitative PCR (qPCR).Attorney Docket No.: GH0253WO

[0243] In some embodiments, the first assay determines the fragmentation profile of the nucleic acids in the first partition. In some embodiments, the fragmentation profile is generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a next generation sequencing assay and the fragmentation profile is generated using data from the next generation sequencing assay. In some embodiments, the second assay determines the modification status of nucleic acids in the second partition. In some embodiments, the second assay comprises a baseconversion based sequencing method. In some embodiments, the base-conversion based sequencing method comprises bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, enzymatic methyl-seq (EM-seq) conversion, Tet-assisted pyridine borane sequencing (TAPS), APOBEC-coupled epigenetic (ACE) conversion, and / or direct methylation sequencing (DM-seq). In some embodiments, the Tet-assisted conversion further comprises a substituted borane reducing agent. In some embodiments, the substituted borane reducing agent comprises 2-picoline borane, borane pyridine, tert-butylamine borane, and / or ammonia borane. In some embodiments, the base-conversion based sequencing method comprises contacting the nucleic acids with a CpG-specific DNA methyltransferase (MTase) or a CpG-specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a deaminase. In some embodiments, the deaminase is a dsDNA deaminase. In some embodiments, the deaminase is a methyl insensitive deaminase. In some embodiments, the methyl insensitive deaminase is an APOBEC enzyme. In some embodiments, the APOBEC enzyme is APOBEC3 A. In some embodiments, the deaminase is a methyl sensitive deaminase. In some embodiments, the methyl sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modificationsensitive DNA deaminase A (MsddA)-like deaminase. In some embodiments, the second assay comprises generating sub-partitions of nucleic acids according to the modification status of the nucleic acids, wherein at least one of the sub-partitions is enriched for nucleic acids with a modification of interest, wherein generating subpartitions comprises providing a binding agent that preferentially binds to nucleic acids within the second partition that contain the modification of interest.

[0244] In some embodiments, the method further comprises separating the nucleic acids bound to the binding agent from the unbound nucleic acids to produce an unboundAttorney Docket No.: GH0253WO sub-partition. In some embodiments, the method further comprises subjecting nucleic acids bound to the binding agent to provide a sub-partition containing nucleic acids comprising the modification. In some embodiments, bound nucleic acids are subjected to a plurality of conditions which increasingly favor elution of the nucleic acids bound to the binding agent to provide multiple sub-partitions wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the modification compared to the nucleic acids eluted in the last of the plurality of conditions.

[0245] In some embodiments, the binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof. In some embodiments, further comprises quantifying the level of modifications, optionally using quantitative PCR (qPCR). In some embodiments, the quantification is used to provide a modification score. In some embodiments, the modification is a methylated nucleotide. In some embodiments, the modification is 5- methylcytosine (5mC).

[0246] In some embodiments, the second assay detects somatic mutations within the nucleic acid population. In some embodiments, the second assay comprises hybrid capture of nucleic acids comprising genomic regions of interest. In some embodiments, the second assay comprises targeted amplification of amplicons containing genomic regions of interest. In some embodiments, the second assay comprises next generation sequencing (NGS) or qPCR.

[0247] In some embodiments, the method further comprises preparing one or more sequencing libraries from at least a portion of the nucleic acids in the first partition and / or the second partition, further comprising sequencing the one or more sequencing libraries. In some embodiments, the nucleic acid population comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

[0248] The present disclosure provides a method of analysing a nucleic acid population comprising nucleic acids of different lengths, wherein the method comprises: (a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition; (b) performing a first assay on the first partition, wherein:Attorney Docket No.: GH0253WO(i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or (ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and (c) performing a second assay on the second partition, wherein: (i) the second assay determines the modification status of nucleic acids in the second partition; and / or (ii) the second assay detects somatic mutations of nucleic acids in the second partition.

[0249] In some embodiments, the nucleic acid population is obtained using a sample preparation method optimized for extracting longer nucleic acids. In some embodiments, the nucleic acid population is partitioned using in vitro size selection.

[0250] In some embodiments, the nucleic acids in the first partition are at least 180 bp in length. In some embodiments the nucleic acids in the second partition are less than 180 bp in length.

[0251] In some embodiments, the first assay and / or the second assay are untargeted. In some embodiments, the first assay and / or the second assay are targeted such that the first partition and / or the second partition are enriched for nucleic acids derived from targeted genomic regions.

[0252] In some embodiments, enrichment for nucleic acids derived from targeted genomic regions is performed using hybrid capture or targeted amplification.

[0253] In some embodiments, the first assay determines active chromatin regions of nucleic acids in the first partition. In some embodiments, the first assay comprises identification of genomic features within the nucleic acids of the first partition. In some embodiments, the genomic features include promoters and / or exons. In some embodiments, the method further comprises using the identified genomic features to identify regions of active transcription. In some embodiments, the method further comprises using the identified genomic features to infer gene expression patterns.

[0254] In some embodiments, the first assay comprises a next generation sequencing assay. In some embodiments, the first assay comprises qPCR.

[0255] In some embodiments, the first assay determines the fragmentation profile of the nucleic acids in the first partition. In some embodiments, the fragmentation profile is generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a next generation sequencing assay and the fragmentation profile is generated using data from the next generation sequencing assay.Attorney Docket No.: GH0253WO

[0256] In some embodiments, the second assay determines the modification status of nucleic acids in the second partition. In some embodiments, the second assay comprises a base-conversion based sequencing method, such as methods which comprise bisulfite conversion, oxidative bisulfite conversion, Tet-assisted bisulfite conversion, EM-seq, TAPS conversion, ACE-seq and direct-methylation sequencing (DM-seq). In some embodiments, the second assay comprises generating sub-partitions of nucleic acids according to the modification status of the nucleic acids, wherein at least one of the subpartitions is enriched for nucleic acids with the modification of interest, wherein generating sub-partitions comprises providing a binding agent that preferentially binds to nucleic acids within the second partition that contain the modification of interest.

[0257] In some embodiments, the method further comprises separating the nucleic acids bound to the binding agent from the unbound nucleic acids to produce an unbound sub-partition. In some embodiments, the method further comprises subjecting nucleic acids bound to the binding agent to conditions which favour elution of the nucleic acids bound to the binding agent to provide a sub-partition containing nucleic acids comprising the modification. In some embodiments, bound nucleic acids are subjected to a plurality of conditions which increasingly favour elution of the nucleic acids bound to the binding agent to provide multiple sub-partitions wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the modification compared to the nucleic acids eluted in the last of the plurality of conditions.

[0258] In some embodiments, the binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody, or an antigen-binding fragment thereof. In some embodiments, nucleic acids comprising the modification are quantified, optionally using quantitative-PCR. In some embodiments, the quantification is used to provide a modification score. In some embodiments, the modification is methylation. In some embodiments, the modification is 5-methylcytosine (5mC).

[0259] In some embodiments, the second assay detects somatic mutations within the nucleic acid population. In some embodiments, the second assay comprises hybrid capture of nucleic acids comprising genomic regions of interest. In some embodiments, the second assay comprises targeted amplification of amplicons containing genomic regions of interest. In some embodiments, the second assay comprises next generation sequencing or qPCR.Attorney Docket No.: GH0253WO

[0260] In some embodiments, the method further comprises preparing one or more sequencing libraries from at least a subset of the nucleic acids in the first partition and / or the second partition, wherein the one or more sequencing libraries are then subjected to nucleic acid sequencing. In some embodiments, the nucleic acid population comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

[0261] The disclosed methods can be combined with analysis of one or more additional biomarkers. In some embodiments, the disclosed methods are combined with one or more methods, such as but not limited to, methods for assessing DNA methylation patterns, DNA mutations (such as somatic mutations), nucleic acid fragmentation patterns, non-coding RNA (such as micro RNAs (miRNAs), ribosomal RNAs, transfer RNAs, small nucleolar RNAs (snow RNAs), and / or small nuclear RNAs (snRNAs)) levels, and / or cell type proportions / levels, cellular locations, and / or structural modifications of one or more proteins (such as in a sample from a subject). In some embodiments, the disclosed methods are combined with one or more analyses of genetic variations including mutations, rare mutations, indels, rearrangements, copy number variations, transversions, translocations, recombinations, inversion, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structure alterations, gene fusions, chromosome fusions, gene truncations, gene amplification, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and / or abnormal changes in nucleic acid 5-methylcytosine.B. Size-based Partitioning

[0262] In some embodiments, a heterogeneous nucleic acid sample is separated. In some embodiments, separating comprises partitioning. In some instances, a heterogeneous nucleic acid sample is partitioned into two or more partitions (subsamples). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristics, and tagged using differential tags that are distinguished from other partitions and partitioning means. In some embodiments, the separating comprises partitioning the DNA in the sample into aAttorney Docket No.: GH0253WO plurality of partitioned subsamples. In some embodiments, the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample.

[0263] The present disclosure provides a method for analyzing a nucleic acid population, wherein the method comprises separating the nucleic acid population to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition. In some embodiments, the nucleic acid population is partitioned according to size of the nucleic acids in the population. In some embodiments, the nucleic acid population is partitioned using in vitro size selection.

[0264] In some embodiments, in vitro size selection allows for the separation of a nucleic acid sample into partitions according to nucleic acid length. In some embodiments, nucleic acids of different lengths within a sample provide information about different properties of the original nucleic acid population. Therefore, partitioning the nucleic acid population into partitions of predetermined nucleic acid length ranges can allow for the resulting partitions to be probed for features for which the predetermined nucleic acid length range can demonstrate. Enrichment of nucleic acids within determined nucleic acid length ranges within partitions allows limited samples to be assayed for a wide range of features.

[0265] In some embodiments, the isolation of shorter nucleic acids can increase the mutant allele frequency of the partition compared to the original nucleic acid population. In some embodiments, the isolation of shorter nucleic acids can be utilized to detect somatic mutations in the original sample. Enrichment of a partition with shorter nucleic acids can allow for the detection of somatic mutations. In some embodiments, at least one partition can be assayed for further features of the nucleic acids within the partition, such as determining the active chromatin regions and / or the fragmentation profiles.

[0266] In vitro size selection can occur after extraction of the nucleic acids from the sample. In vitro size selection an occur prior to, or subsequent to, the sample preparation step. In vitro size selection an occur prior to, or subsequent to, library preparation. In vitro size selection an occur prior to, or subsequent to, at least one amplification step. In vitro size selection an occur prior to, or subsequent to, to aAttorney Docket No.: GH0253WO targeted amplification step. In vitro size selection an occur prior to, or subsequent to, a hybrid capture step.

[0267] Methods of in vitro size selection are well known in the art. In vitro size selection of nucleic acid population allows for the extraction of nucleic acids of a predetermined nucleic acid length range from a nucleic acid population. Resulting partitions can be further assayed. Assays can be tailored to features known to be demonstrated at higher resolution in the nucleic acid length range of a partition compared to the nucleic acid population as a whole.

[0268] Methods for in vitro size selection are well known to in the art. At a basic level, polyacrylamide gel electrophoresis can separate the original nucleic acid population into partitions depending on nucleic acid length. Partitions can be extracted and assayed. Larger sample sizes can be partitioned using commercially available kits. In some instances, commercially available kits for nucleic acid size selection make use of microfluidic devices, such as Pippin HT (Sage Biosciences), further details of size selection workflows can be found in Moulier et al. Science translational medicine.2018; vol. 10,466 and Hellwig et al. PloS one vol. 2018; 13,7 e0197333. In some instances, commercially available kits for nucleic acid size selection make use of SPRI beads, further details of which can be found in Ishda et al. JGH Open, 2020; 4(5).

[0269] In some embodiments, the method comprises separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition, wherein the nucleic acids in the first partition are at least 180 bp in length. In some embodiments the method comprises separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition, wherein the nucleic acids in the second partition are less 180 bp in length. In some embodiments, the nucleic acids in the first partition are at least 180 bp and the nucleic acids in the second partitions are less than 180 bp in length.

[0270] In some embodiments the nucleic acids in the first partition are at least 180 bp in length. In some embodiments, the nucleic acids in the first partition are at least 150Attorney Docket No.: GH0253WO bp, 155 bp, 160 bp, 165 bp, 170 bp, 175 bp, 180 bp, 185 bp, 190 bp, 195 bp, 200 bp, 205 bp, 210 bp, 215 bp, 220 bp, or 225 bp in length. In some embodiments, the nucleic acids in the first partition are at least 175 bp, 176 bp, 177 bp, 178 bp, 179 bp, 180 bp, 18 Ibp, 182 bp, 183 bp, 184 bp, or 185 bp in length.

[0271] In some embodiments, the nucleic acids in the second partition are less than 180 bp in length. In some embodiments, the nucleic acids in the second partition are less than 150 bp, 155 bp, 160 bp, 165 bp, 170 bp, 175 bp, 180 bp, 185 bp, 190 bp, 195 bp, 200 bp, 205 bp, 210 bp, 215 bp, 220 bp, or 225 bp in length. In some embodiments, the nucleic acids in the second partition are less than 175 bp, 176 bp, 177 bp, 178 bp, 179 bp, 180 bp, 18 Ibp, 182 bp, 183 bp, 184 bp, or 185 bp in length.

[0272] In some embodiments, the average length of nucleic acids in the first partition is longer relative to the average length of nucleic acids in the second partition. In some embodiments, the first partition comprises nucleic acids with an average length that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200% longer, in bp, than the average length of the nucleic acids comprised in the second partition.

[0273] In some embodiments, the second partition comprises nucleic acids from mono-nucleosomal cfDNA. Mono-nucleosomal cfDNA fragments have an approximate length of 158 bp (see, e.g., Ungerer et al. Diagnostics (Basel, Switzerland). 2022; vol. 12,8 1896). Therefore, in some embodiments, the second partition comprises nucleic acids with an average length of approximately 158 bp. In some embodiments, the second partition comprises nucleic acids with an average length of 158 bp and the first partition comprises nucleic acids with an average length that is larger than 158 bp.

[0274] In some embodiments, the second partition comprises nucleic acids of lengths that are within 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 50%, 60%, or 70% of 158 bp. In such embodiments, the first partition comprises nucleic acids of lengths that are 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 50%, 60%, or 70% larger than 158 bp.Attorney Docket No.: GH0253WO

[0275] In some embodiments, the second partition comprises nucleic acids from chromatosomal cfDNA. Chromatosomal cfDNA fragments are known to be approximately 168 bp in length. In some embodiments, the second partition comprises nucleic acids with an average length of 168 bp. In some embodiments, the second partition comprises nucleic acids with an average length of 168 bp and the first partition comprise nucleic acids with an average length larger than 168 bp.

[0276] In some embodiments, the second partition comprises nucleic acids of lengths that are within 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 50%, 60%, or 70% of 168 bp. In such embodiments, the first partition comprises nucleic acids of lengths that are 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 50%, 60%, or 70% larger than 168 bp.

[0277] In some embodiments, the second partition comprises nucleic acids from di- nucleosomal cfDNA, tri-nucleosomal cfDNA, tetra-nucleosomal DNA, or penta- nucleosomal cfDNA. In some embodiments, the second partition comprises nucleic acids from circulating tumor DNA (ctDNA).C. Assay for active chromatin

[0278] In some embodiments, the present disclosure provides methods comprising determining active chromatin regions of nucleic acids. In some embodiments, a first assay determines active chromatin regions of nucleic acids in the first partition. Active chromatin regions of the nucleic acids can be defined as nucleic acids that map to regions of the genome where active transcription is occurring. Active transcription can be a result of histone modifications that facilitate the exposure of the DNA sequence, allowing transcription factors to bind within the region of the genome where active transcription is occurring. In some embodiments, the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition. In some embodiments, the nucleic acids in the first partition are less likely to be associated with mono-nucleosomes or other histone structures that decrease active transcription. Regions of active chromatin are often associated with nucleosome-free regions. Hence,Attorney Docket No.: GH0253WO in some embodiments, the longer nucleic acids of the first partition are associated with nucleosome-free regions.

[0279] In some embodiments, the present disclosure provides methods comprising identifying genomic features within the nucleic acids. In some embodiments, a first assay comprises identifying genomic features within the nucleic acids of a first partition, wherein the first assay determines active chromatin regions of nucleic acids within the said partition. Identified genomic features can be used to identify regions of active transcription. In some embodiments, the genomic features comprise one or any combination of the following: an actively transcribed sequence, a gene body, an exon, a promoter, or a transcription factor binding site. In some embodiments, the genomic feature comprises one or more actively transcribed sequences. In some embodiments, the genomic feature comprises one or more gene bodies. In some embodiments, the genomic feature comprises one or more exons. In some embodiments, the genomic comprises one or more promoters. In some embodiments, the genomic feature comprises one or more transcription factor binding sites. The identified genomic features can be associated with active transcription, wherein they comprise actively transcribed sequences, gene bodies, exons, promoters or other types of transcription factor binding sites. Such genomic features can be found within nucleosome-free regions. In some embodiments, the genomic feature includes a plurality of promoters. In some embodiments, the genomic feature includes a plurality of exons. In some embodiments, the genomic feature includes a promoter and an exon, or a plurality of promoters and a plurality of exons. In some embodiments, the genomic feature includes an enhances. In some embodiments, the genomic feature includes cis regulatory modules, which contain clusters of binding sites for multiple transcription factors. Cis regulatory modules can be found within enhancers or promoters. Other identified genomic features may include, but are not limited to, insulators, silencers, locus control regions, CTCF binding sites, repressor binding sites, or super-enhancers.

[0280] In some embodiments, identified genomic features can be used to infer gene expression patterns. In some embodiments, comparison of the sequences of the nucleic acids of the first partition and their genomic features with known genomic features can be used to infer gene expression patterns. Known genomic and epigenomic data can be compared with that of the nucleic acids of the first partition. Identified genomic featuresAttorney Docket No.: GH0253WO can be mapped to known regulatory interactions to infer the gene expression patterns. Expression profiles or data sets from model systems or the original nucleic acid population can be used in mapping.

[0281] In some embodiments, the first assay can include the comparison of active chromatin data from the first partition with data generated from a next generation sequencing assay performed separately. Such comparisons can be used to infer information about the identified active chromatin regions of the nucleic acids within the first partition or of identified gene expression patterns. In some embodiments, separate assays used may investigate DNA-DNA interactions, DNA-protein interactions, or protein-protein interactions. In some embodiments, assays used to investigate DNA- DNA interactions, DNA-protein interactions, or protein-protein interactions are performed on samples from the same source as the source used in the methods of the present disclosure. Such assays could provide information about genome organization and regulation. These assays may include next generation sequencing based assays.Assays may include, but are not limited to, chromosome conformation capture (3C) and its derivatives including, but not limited to, chromatin immunoprecipitation assay (ChIP), Hi-C, Micro-C, circular chromosome conformation capture (4C), chromosome conformation capture carbon copy (5C), chromatin interaction analysis by paired-end tag sequencing (ChlA-PET) and HiChlP. Other assays include, but are not limited to, nucleosome occupancy and methylome sequencing (NOMe-seq), assay for transposase- accessible chromatin with high-throughput sequencing (ATAC-seq), micrococcal nuclease digestion with deep sequencing (MNase-seq), formaldehyde- assisted isolation of regulatory elements sequencing (FAIRE-seq), or DNase I hypersensitive sites sequencing (DNase-seq).

[0282] In some embodiments, the first assay may comprise a next generation sequencing (NGS) assay. In some embodiments, the first assay may comprise quantitative PCR (qPCR). The assay can be used to detect active chromatin regions within the first partition. As previously described, these assays can be combined with data from the original nucleic acid population, which can be used to investigate gene regulation and gene expression patterns. The first assay can be used to infer gene expression patterns. In some embodiments, the first assay can be used to infer gene regulatory patterns. In some embodiments, the first assay, wherein the first assayAttorney Docket No.: GH0253WO determines active chromatin regions of nucleic acids in the first partition, can be used for the detection of disease, for example for the detection of cancer.D. Fragmentation assay

[0283] In some embodiments, the present disclosure provides methods comprising determining a fragmentation profile of nucleic acids. In some embodiments, a first assay determines the fragmentation profile of nucleic acids in the first partition. In some embodiments, the fragmentation profile is generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a next generation sequencing (NGS) assay, and the fragmentation profile is generated using data from the NGS assay.

[0284] The fragmentation profile of the nucleic acids in the first partition can show the distribution of fragment lengths of the nucleic acids in the first partition. In some embodiments, genome structure can affect fragmentation patterns. In some embodiments, some genomic regions may be more susceptible to fragmentation than other regions. The fragmentation patterns of a genomic region can be indicative of disease, such as cancer, wherein the genomic structure has been altered in the disease mechanism. In some embodiments, fragmentation profile, particularly the fragmentation profile of a targeted region, can provide insight into genomic structure and be indicative of disease.

[0285] In some embodiments, the first assay comprises a NGS assay wherein the fragmentation profile is generated using data from the NGS assay. In some embodiments, the nucleic acids of the first partition may undergo next generation sequencing and be mapped to a reference genome. The length of the mapped reads can be used for generating a fragmentation profile of the nucleic acids in the first partition. The mapped reads can be sorted into bins according to their genomic location and length. A fragmentation profile for targeted regions of the genome may be generated using the mapped reads of nucleic acids in the first partition.

[0286] The fragmentation profile of nucleic acids in the first partition may be generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a NGS assay and the fragmentation profile is generated using data from the NGS assay. In some embodiments, the fragmentationAttorney Docket No.: GH0253WO profile of nucleic acids in the first partition may be generated based on the length distribution of nucleic acids in the first partition. The fragmentation profile of nucleic acids in the first partition may be generated using the mean, median, or mode lengths of nucleic acids. In some embodiments, the fragmentation profile of nucleic acids in the first partition is generated by calculating the frequency of fragment in a defined range of lengths.

[0287] In some embodiments, a fragmentation profile of nucleic acids in the first partition is generated using a fragmentation length distribution curve. The fragment length distribution curve may be generated using the frequency of each fragment length.

[0288] In some embodiments, the fragmentation profile of nucleic acids in the first partition may be mapped to the genome. Sequence reads of the nucleic acids in the first partition can be mapped and a fragmentation profile can be generated for a given locus or a plurality of given loci. In some embodiments, the fragmentation profile of nucleic acids in the first partition can be used to generate a model for the prediction of the fragmentation profile for a given genomic region. In some embodiments, a machine learning model can be trained on the fragmentation profile of the nucleic acids in the first partition.

[0289] In some embodiments, the fragmentation profile of the nucleic acids in the first partition can be used in the detection of disease, for example in the detection of cancer. Statistical models or machine learning models generated using the fragmentation profile of the nucleic acids in the first partition can be used in the detection of disease, for example in the detection of cancer.E. Modification assay and modification-based partitioning

[0290] The present disclosure provides methods comprising determining the modification status of nucleic acids. In some embodiments, the second assay determines the modification status of nucleic acids in the second partition.

[0291] In some embodiments, a heterogeneous nucleic acid sample, such as the second partition, is separated. In some embodiments, separating comprises partitioning (e.g., further partitioning). In some instances, a heterogeneous nucleic acid sample, such as the second partition, is (further) partitioned into two or more (further) partitions (subsamples). Any partitioning described in this section may be performed as a furtherAttorney Docket No.: GH0253WO partitioning of the second partition provided by separating the nucleic acid population into at least two partitions according to nucleic acid length. In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on one or more different characteristics, and tagged using differential tags that are distinguished from other partitions and partitioning means. In some embodiments, the separating comprises partitioning the DNA in the sample into a plurality of partitioned subsamples. In some embodiments, the plurality of partitioned subsamples comprises a first partitioned subsample and a second partitioned subsample.

[0292] In some embodiments, the second assay comprises generating sub -partitions of nucleic acids according to the modification status of the nucleic acids, wherein at least one of the sub-partitions is enriched for nucleic acids with the modification of interest, wherein generating sub-partitions comprises providing a binding agent that preferentially binds to nucleic acids within the second partition that contain the modification of interest. In some embodiments, the nucleic acids comprising the modification are quantified. Quantification of the modification in the second partition can be determined, for example, using quantitative PCR (qPCR). The quantified value of the modifications in the second partition can be used to provide a modification score for the second partition.

[0293] Sub-partitions of the second partition can be sub-partitioned based on the modification status of nucleic acids in the second partition. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically subpartitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated.

[0294] In some embodiments, the second partition is sub-partitioned into two or more sub-partitions (e.g., at least 3, 4, 5, 6 or 7 sub-partitions). The agents used to subpartition the second partition can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech. 2010; 28: 1106-1114; Song et al., Nat Biotech. 2011; 29: 68-72), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, theAttorney Docket No.: GH0253WO agent used is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase may be a “converted nucleobase,” meaning that its base pairing specificity was changed by a procedure. For example, certain procedures convert unmethylated or unmodified cytosine to dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination, resulting in uracil (considered a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of sub-partitioning agents include antibodies, such as antibodies that recognize a modified nucleobase, which may be a modified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the binding agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5- carboxylcytosine (5caC). Alternative sub-partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs), including proteins such as MeCP2, MBD2, and antibodies preferentially binding to 5-methylcytosine. Where an antibody is used to immunoprecipitate methylated DNA, the methylated DNA may be recovered in single-stranded form. In such embodiments, a second strand can be synthesized. Hypermethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation sensitive nuclease that does not cleave hemimethylated DNA, such as Hpall, BstUI, or Hin6i. Alternatively or in addition, hypomethylated (and optionally intermediately methylated) subsamples may then be contacted with a methylation dependent nuclease that cleaves hemi-methylated DNA.

[0295] Additional, non-limiting examples of sub-partitioning agents are histone binding proteins which can separate nucleic acids bound to histones from free or unbound nucleic acids. Examples of histone binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48 and SANT domain peptides.

[0296] The binding agents used in the disclosed methods preferentially bind nucleic acids comprising the first type of modification, or derivative thereof, over nucleic acids without the modification of interest for instance with an affinity at least 50X, at least 100X or at least 1000X greater compared to nucleic acids that do not comprise the modification of interest.Attorney Docket No.: GH0253WO

[0297] In some embodiments, the modification is methylation. In some embodiments, the modification is 5-methylcytosine (5mC). Wherein the modification is 5mC, the corresponding binding agent may comprise an MBD, such as methyl-CpG binding domain protein 1 (MBD1) or MBD2. In alternative embodiments, the corresponding binding agent may be an anti-5mC antibody or an antigen-binding fragment thereof.

[0298] Where the modification of interest is methylation, or specifically 5mC, the second partition is subjected to a methylation-based sub-partitioning assay, wherein the methylation-based sub-partitioning assay sub-partitions nucleic acids using methyl- binding domain (MBD). In such methods, the methylation-based sub-partitioning assay can form a hypermethylated sub-partition and / or a hypomethylated sub-partition. After sub-partitioning, one or more of the resulting sub-partitions can be analyzed by the methods disclosed herein to determine a quantitative measure indicative of a number of nucleic acids in the one or more sub-partitions that map to a genomic region. In some embodiments, the resulting sub-partitions analyzed can include a hypermethylated subpartition obtained from the methylation-based sub-partitioning assay. In some embodiments, the resulting sub-partitions analyzed can include a hypomethylated subpartition obtained from the methylation-based sub-partitioning assay.

[0299] In some embodiments, the method can comprise determining a quantitative measure indicative of a number of nucleic acids in the hypermethylated or hypomethylated sub-partition derived from a genomic region in the sample. In some embodiments, the quantitative measure can be determined using qPCR. In some embodiments, the quantitative measure can be calculated by determining a normalized quantitative measure at one or more genomic regions and determining a methylation level at that genomic region based on the normalized quantitative measure. The methylation level can be determined, for example, by comparing the normalized quantitative measure in the hypermethylated sub-partition to the normalized quantitative measure from the hypomethylated sub-partition. The methylation score can be determined, for example, by comparing the normalized quantitative measure in the hypermethylated sub-partition and / or the normalized quantitative measure from the hypomethylated sub-partition to a reference value. The reference value may be, for example, derived from a normalized quantitative measure of a control genomic regionAttorney Docket No.: GH0253WO from the same sub-partition. The normalized quantitative measures can be determined by, for example, qPCR.

[0300] In some embodiments, sub-partitioning can comprise both binary subpartitioning and sub-partitioning based on degree / level of modifications. For example, methylated fragments can be sub-partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be sub-partitioned from unmethylated fragments using methyl binding domain proteins (e.g., MethylMiner™ Methylated DNA Enrichment Kit (ThermoFisher Scientific)). Subsequently, additional subpartitioning may involve eluting fragments having different levels of methylation by adjusting the salt concentration in a solution with the methyl binding domain and bound fragments. As salt concentration increases, fragments having greater methylation levels are eluted.

[0301] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversion such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable approach. For example, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. For example, a methylated DNA binding protein (e.g., an MBD such as MBD2, MBD4, or MeCP2) or an antibody specific for 5-methylcytosine (as in MeDIP) can be used to partition the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, a DNA fragmentation pattern can be determined based on endpoints and / or centerpoints of DNA molecules, such as cfDNA molecules.

[0302] In some instances, the final partitions are enriched in nucleic acids having different extents of modifications (overrepresentative or underrepresentative of modifications). Overrepresentation and underrepresentation can be defined by the number of modifications bom by a nucleic acid relative to the median number of modifications per strand in a population. For example, if the median number of 5- methylcytosine residues in nucleic acid in a sample is 2, a nucleic acid including more than two 5-methylcytosine residues is overrepresented in this modification and a nucleic acid with 1 or zero 5-methylcytosine residues is underrepresented. The effect of affinityAttorney Docket No.: GH0253WO separation is to enrich for nucleic acids overrepresented in a modification in a bound phase and for nucleic acids underrepresented in a modification in an unbound phase (i.e. in solution). The nucleic acids in the bound phase can be eluted before subsequent processing.

[0303] Various levels of methylation can be sub-partitioned using sequential elutions. For example, a hypom ethylated sub-partition (no methylation) can be separated from a methylated sub-partition by contacting the nucleic acid population with MBD, such as MBD attached to magnetic beads. The beads can be used to separate out the methylated nucleic acids from the nonmethylated nucleic acids. Subsequently, one or more elution steps are performed sequentially to elute nucleic acids having different levels of methylation. For example, a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or higher, e.g., at least 150 mM, at least 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or 2000 mM. After such methylated nucleic acids are eluted, magnetic separation can once again be used to separate higher levels of methylated nucleic acids from those with lower levels of methylation. The elution and magnetic separation steps can be repeated to create various sub-partitions such as a hypomethylated sub-partition (enriched in nucleic acids comprising no methylation), a methylated sub-partition (enriched in nucleic acids comprising low levels of methylation), and a hypermethylated subpartition (enriched in nucleic acids comprising high levels of methylation). Any one or more sub-partitions can then be analyzed using the methods disclosed herein.

[0304] In some methods, nucleic acids bound to a binding-agent used for affinity separation-based sub-partitioning are subjected to a wash step. The wash step washes off nucleic acids weakly bound to the affinity agent. Such nucleic acids can be enriched in nucleic acids having the modification to an extent close to the mean or median (i.e. intermediate between nucleic acids remaining bound to the solid phase and nucleic acids not binding to the solid phase on initial contacting of the sample with the agent).

[0305] In some embodiments, the partitioning (e.g., further partitioning of the second partition) comprises partitioning at least a portion of the polynucleotides into at least two partitions based on at least one epigenetic feature, thereby generating partitioning polynucleotides. In some embodiments, the partitioning comprises partitioning at least a portion of the polynucleotides into at least two partitions based onAttorney Docket No.: GH0253WO at least one somatic variation, thereby generating partitioning polynucleotides. In some embodiments, the first partitioned subsample comprises methylated DNA in a greater proportion than the second partitioned subsample.

[0306] In some embodiments, the partitioning (e.g., further partitioning of the second partition) uses a label to partition at least a portion of the polynucleotides into at least two partitions based on at least one epigenetic feature, thereby generating partitioned polynucleotides. In some embodiments, the partitioning uses a label to partition at least a portion of the polynucleotides into at least two partitions based on at least one somatic variation, thereby generating partitioned polynucleotides.

[0307] In some embodiments, a sample, such as the second partition, is (further) partitioned into at least a first subsample and a second subsample. This may be accomplished simply by dividing the library into identical or substantially identical subsamples. Alternatively, in some methods, different DNA (e.g., sequence-variable target regions, and epigenetic target regions) can be partitioned based on one or more characteristics of the DNA. Detecting aberrant features in DNA (whether sequencebased, epigenetic, or both) while also detecting target regions comprising sequencevariable target regions and / or epigenetic target regions may provide greater specificity and / or sensitivity for identifying an abnormal state than detecting the DNA features alone or levels of one or more post-translationally modified proteins alone.

[0308] In some embodiments, the first subsample comprises sequence-variable target regions and / or epigenetic target regions in a greater proportion than the second subsample. In some embodiments, the second subsample comprises sequence-variable target regions and / or epigenetic target regions in a greater proportion than the first subsample.

[0309] In some embodiments, the partitioning the nucleic acids (e.g., DNA such cfDNA, or further partitioning the second partition) into a plurality of subsamples comprises contacting the nucleic acids (e.g., DNA such cfDNA) with an agent that recognizes methyl cytosine in the DNA. The partitioning step can occur prior to or after capturing an epigenetic target region set of nucleic acids (e.g., DNA such cfDNA) or a sequence-variable target region of the nucleic acids (e.g., DNA such cfDNA). The partitioning step can occur prior to capturing an epigenetic target region set of nucleic acids (e.g., DNA such cfDNA) or a sequence- variable target region of the DNA. TheAttorney Docket No.: GH0253WO partitioning step can occur prior to or after capturing an epigenetic target region set of nucleic acids (e.g., DNA such cfDNA) or a sequence-variable target region of the nucleic acids (e.g., DNA such cfDNA) and prior to or after sequencing the nucleic acids (e.g., DNA such cfDNA). The partitioning step can occur after capturing an epigenetic target region set of nucleic acids (e.g., DNA such cfDNA) or a sequence-variable target regions of the DNA and prior to sequencing the nucleic acids (e.g., DNA such cfDNA).

[0310] Disclosed methods herein can comprise analyzing nucleic acids (e.g., DNA such cfDNA) in a sample. In some embodiments described herein, the disclosed methods comprise partitioning nucleic acids (e.g., DNA such cfDNA). In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the nucleic acids (e.g., DNA such cfDNA).

[0311] In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0312] In some embodiments, determining the modification status of nucleic acids (e.g., in the second partition) comprises methylation profiling, which can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based on extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation.

[0313] In some embodiments, the partitioning comprises contacting the nucleic acids (e.g., DNA such cfDNA) with an agent that recognizes a modification associated with (e.g., in) the nucleic acids (e.g., DNA such cfDNA). In some embodiments, the agent that recognizes the modification is an antibody or a methyl binding domainAttorney Docket No.: GH0253WO(MBD) protein. In some embodiments, the agent is immobilized on a solid support. In some embodiments, the solid support comprises a bead. In some embodiments, the partitioning comprises immunoprecipitation, e.g., using the agent that recognizes the modification, such as an antibody or an MBD protein, immobilized on solid support.

[0314] In some embodiments, the partitioning comprises precipitating the methylated nucleic acids (e.g., DNA such cfDNA). In some embodiments, the partitioning comprises precipitating the methylated nucleic acids (e.g., DNA such cfDNA) to separate it from the unmethylated nucleic acids (e.g., DNA such cfDNA). In some embodiments, the precipitating the methylated nucleic acids (e.g., DNA such cfDNA) can be performed using any pair of binding partners. In some embodiments, one of the binding partners may be linked to the MBD protein or antibody, and the other binding partner may be linked to a solid support. In some embodiments, the binding partner comprises biotin and streptavidin. In some embodiments, the biotin may be linked to the MBD protein, and the streptavidin may be linked to a solid support. In some embodiments, the MBD protein is linked to a solid support, optionally using any pair of binding partners. In some embodiments, the partitioning comprises immunoprecipitating the methylated nucleic acids (e.g., DNA such cfDNA). In some embodiments, the partitioning comprises immunoprecipitating the methylated DNA separately from the unmethylated nucleic acids (e.g., DNA such cfDNA).

[0315] In some embodiments, the modification is methylation, and in some such embodiments, the partitioning comprises partitioning on the basis of methylation level. In some such embodiments, the agent is a methyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5-methylcytosine. In some such embodiments, the agent is a hydroxymethyl binding reagent. In some embodiments, the methyl binding reagent specifically recognizes 5- hydroxymethylcytosine, biotinylated 5-hydroxymethylcytosine, glucosylated 5- hydroxymethylcytosine, or sulfonylated 5-hydroxymethylcytosine. In some embodiments, the partitioning comprises partitioning on the basis of binding to a protein comprising contacting the sample comprising the DNA with a binding reagent specific for the protein. In some such embodiments, binding reagent specifically binds a methylated protein or an acetylated protein, such as a methylated or acetylated histone, or an unmethylated protein or an unacetylated protein such as an unmethylated orAttorney Docket No.: GH0253WO unacetylated histone. In some embodiments, the binding reagent specifically binds an unmethylated or unacetylated protein epitope.

[0316] In some embodiments, the modification is hydroxymethylation, and in some such embodiments, the partitioning comprises partitioning on the basis of hydroxymethylation level. In some such embodiments, the agent is a hydroxymethyl binding reagent, such as an antibody. In some embodiments, the hydroxymethyl binding reagent (e.g., antibody) specifically recognizes 5-hydroxymethylcytosine (5-hmC). In some embodiments, a modification such as hydroxymethylation is labeled (e.g., biotinylated, glucosylated, or sulfonated) before being contacted with an agent that recognizes the labeled form of the modification. For example, 5-hmC can be enzymatically glucosylated and then partitioned based on binding to J-binding protein 1. Exemplary methods of labeling and / or partitioning 5-hmC are provided, e.g., in Song et al., Nat. Biotech. 29:68-72 (2010); Ko et al., Nature 468:839-843 (2010); and Robertson et al., Nucleic Acids Res. 39:e55 (2011).

[0317] Where immunoprecipitation is used and involves an antibody that recognizes single-stranded DNA, the DNA may be converted to double-stranded form by complementary strand synthesis before a subsequent step. Such synthesis may use an adapter as a primer binding site, or can use random priming.

[0318] Partitioning nucleic acid molecules in a sample can increase a rare signal, e.g., by enriching rare nucleic acid molecules that are more prevalent in one partition of the sample. For example, a genetic variation present in epigenetic target regions and / or sequence-variable target regions, can be more easily detected by partitioning a sample into a subsample comprising epigenetic target regions or sequence-variable target regions. By analyzing multiple partitions of a sample, a multi-dimensional analysis of a single molecule can be performed, and hence, greater sensitivity can be achieved. Partitioning may include physically partitioning nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions or subsamples based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic, or combination thereof that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cellAttorney Docket No.: GH0253WO free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0319] In some embodiments, hypermethylation and / or hypomethylation variable epigenetic target regions are analyzed to determine whether they show differential methylation characteristic of tumor cells or cells of a type that does not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or particular immune cell types.

[0320] In some instances, heterogenous DNA in a sample can be partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristic (examples provided herein) and tagged using differential tags that are distinguished from other partitions and partitioning means. In other instances, the differentially tagged partitions are separately sequenced.

[0321] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. After sequencing, analysis of reads can be performed on a partiti on-by-partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis to detect genetic variants can be performed on a partition-by-partition level, as well as whole nucleic acid population level. For example, analysis can include in silico analysis to determine genetic variants, such as copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), and / or fusions in nucleic acids in each partition. In some instances, in silico analysis can include analysis to determine epigenetic variation (one or more of methylation chromatin structure, etc.). Analysis can include in silico using sequence information, genomic coordinates length, coverage, and / or copy number. For example, coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Tags are used to sort reads from different partitions. Higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or nucleosome depleted region (NDR).Attorney Docket No.: GH0253WO

[0322] Examples of characteristics that can be used for partitioning include methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitioning based on a cytosine modification (e.g., cytosine methylation) or methylation generally is performed and is optionally combined with at least one additional partitioning step, which may be based on any of the foregoing characteristics or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with base modification and without one or more base modifications, including e.g., one or more sequence-variable target regions or one or more epigenetic modifications. Examples of base modifications are described elsewhere herein. Alternatively or additionally, a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively or additionally, a heterogeneous population of nucleic acids may be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned based on nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).

[0323] In some cases, different procedures are applied to different partitions to determine different characteristics of the initial sample. In some embodiments, the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure described herein. In cases where the method comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0324] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the number of sequencing runs needed and avoid unnecessary cost) and sequencing molecules, the partition tags identify the source partition. In another embodiment,Attorney Docket No.: GH0253WO different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35 barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0325] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to the molecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0326] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0327] While tags may be attached to molecules already partitioned based on one or more characteristics, the final tagged molecules in the library may no longer possess that characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to a molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.

[0328] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.Attorney Docket No.: GH0253WO

[0329] After sequencing, analysis of reads can be performed on a partition-by- partition level, as well as a whole DNA population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation, chromatin structure, etc.) using sequence information, genomic coordinates length, coverage, and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in a genomic region, while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0330] The affinity separation results in at least two, and sometimes three or more partitions of nucleic acids with different extents of a modification. While the partitions are still separate, the nucleic acids of at least one partition, and usually two or three (or more) partitions are linked to nucleic acid tags, usually provided as components of adapters, with the nucleic acids in different partitions receiving different tags that distinguish members of one partition from another. The tags linked to nucleic acid molecules of the same partition can be the same or different from one another. But if different from one another, the tags may have part of their code in common so as to identify the molecules to which they are attached as being of a particular partition.

[0331] For further details regarding partitioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0332] In some embodiments, the second assay can determine the modification of nucleic acids in the second partition wherein the assay can be used to detect disease, for example the second assay can be used for the detection of cancer.

[0333] In some embodiments, nucleic acid molecules (e.g., in the second partition) can be (further) partitioned into different partitions based on the nucleic acid molecules that are bound to a specific protein or a fragment thereof and those that are not bound to that specific protein or fragment thereof.

[0334] Nucleic acid molecules can be partitioned based on DNA-protein binding. Protein-DNA complexes can be partitioned based on a specific property of a protein. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation) or enzymatic activity. Examples of proteins which may bind to DNA and serve as a basis for fractionation may include, but are not limited to,Attorney Docket No.: GH0253WO protein A and protein G. Any suitable method can be used to partition the nucleic acid molecules based on protein bound regions. Examples of methods used to partition nucleic acid molecules based on protein bound regions include, but are not limited to, SDS-PAGE, chromatin-immuno-precipitation (ChIP), heparin chromatography, and asymmetrical field flow fractionation (AF4).

[0335] In some embodiments, the partitioning (e.g., further partitioning of the second partition) comprises contacting the DNA with a methylation sensitive restriction enzyme (MSRE) and / or a methylation dependent restriction enzyme (MDRE). Following the treatment of the DNA with a MSRE or a MDRE, the DNA may be partitioned based on size to generate hypermethylated (longest DNA molecules following MSRE treatment and shortest DNA fragments following MDRE treatment), intermediate (intermediate length DNA molecules following MSRE or MDRE treatment), and hypomethylated (shortest DNA molecules following MSRE treatment and longest DNA fragments following MDRE treatment) subsamples.

[0336] In some embodiments, the partitioning (e.g., further partitioning of the second partition) is performed by contacting the nucleic acids with a methyl binding domain (“MBD”) of a methyl binding protein (“MBP”). In some such embodiments, the nucleic acids are contacted with an entire MBP. In some embodiments, an MBD binds to 5-methylcytosine (5mC), and an MBP comprises an MBD and is referred to interchangeably herein as a methyl binding protein or a methyl binding domain protein. In some embodiments, MBD is coupled to paramagnetic beads, such as Dynabeads® M-280 Streptavidin via a biotin linker. Partitioning into fractions with different extents of methylation can be performed by eluting fractions by increasing the NaCl concentration.

[0337] In some embodiments, bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This may be performed instead of or in addition to elution steps using NaCl as discussed above.

[0338] Examples of agents that recognize a modified nucleobase contemplated herein include, but are not limited to:(a) MeCP2 is a protein that preferentially binds to 5-methyl-cytosine over unmodified cytosine.Attorney Docket No.: GH0253WO(b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 preferentially bind to 5- hydroxymethyl-cytosine over unmodified cytosine.(c) F0XK1, F0XK2, FOXP1, FOXP4 and F0XI3 preferably bind to 5-formyl-cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)).(d) Antibodies specific to one or more methylated or modified nucleobases or conversion products thereof, such as 5mC, 5-caC, or DHU.

[0339] In general, elution is a function of the number of modifications, such as the number of methylated sites per molecule, with molecules having more methylation eluting under increased salt concentrations. To elute the DNA into distinct populations based on the extent of methylation, one can use a series of elution buffers of increasing NaCl concentration. Salt concentration can range from about 100 nm to about 2500 mM NaCl. In one embodiment, the process results in three (3) partitions. Molecules are contacted with a solution at a first salt concentration and comprising a molecule comprising an agent that recognizes a modified nucleobase, which molecule can be attached to a capture moiety, such as streptavidin. At the first salt concentration a population of molecules will bind to the agent and a population will remain unbound. The unbound population can be separated as a “hypom ethylated” population. For example, a first partition enriched in hypomethylated form of DNA is that which remains unbound at a low salt concentration, e.g., 100 mM or 160 mM. A second partition enriched in intermediate methylated DNA is eluted using an intermediate salt concentration, e.g., between 100 mM and 2000 mM concentration. This is also separated from the sample. A third partition enriched in hypermethylated form of DNA is eluted using a high salt concentration, e.g., at least about 2000 mM.

[0340] In some embodiments, a monoclonal antibody raised against 5- methylcytidine (5mC) is used to purify methylated DNA. DNA is denatured, e.g., at 95°C in order to yield single-stranded DNA fragments. Protein G coupled to standard or magnetic beads as well as washes following incubation with the anti-5mC antibody are used to immunoprecipitate DNA bound to the antibody. Such DNA may then be eluted. Partitions may comprise unprecipitated DNA and one or more partitions eluted from the beads.

[0341] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.Attorney Docket No.: GH0253WO

[0342] Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated. Accordingly, in some embodiments, the DNA contacted with targetspecific probes specific for members of an epigenetic target region set comprising a plurality of target regions that are both type-specific differentially methylated regions and copy number variants comprises at least a portion of a hypermethylated partition. The DNA from or comprising at least a portion of the hypermethylated partition may or may not be combined with DNA from or comprising at least a portion of one or more other partitions, such as an intermediate partition or a hypomethylated partition.

[0343] In some cases, different procedures are applied to different partitions to determine different characteristics of the initial sample. In some embodiments, the DNA of at least one partition is subjected to an end repair and sequencing procedure described herein. In some embodiments at least one partition is not subjected to the end repair and sequencing procedure according to the methods of the disclosure described herein. In cases where the sequencing procedure comprises a conversion procedure, corresponding sequences from the converted and non-converted partitions can be compared to identify single nucleotides that have undergone conversion and therefore identify corresponding modified nucleosides in the initial sample.

[0344] Disclosed methods herein can comprise analyzing DNA in a sample. In some embodiments described herein, the disclosed methods comprise partitioning DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated and whether certain hypermethylated regions overlap with regions with copy number variants. In some embodiments, a first subsample or aliquot of a sample is subjected to steps for making capture probes as described elsewhere herein and a second subsample or aliquot of a sample is subjected to partitioning. In some embodiments, a sample or subsample or aliquot thereof is subjected to partitioning and differential tagging, followed by a capture step using capture probes for rearranged sequences and optionally additional capture probes, e.g., for sequence-variable and / or epigenetic target regions.

[0345] Methylation profiling can involve determining methylation patterns across different regions of the genome. For example, after partitioning molecules based onAttorney Docket No.: GH0253WO extent of methylation (e.g., relative number of methylated nucleobases per molecule) and sequencing, the sequences of molecules in the different partitions can be mapped to a reference genome. This can show regions of the genome that, compared with other regions, are more highly methylated or are less highly methylated. In this way, genomic regions, in contrast to individual molecules, may differ in their extent of methylation. In some embodiments, the nucleic acid molecules (from the sample of polynucleotides) may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”). Tags may be incorporated into or otherwise joined to adapters by chemical synthesis, ligation (e.g., blunt-end ligation or sticky-end ligation), or overlap extension polymerase chain reaction (PCR), among other methods. Such adapters may be ultimately joined to the target nucleic acid molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) are generally applied to introduce sample indexes to a nucleic acid molecule using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after sequence capturing steps are performed. In some embodiments, only the molecular barcodes are introduced prior to probe capturing and the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, both the molecular barcodes and the sample indexes are introduced prior to performing probe-based capturing steps. In some embodiments, the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, molecular barcodes are incorporated to the nucleic acid molecules (e.g. cfDNA molecules) in a sample through adapters via ligation (e.g., blunt-end ligation or sticky-end ligation). In some embodiments, sample indexes are incorporated to the nucleic acid molecules (e.g. cfDNA molecules) in a sample through overlap extension polymerase chain reaction (PCR). Typically, sequence capturing protocols involve introducing a single-stranded nucleic acid molecule complementary to a targeted nucleic acid sequence, e.g., a coding sequence of a genomic region and mutation of such region is associated with a cancer type.Attorney Docket No.: GH0253WO

[0346] In some embodiments, the tags may be located at one end or at both ends of the sample nucleic acid molecule. In some embodiments, tags are predetermined or random or semi-random sequence oligonucleotides. In some embodiments, the tags may be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length. The tags may be linked to sample nucleic acids randomly or non-randomly.

[0347] In some embodiments, each sample is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or sub-sample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation) to individual molecules such that the combination of the molecular barcode and the sequence it may be attached to creates a unique sequence that may be individually tracked. Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original nucleic acid molecule in the sample, start and stop genomic positions corresponding to the sequence of the original nucleic acid molecule in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original nucleic acid molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a uniqueAttorney Docket No.: GH0253WO identity, may thereby permit subsequent identification of fragments from the parent strand, and / or a complementary strand.

[0348] In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule. Alternatively, from about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20- 50 x 20-50 molecular barcode sequences (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the target molecule) can be used.Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of molecules have the same combinations of molecular barcodes.

[0349] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only endogenous sequence information (e.g., start and / or stop positions, sub-sequences of one or both ends of a sequence, and / or lengths).

[0350] In certain embodiments described herein, a population of different forms of nucleic acids (e.g., hypermethylated and hypomethylated DNA in a sample) can be physically partitioned to analysis, e.g., sequencing, or tagging and sequencing. This approach can be used to determine, for example, whether hypermethylation variable epigenetic target regions show hypermethylation characteristic of tumor cells or hypomethylation variable epigenetic target regions show hypomethylation characteristic of tumor cells. Additionally, by partitioning a heterogeneous nucleic acid population, one may increase rare signals, e.g., by enriching rare nucleic acid molecules that areAttorney Docket No.: GH0253WO more prevalent in one fraction (or partition) of the population. For example, a genetic variation present in hyper-methylated DNA but less (or not) in hypomethylated DNA can be more easily detected by partitioning a sample into hyper-methylated and hypo- methylated nucleic acid molecules. By analyzing multiple fractions of a sample, a multidimensional analysis of a single locus of a genome or species of nucleic acid can be performed and hence, greater sensitivity can be achieved.

[0351] In some instances, a heterogeneous nucleic acid sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6 or 7 partitions). In some embodiments, each partition is differentially tagged - i.e., each partition can have a different set of molecular barcodes. Tagged partitions can then be pooled together for collective sample prep and / or sequencing. The partitioning-tagging-pooling steps can occur more than once, with each round of partitioning occurring based on a different characteristic (such as any of the exemplary characteristics provided herein) and tagged using differential tags that are distinguished from other partitions and partitioning means.

[0352] Examples of characteristics that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins that bind to DNA. Resulting partitions can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids with one or more epigenetic modifications and without the one or more epigenetic modifications. Examples of epigenetic modifications include presence or absence of methylation; level of methylation; type of methylation (e.g., 5- methylcytosine versus other types of methylation, such as adenine methylation and / or cytosine hydroxymethylation); and association and level of association with one or more proteins, such as histones. Alternatively, or additionally, a heterogeneous population of nucleic acids can be partitioned into nucleic acid molecules associated with nucleosomes and nucleic acid molecules devoid of nucleosomes. Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned into singlestranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively, or additionally, a heterogeneous population of nucleic acids may be partitioned based onAttorney Docket No.: GH0253WO nucleic acid length (e.g., molecules of up to 160 bp and molecules having a length of greater than 160 bp).

[0353] In some instances, each partition (representative of a different nucleic acid form) is differentially tagged with molecular barcodes, and the partitions are pooled together prior to sequencing. In other instances, the different forms are separately sequenced. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated from the set of tags used to label other partitions. In some embodiments, a tag can be multifunctional - i.e., it can simultaneously act as a molecular identifier (i.e., molecular barcode), partition identifier (i.e., partition tag) and sample identifier (i.e., sample index). For example, if there are four DNA samples and each DNA sample is partitioned into three partitions, then the DNA molecules in each of the twelve partitions (i.e., twelve partitions for the four DNA samples in total) can be tagged with a separate set of tags such that the tag sequence attached to the DNA molecule reveals the identity of the DNA molecule, the partition it belongs to and the sample from which it was originated. In some embodiments, a tag can be used both as a molecular barcode and as a partition tag. For example, if a DNA sample is partitioned into three partitions, then DNA molecule in each partition is tagged with a separated set of tags such that the tag sequence attached to a DNA molecule reveals the identity of the DNA molecule and the partition it belongs to. In some embodiments, a tag can be used both as a molecular barcode and as a sample index. For example, if there are four DNA samples, then DNA molecules in each sample with be tagged with a separate set of tags that can be distinguishable from each sample such that the tag sequence attached to the DNA molecule serves as a molecule identifier and as a sample identifier.

[0354] In one embodiment, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions and sequencing molecules, the partition tags identify the source partition. In another embodiment, different partitions are tagged with different sets of molecular tags, e.g., comprised of a pair of barcodes. In this way, each molecular barcode indicates the source partition as well as being useful to distinguish molecules within a partition. For example, a first set of 35Attorney Docket No.: GH0253WO barcodes can be used to tag molecules in a first partition, while a second set of 35 barcodes can be used tag molecules in a second partition.

[0355] In some embodiments, after partitioning and tagging with partition tags, the molecules may be pooled for sequencing in a single run. In some embodiments, a sample tag is added to the molecules, e.g., in a step subsequent to addition of partition tags and pooling. Sample tags can facilitate pooling material generated from multiple samples for sequencing in a single sequencing run.

[0356] Alternatively, in some embodiments, partition tags may be correlated to the sample as well as the partition. As a simple example, a first tag can indicate a first partition of a first sample; a second tag can indicate a second partition of the first sample; a third tag can indicate a first partition of a second sample; and a fourth tag can indicate a second partition of the second sample.

[0357] While tags may be attached to molecules already partitioned based on one or more epigenetic characteristics, the final tagged molecules in the library may no longer possess that epigenetic characteristic. For example, while single stranded DNA molecules may be partitioned and tagged, the final tagged molecules in the library are likely to be double stranded. Similarly, while DNA may be subject to partition based on different levels of methylation, in the final library, tagged molecules derived from these molecules are likely to be unmethylated. Accordingly, the tag attached to molecule in the library typically indicates the characteristic of the “parent molecule” from which the ultimate tagged molecule is derived, not necessarily to characteristic of the tagged molecule, itself.

[0358] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label molecules in the third partition. Differentially tagged partitions can be pooled prior to sequencing. Differentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0359] After sequencing, analysis of reads to detect genetic variants can be performed on a partition-by-partition level, as well as a whole nucleic acid population level. Tags are used to sort reads from different partitions. Analysis can include in silico analysis to determine genetic and epigenetic variation (one or more of methylation,Attorney Docket No.: GH0253WO chromatin structure, etc.) using sequence information, genomic coordinates length, coverage and / or copy number. In some embodiments, higher coverage can correlate with higher nucleosome occupancy in genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).F. Subjecting the sample to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase; Conversion-based sequencing

[0360] Methods disclosed herein may comprise a step of subjecting a sample (e.g., any or each of the samples obtained or used in a method disclosed herein) to a procedure that affects a first nucleobase in the nucleic acids (e.g., DNA such as cfDNA) differently from a second nucleobase. Methods disclosed herein may comprise a step of subjecting a sample from a subject (e.g., any or each of the samples obtained or used in a method disclosed herein) to a procedure that affects a first nucleobase in the nucleic acids (e.g., DNA such as cfDNA) differently from a second nucleobase. In some embodiments, the second assay performed on the second partition comprises the procedure that affects a first nucleobase in the nucleic acids (e.g., DNA such as cfDNA) differently from a second nucleobase. Such procedures can be useful for distinguishing modified and unmodified forms of a nucleobase, such as methylated and unmethylated cytosine, or different modified forms of a nucleobase, such as hydroxymethylated cytosine and cytosine that is methylated but not hydroxymethylated.

[0361] In some embodiments, the second assay determines the modification status of nucleic acids in the second partition. In some embodiments, the second assay comprises a methylation-sensitive conversion method. In some embodiments, the second assay comprises a base-conversion based sequencing method, such as methods which comprise bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, enzymatic methyl-seq (EM-seq) conversion, Tet-assisted pyridine borane sequencing (TAPS), APOBEC-coupled epigenetic (ACE) conversion, and / or direct methylation sequencing (DM-seq). The disclosed methods can then be used to determine the modification status of a nucleic acid population. In some embodiments, the method comprises quantifying modifications of the nucleic acid molecules within the nucleic acid population. Quantification of the modification (e.g., in the second partition) can be determined, for example, using quantitative PCR (qPCR). qPCR can be employed to determine the modification status of nucleic acids by utilizingAttorney Docket No.: GH0253WO primers and probes that specifically discriminate between modified and unmodified nucleic acid sequences. Such methods can rely on the differential binding affinities of oligonucleotide probes or primers to regions containing modifications, such as methylated or chemically altered bases, compared to their unmodified counterparts. For example, bisulfite treatment can be used to convert unmethylated cytosines to uracils while leaving methylated cytosines unchanged, creating a detectable difference in the DNA sequence that can be amplified and quantified using qPCR. The quantified value of the modifications in the second partition can be used to provide a modification score for the second partition.

[0362] Such conversion procedures can comprise subjecting the nucleic acids (e.g., DNA such as cfDNA) to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the nucleic acids (e.g., DNA such as cfDNA). In some embodiments, methods disclosed herein comprise a step of subjecting nucleic acids (e.g., DNA such as cfDNA), or a subsample thereof, to a procedure that affects a first nucleobase in the nucleic acids (e.g., DNA such as cfDNA) differently from a second nucleobase in the nucleic acids (e.g., DNA such as cfDNA), wherein the first nucleobase is a modified or unmodified nucleobase, the second nucleobase is a modified or unmodified nucleobase different from the first nucleobase, and the first nucleobase and the second nucleobase have the same base pairing specificity. In some embodiments, the procedure chemically converts the first or second nucleobase such that the base pairing specificity of the converted nucleobase is altered. In some embodiments, nucleic acids (e.g., DNA such as cfDNA) are subjected to a procedure that affects a first nucleobase in the nucleic acids (e.g., DNA such as cfDNA) differently from a second nucleobase in the nucleic acids (e.g., DNA such as cfDNA) before library preparation using the nucleic acids (e.g., DNA such as cfDNA), before a first amplification of the nucleic acids (e.g., DNA such as cfDNA), and / or before the ligation of adapters to the nucleic acids (e.g., DNA such as cfDNA). In certain embodiments, the nucleic acids (e.g., DNA such as cfDNA) are subjected to the procedure before or after contacting the DNA with a methylation-sensitive nuclease. In certain embodiments, the nucleic acids (e.g., DNA such as cfDNA) are subjected to the procedure before contacting the DNA with a methylation-sensitive nuclease. In certainAttorney Docket No.: GH0253WO embodiments, the nucleic acids (e.g., DNA such as cfDNA) are subjected to the procedure after contacting the DNA with a methylation-sensitive nuclease.

[0363] The methods disclosed herein can comprise contacting the nucleic acids (e.g., DNA such as cfDNA) with a methyl-sensitive deaminase, thereby providing a converted sample in which unmethylated CpGs in the nucleic acids (e.g., DNA such as cfDNA) are converted to UpGs. This step may occur after one or more of contacting nucleic acids (e.g., DNA such as cfDNA) in a sample with an mCpG-binding protein, eluting mCpG-dense DNA from the mCpG-binding protein, an end-repair step, and / or a tagging (e.g., adapter ligation step) and may also occur before one or more of an amplification step, a capture or enrichment step, and a sequencing step. In some embodiments, the contacting the nucleic acids (e.g., DNA such as cfDNA) with a methyl-sensitive deaminase occurs after the separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition. In some embodiments, the contacting the nucleic acids (e.g., DNA such as cfDNA) with a methyl-sensitive deaminase occurs after performing an assay on the second partition. In some embodiments, the second partition comprises nucleic acids that have a lower average length relative to the nucleic acids in the first partition. The methods disclosed herein can comprise contacting nucleic acids (e.g., DNA such as cfDNA) in a sample with an mCpG-binding protein, thereby providing mCpG protein-bound nucleic acids (e.g., mCpG protein-bound DNA such as mCpG protein-bound cfDNA). The methods disclosed herein can comprise separating mCpG protein-bound nucleic acids (e.g., mCpG protein-bound DNA such as mCpG protein-bound cfDNA) from unbound nucleic acids (e.g., unbound DNA such as unbound cfDNA) to yield mCpG-dense nucleic acids (e.g., mCpG-dense DNA such as mCpG-dense cfDNA) before contacting the mCpG-dense nucleic acids (e.g., mCpG-dense DNA such as mCpG-dense cfDNA) with a methyl-sensitive deaminase. In some embodiments, the separating the mCpG- binding protein comprises eluting mCpG protein-bound nucleic acids (e.g., mCpG protein-bound DNA such as mCpG protein-bound cfDNA) from the nucleic acids (e.g., DNA such as cfDNA) unbound to the mCpG-binding protein. In some embodiments, the mCpG-binding protein is removed from the mCpG-dense nucleic acids (e.g.,Attomey Docket No.: GH0253WO mCpG-dense DNA such as mCpG-dense cfDNA) after the separating. In some embodiments, the mCpG-binding domain protein comprises a methyl-binding domain protein including, for example, mCpG-binding domain 4 (MBD4), mCpG-binding domain 2 (MBD2), mCpG-binding domain 1 (MBD1), or methyl CpG binding protein 2 (MeCP2). In some embodiments, the CpG-binding domain protein comprises MBD3.

[0364] In some embodiments, if the first nucleobase is a modified or unmodified adenine, then the second nucleobase is a modified or unmodified adenine; if the first nucleobase is a modified or unmodified cytosine, then the second nucleobase is a modified or unmodified cytosine; if the first nucleobase is a modified or unmodified guanine, then the second nucleobase is a modified or unmodified guanine; and if the first nucleobase is a modified or unmodified thymine, then the second nucleobase is a modified or unmodified thymine (where modified and unmodified uracil are encompassed within modified thymine for the purpose of this step). Such a procedure can be used to identify nucleotides in the subsample that have or lack certain modifications, such as methylation.

[0365] In some embodiments, the first nucleobase is a modified or unmodified cytosine, then the second nucleobase is a modified or unmodified cytosine. In some embodiments, the first nucleobase is a modified cytosine, then the second nucleobase is an unmodified cytosine. In some embodiments, the first nucleobase is an unmodified cytosine, then the second nucleobase is a modified cytosine. For example, first nucleobase may comprise unmodified cytosine (C) and the second nucleobase may comprise one or more of 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC). Alternatively, the second nucleobase may comprise C and the first nucleobase may comprise one or more of mC and hmC. Other combinations are also possible, as indicated, e.g., in the Summary above and the following discussion, such as where one of the first and second nucleobases comprises mC and the other comprises hmC.

[0366] In some embodiments, the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is a conversion. In some embodiments, the procedure that affects a first nucleobase of the DNA differently from a second nucleobase of the DNA is methylation-sensitive conversion. The methods disclosed herein can comprise contacting DNA in a sample with a deaminase, thereby providing a converted sample. In some embodiments, the deaminase is a methyl-Attorney Docket No.: GH0253WO sensitive deaminase or a methyl-insensitive deaminase. In some embodiments, the deaminase is a dsDNA deaminase and / or a ssDNA deaminase. This step of contacting the DNA in the sample with a deaminase can be referred to as, or be included in, a conversion procedure, such as any of the conversion procedures described elsewhere herein. For an exemplary description of conversion using a deaminase, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090. In some embodiments, the DNA in the converted sample is then sequenced, and a level or methylation at one or more differentially methylated regions of the DNA is quantified, or a variation of the copy number at one or more regions of the DNA is quantified.

[0367] Table 1 summarizes exemplary methods of deamination with the type of modified bases detectable with these methods. These are described in more detail below.

[0368] As outlined below, there are various methods of detecting and / or identifying modified nucleosides that rely on a conversion procedure that changes the base-pairing specificity of a nucleoside, based on the modification status of the nucleosides. These changes of base-pairing specificity can then be detected, and thus the modification status of the nucleoside inferred, by sequencing.

[0369] In some embodiments, the conversion procedure used in the methods of the disclosure is one that changes the base pairing specificity of a modified nucleoside (e.g. methylated cytosine) but does not change the base pairing specificity of theAttorney Docket No.: GH0253WO corresponding unmodified nucleoside (e.g. cytosine) or does not change the base pairing specificity of any un-modified nucleoside (e.g. cytosine, adenosine, guanosine and thymidine (or uracil)). Advantages of methods that do not convert the base-pairing specificity of unmodified nucleosides include reduced loss of sequence complexity, higher sequencing efficiency and reduced alignment losses. Additionally, methods such as TAPS may in some cases be preferred over methods such as bisulfite sequencing and EM-seq because they are less destructive (especially important for low yield samples such as cfDNA or FFPE samples) and do not require denaturation, meaning that nonconversion errors are theoretically more likely to be random. In methods that require denaturation for conversion, failure to denature a DNA molecule will result in nonconversion of all bases in the DNA molecule. As biological changes in methylation are predominantly concerted to a localized regions of interest, these non-random (localized) non-conversion events can appear as false negatives (non-methylated regions). Random non-conversion methods can maximally affect a low percent of bases within a region, and thus the specificity of methylation change detection can be maximized (reduce false positives) by placing a threshold on percentage of bases within a region that are methylated / non-methylated. Hence, in some cases, a conversion procedure that does not involve denaturation can be preferred.

[0370] In other cases, the conversion procedure that can be used in the methods of the disclosure is one that changes the base pairing specificity of an unmodified nucleoside (e.g. cytosine) but does not change the base pairing specificity of the corresponding modified nucleoside (e.g. methylated cytosine such as 5hmC and / or 5mC). Such methods include, for example, bisulfite sequencing.

[0371] The skilled person can select a suitable method according to their needs, including which nucleoside modifications are to be detected and / or identified and which type of modified base is used in the end repair reaction.

[0372] Hence, in these embodiments, the end repair reaction can be performed with dNTPs, wherein the at least one type of dNTP comprises a 5mC or 5hmC, and regions of the end-repaired DNA synthesized during the end repair reaction can be identified as those regions comprising 5mC or 5hmC (via T being called at positions which are C in the reference) at non-CpG positions. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), described in further detail in Liu et al. 2019, supra. In thisAttorney Docket No.: GH0253WO method Tet enzyme is used to progressively oxidize 5mC and 5hmC to 5fC or 5caC, then pyridine borane deaminates 5fC, 5CaC to DHU, amplified as T.

[0373] In some embodiments, the conversion procedure converts modified nucleosides. In some embodiments, the conversion procedure which converts modified nucleosides comprises Tet-assisted conversion with a substituted borane reducing agent. In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. In Tet-assisted pic-borane conversion with a substituted borane reducing agent conversion, a TET protein is used to convert mC and hmC to caC, without affecting unmodified C. caC, and fC if present, are then converted to dihydrouracil (DHU) by treatment with 2-picoline borane (pic-borane) or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane, or ammonia borane, also without affecting unmodified C. See, e.g., Liu et al., Nature Biotechnology 2019; 37:424-429 (e.g., at Supplementary Fig. 1 and Supplementary Note 7). DHU is read as a T in sequencing. Thus, when this type of conversion is used, the first nucleobase comprises one or more of mC, fC, caC, or hmC, and the second nucleobase comprises unmodified cytosine. Sequencing of the converted DNA identifies positions that are read as cytosine as being unmodified C positions.Meanwhile, positions that are read as T are identified as being T, mC, fC, caC, or hmC. Performing TAP conversion, such as on a DNA sample as described herein, thus facilitates identifying positions containing unmodified C using the sequence reads obtained from the sample. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), described in further detail in Liu et al. 2019, supra.

[0374] Alternatively, protection of 5hmC (e.g., using PGT or 5- hydroxymethylcytosine carbamoyltransferase) can be combined with Tet-assisted conversion with a substituted borane reducing agent, e.g. as described above. In this method (TAPS-P), 5hmC can be protected from conversion, for example through glucosylation using P-glucosyl transferase (PGT), forming (forming 5- glucosylhydroxymethylcytosine) 5ghmC, or through carbamoylation using 5- hydroxymethylcytosine carbamoyltransferase, forming 5cmC. This is described in Yu etAttorney Docket No.: GH0253WO al., Cell 2012; 149: 1368-80. Treatment with a TET protein such as mTetl then converts 5mC to 5caC but does not convert C, 5ghmC, or 5cmC. 5caC is then converted to DHU by treatment with pic-borane or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane, or ammonia borane, also without affecting ghmC, 5cmC, or unmodified C. Thus, when Tet-assisted conversion with a substituted borane reducing agent is used, the first nucleobase comprises mC, and the second nucleobase comprises one or more of unmodified cytosine or hmC, such as unmodified cytosine and optionally hmC, fC, and / or caC. Sequencing of the converted DNA identifies positions that are read as cytosine as being either 5hmC or unmodified C positions. Meanwhile, positions that are read as T are identified as being T, 5fC, 5caC, or 5mC. Performing TAPSP conversion on a sample as described herein thus facilitates distinguishing positions containing unmodified C or 5hmC on the one hand from positions containing 5mC using the sequence reads obtained. Hence, in these embodiments, the end repair reaction can be performed with dNTPs, wherein the at least one type of dNTP comprises a 5mC, and regions synthesized during the end repair reaction can be identified as those regions comprising 5mC (via T being called at positions which are C in the reference) at non-CpG positions. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37:424-429. 5-hydroxymethylcytosine carbamoyltransferase is described in Yang et al., Bio-protocol, 2023; 12(17): e4496.

[0375] In some embodiments, the conversion procedure converts modified nucleosides. In some embodiments, the conversion procedure which converts modified nucleosides comprises chemical-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, borane pyridine or ammonia borane. In chemical-assisted conversion with a substituted borane reducing agent, an oxidizing agent such as potassium perruthenate (KRuCU) (also suitable for use in ox-BS conversion) is used to specifically oxidize 5hmC to 5fC. Treatment with pic-borane or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane, or ammonia borane converts 5fC and 5caC to DHU but does not affect 5mC or unmodified C. Thus, when this type of conversion is used, the first nucleobase comprises one or more of hmC, fC, and caC, and the second nucleobase comprises oneAttorney Docket No.: GH0253WO or more of unmodified cytosine or mC, such as unmodified cytosine and optionally mC. Sequencing of the converted DNA identifies positions that are read as cytosine as being either 5mC or unmodified C positions. Meanwhile, positions that are read as T are identified as being T, 5fC, 5caC, or 5hmC. Performing this type of conversion as described herein thus facilitates distinguishing positions containing unmodified C or 5mC on the one hand from positions containing 5hmC using the sequence reads obtained. Hence, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP comprises a 5hmC, and regions synthesized during the end repair reaction can be identified as those regions comprising 5hmC (via T being called at positions which are C in the reference) at non-CpG positions. For an exemplary description of this type of conversion, see, e.g, Liu et al., Nature Biotechnology 2019; 37:424-429.

[0376] Exemplary conversion procedures that change the base-pairing specificity of modified cytosines have been described. However, the methods described herein could in principle use any modified nucleoside and suitable conversion procedure (i.e. singlebase epigenetic conversion assay) that changes the base-pairing specificity of the modified nucleoside and thereby allows the modified base to be distinguished from the corresponding unmodified nucleoside and / or other types of modification when sequenced. For example, any conversion procedure could be used allowing any one of N6-methyladenine (6mA), N6-hydroxymethyladenine (6hmA), or N6-formyladenine (6fA) to be distinguished from unmodified adenosine.

[0377] In some embodiments, the conversion procedure converts unmodified nucleosides. In some embodiments, the conversion procedure which converts unmodified nucleosides comprises bisulfite conversion. In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises bisulfite conversion. Treatment with bisulfite converts unmodified cytosine and certain modified cytosine nucleotides (e.g., 5-formyl cytosine (fC) or 5-carboxylcytosine (caC)) to uracil whereas other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted. Thus, where bisulfite conversion is used, the first nucleobase comprises one or more of unmodified cytosine, 5-formyl cytosine, 5-carboxylcytosine, or other cytosine forms affected by bisulfite, and the second nucleobase may comprise one or more of 5-methyl cytosineAttorney Docket No.: GH0253WO(mC) and 5-hydroxymethylcytosine (hmC), such as mC and optionally hmC. Sequencing of bisulfite-treated DNA identifies positions that are read as cytosine as being mC or hmC positions. Meanwhile, positions that are read as T are identified as being T or a bisulfite-susceptible form of C, such as unmodified cytosine, 5-formyl cytosine, or 5-carboxylcytosine. Performing bisulfite conversion, such as on a DNA sample as described herein, thus facilitates identifying positions containing mC or hmC using the sequence reads obtained from the exemplary sample. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018; 9: 5068.

[0378] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises oxidative bisulfite (Ox-BS) conversion. This procedure first converts hmC to fC, which is bisulfite susceptible, followed by bisulfite conversion. Thus, when oxidative bisulfite conversion is used, the first nucleobase comprises one or more of unmodified cytosine, fC, caC, hmC, or other cytosine forms affected by bisulfite, and the second nucleobase comprises mC. Sequencing of Ox-BS converted DNA identifies positions that are read as cytosine as being mC positions. Meanwhile, positions that are read as T are identified as being T, hmC, or a bisulfite-susceptible form of C, such as unmodified cytosine, fC, or hmC. Performing Ox-BS conversion, such as on a DNA sample as described herein, thus facilitates identifying positions containing mC using the sequence reads obtained from the sample. For an exemplary description of oxidative bisulfite conversion, see, e.g., Booth et al., Science 2012; 336: 934-937.

[0379] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises Tet-assisted bisulfite (TAB) conversion. In TAB conversion, hmC is protected from conversion and mC is oxidized in advance of bisulfite treatment, so that positions originally occupied by mC are converted to U while positions originally occupied by hmC remain as a protected form of cytosine. For example, as described in Yu et al., Cell 2012; 149: 1368-80, [3- glucosyl transferase can be used to protect hmC (forming 5- glucosylhydroxymethylcytosine (ghmC)), then a TET protein such as mTetl can be used to convert mC to caC, and then bisulfite treatment can be used to convert C and caC to U while ghmC remains unaffected.Attorney Docket No.: GH0253WO

[0380] Alternatively, a carbamoyltransferase enzyme, such as 5- hydroxymethylcytosine carbamoyltransferase as described in Yang et al., Bio-protocol, 2023; 12(17): e4496, can be used to protect hmC (by converting hmC to 5- carbamoyloxymethylcytosine (5cmC)), then a TET protein such as mTetl can be used to convert mC to caC, and then bisulfite treatment can be used to convert C and caC to U while 5cmC remains unaffected. Thus, when TAB conversion is used, the first nucleobase comprises one or more of unmodified cytosine, fC, caC, mC, or other cytosine forms affected by bisulfite, and the second nucleobase comprises hmC. Sequencing of TAB -converted DNA identifies positions that are read as cytosine as being hmC positions. Meanwhile, positions that are read as T are identified as being T, mC, or a bi sulfite-susceptible form of C, such as unmodified cytosine, fC, or caC. Performing TAB conversion, such as on a DNA sample as described herein, thus facilitates identifying positions containing hmC using the sequence reads obtained from the sample. Hence, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP comprises a 5hmC, and regions synthesized during the end repair reaction can be identified as those regions comprising 5hmC (via C being called at these positions) at non-CpG positions.

[0381] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises chemical-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. In chemical-assisted conversion with a substituted borane reducing agent, an oxidizing agent such as potassium perruthenate (KRuCL) (also suitable for use in ox-BS conversion) is used to specifically oxidize hmC to fC. Treatment with pic- borane or another substituted borane reducing agent such as borane pyridine, tert- butylamine borane, or ammonia borane converts fC and caC to DHU but does not affect mC or unmodified C. Thus, when this type of conversion is used, the first nucleobase comprises one or more of hmC, fC, and caC, and the second nucleobase comprises one or more of unmodified cytosine or mC, such as unmodified cytosine and optionally mC. Sequencing of the converted DNA identifies positions that are read as cytosine as being either mC or unmodified C positions. Meanwhile, positions that are read as T are identified as being T, fC, caC, or hmC. Performing this type of conversion, such as on aAttorney Docket No.: GH0253WODNA sample as described herein, thus facilitates distinguishing positions containing unmodified C or mC on the one hand from positions containing hmC using the sequence reads obtained from the sample. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37:424-429. 5- hydroxymethylcytosine carbamoyltransferase is described in Yang et al., Bio-protocol, 2023; 12(17): e4496.

[0382] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises APOBEC-coupled epigenetic (ACE) conversion. In ACE conversion, an AID / APOBEC family DNA deaminase enzyme such as APOBEC3 A (A3 A) is used to deaminate unmodified cytosine and mC without deaminating hmC, fC, or caC. Thus, when ACE conversion is used, the first nucleobase comprises unmodified C and / or mC (e.g., unmodified C and optionally mC), and the second nucleobase comprises hmC. Sequencing of ACE- converted DNA identifies positions that are read as cytosine as being hmC, fC, or caC positions. Meanwhile, positions that are read as T are identified as being T, unmodified C, or mC. Performing ACE conversion on a DNA sample as described herein thus facilitates distinguishing positions containing hmC from positions containing mC or unmodified C using the sequence reads obtained from the sample. For an exemplary description of ACE conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0383] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises enzymatic conversion of the first nucleobase, e.g., as in EM-Seq. See, e.g., Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv, DOI: 10.1101 / 2019.12.20.884692, available at www.biorxiv.org / content / 10.1101 / 2019.12.20.884692vl . For example, TET2 and T4- PGT or 5 -hydroxymethyl cytosine carbamoyltransferase (described in Yang et al., Bioprotocol, 2023; 12(17): e4496) can be used to convert 5mC and 5hmC into substrates that cannot be deaminated by a deaminase (e.g., APOBEC3 A), and then a deaminase (e.g., APOBEC3 A) can be used to deaminate unmodified cytosines converting them to uracils.Attorney Docket No.: GH0253WO

[0384] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises enzymatic conversion of the first nucleobase using a non-specific, modification-sensitive doublestranded DNA deaminase, e.g., as in SEM-seq. See, e.g., Vaisvila et al. bioRxiv. 2023; DOI: 10.1101 / 2023.06.29.547047, available at https: / / www.biorxiv.org / content / 10.1101 / 2023.06.29.547047vl. SEM-Seq employs a non-specific, modification-sensitive double-stranded DNA deaminase (MsddA) in a nondestructive single-enzyme 5-methylctyosine sequencing (SEM-seq) method that deaminates unmodified cytosines. Accordingly, SEM-seq does not require the TET2 and T4-PGT or 5-hydroxymethylcytosine carbamoyltransferase protection and denaturing steps that are of use, e.g., in APOEC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5caC). In SEM-seq, unmodified cytosines in the DNA are deaminated to uracil and is read as “T” during sequencing. Modified cytosines (e.g., 5mC) are not converted and are read as “C” during sequencing. Cytosines that are read as thymines are identified as unmodified (e.g., unmethylated) cytosines or as thymines in the DNA. Performing SEM-seq conversion thus facilitates identifying positions containing 5mC using the sequence reads obtained. In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises enzymatic conversion of the first nucleobase using MsddA or a modification-sensitive DNA deaminase A (MsddA)-like deaminase. For an exemplary description of MsddA and MsddA-like deaminases, see, e.g., Vaisvila et al. Mol Cell. 2024 Mar 7;84(5):854- 866. e7, which illustrates in Fig. 2A-C that MsddA-like deaminases have reduced activity on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine in dsDNA, e.g., a reduction of about 75%, 80%, or more on each of 5mC, 5hmC, and 5gmC relative to unmodified cytosine (e.g., using assay conditions as described in Vaisvila et al., such as analysis of deamination of C in E. coli or lambda dem- DNA, deamination of 5mC in XP12 phage DNA, deamination of 5hmC in a C-hydroxymethylated adenovirus PCR fragment or fully C-hydroxymethylated T4147 phage DNA, and deamination of 5gmC in alpha-glucosyltransf erase knockout (AGT-) T4 phage DNA). Deamination can be performed by contacting substrate DNA with deaminase and analyzed using NGS as follows: 50 ng of unmodified E. coli C2566 genomic DNA canAttorney Docket No.: GH0253WO be combined with the control DNAs (about 1 ng of Lambda, XP12, and T4147, and 0.1 ng of the 5hmC Adenovirus PCR fragment), sheared to about 300 bp and ligated to pyrrolo-dC adapters with 1 uL of in vitro synthesized deaminase (e.g., synthesized using the PURExpress In Vitro Protein Synthesis kit (NEB, Ipswich, MA) following manufacturer’s recommendations with 100-400 ng of PCR fragment template DNA containing codon-optimized deaminase coding sequence and T7 promoter and terminator). Exemplary deamination reaction conditions are 50 mM Bis-Tris pH 6.0, 0.1% Triton X-100 for 1 hour at 37 degrees C. After the deamination reaction, 1 uL of Therm olabile Proteinase K (NEB, Ipswich, MA) can be added and incubated for 30 min at 37 degrees C and then the Proteinase K can be heat inactivated at 60 degrees C for 10 minutes. The deaminated product can then be used for library amplification using the NEBNext Q5U Master Mix (New England Biolabs, Ipswich, MA, USA) with 5mMof NEBNext Unique Dual Index Primers. The resulting library can be purified using IX NEBNext Sample Purification Beads according to the manufacturer’s instructions and the purified library can be analyzed and quantified by an Agilent Bioanalyzer 2100 DNA Highsensitivity chip. The libraries can be sequenced using the Illumina NextSeq and NovaSeq platforms. Paired-end sequencing of 75 cycles (2 x 75 bp) can be performed for all the sequencing runs. Base calling and demultiplexing can be carried out with the standard Illumina pipeline.

[0385] In some embodiments, the procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA comprises separating DNA originally comprising the first nucleobase from DNA not originally comprising the first nucleobase. In some such embodiments, the first nucleobase is hmC. DNA originally comprising the first nucleobase may be separated from other DNA using a labeling procedure comprising biotinylating positions that originally comprised the first nucleobase. In some embodiments, the first nucleobase is first derivatized with an azide- containing moiety, such as a glucosyl -azide containing moiety. The azide-containing moiety then may serve as a reagent for attaching biotin, e.g., through Huisgen cycloaddition chemistry. Then, the DNA originally comprising the first nucleobase, now biotinylated, can be separated from DNA not originally comprising the first nucleobase using a biotin-binding agent, such as avidin, neutravidin (deglycosylated avidin with an isoelectric point of about 6.3), or streptavidin. An example of aAttorney Docket No.: GH0253WO procedure for separating DNA originally comprising the first nucleobase from DNA not originally comprising the first nucleobase is hmC-seal, which labels hmC to form P-6- azide-glucosyl-5-hydroxymethylcytosine and then attaches a biotin moiety through Huisgen cycloaddition, followed by separation of the biotinylated DNA from other DNA using a biotin-binding agent. For an exemplary description of hmC-seal, see, e.g., Han et al., Mol. Cell 2016; 63: 711-719. This approach is useful for identifying fragments that include one or more hmC nucleobases.

[0386] In some embodiments, the conversion procedure converts modified nucleosides. In some embodiments, the conversion procedure which converts modified nucleosides comprises enzymatic conversion, such as DM-seq, for example, as described in Wang et al., Nat Chem Biol. 2023, 19(8): 1004-1012 and WO2023 / 288222A1. In DM-seq, unmodified cytosines in the DNA are enzymatically protected from a subsequent deamination step wherein 5mC in 5mCpG is converted to T. The enzymatically protected unmodified (e.g., unmethylated) cytosines are not converted and are read as “C” during sequencing. Cytosines that are read as thymines (in a CpG context) are identified as methylated cytosines in the DNA. For an exemplary description of mCpG binding domain proteins, see, e.g., Du et al., Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics. 2015;7(6):1051-73.

[0387] Thus, when this type of conversion is used, the first nucleobase comprises unmodified (such as unmethylated) cytosine, and the second nucleobase comprises modified (such as methylated) cytosine. Sequencing of the converted DNA identifies positions that are read as cytosine as being unmodified C positions. Meanwhile, positions that are read as T are identified as being T or 5mC. Performing DM-seq conversion thus facilitates identifying positions containing 5mC using the sequence reads obtained.

[0388] Exemplary cytosine deaminases for use herein include APOBEC enzymes, for example, APOBEC3 A. Generally, AID / APOBEC family DNA deaminase enzymes such as APOBEC3 A (A3 A) are used to deaminate (unprotected) unmodified cytosine and 5mC. For an exemplary description of APOBEC enzymes, see, e.g., Gajula et al., Nucleic Acids Res. 2014 Sep;42(15):9964-75 and Schutsky et al., Nucleic Acids Res. 2017 Jul 27;45(13):7655-7665. For an exemplary description of APOBEC conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.Attorney Docket No.: GH0253WO

[0389] The enzymatic protection of unmodified cytosines in the DNA comprises addition of a protective group to the unmodified cytosines. Such protective groups can comprise an alkyl group, an alkyne group, a carboxyl group, a carboxyalkyl group, an amino group, a hydroxymethyl group, a glucosyl group, a glucosylhydroxymethyl group, an isopropyl group, or a dye. For example, DNA can be treated with a methyltransferase, such as a CpG-specific methyltransferase, which adds the protective group to unmodified cytosines. The term methyltransferase is used broadly herein to refer to enzymes capable of transferring a methyl or substituted methyl (e.g., carboxymethyl) to a substrate (e.g., a cytosine in a nucleic acid). In some embodiments, the DNA is contacted with a CpG-specific DNA methyltransferase (MTase), such as a CpG-specific carboxymethyltransferase (CxMTase), and a substituted methyl donor, such as a carboxymethyl donor (e.g., carboxymethyl-S- adenosyl-L-methionine). See, e.g., WO2021 / 236778A2. In particular embodiments, the CxMTase can facilitate the addition of a protective carboxymethyl group to an unmethylated cytosine. In some embodiments, the unmethylated cytosine is unmodified cytosine. The carboxymethyl group can prevent deamination of the cytosine during a deamination step (such as a deamination step using an APOBEC enzyme, such as A3 A). Substituted methyl or carboxymethyl donors useful in the disclosed methods include but are not limited to, S-adenosyl-L-methionine (SAM) analogs, optionally wherein the SAM analog is carboxy-S-adenosyl-L-methionine (CxSAM). SAM analogs are described, for example, in WO2022 / 197593A1. The MTase may be, for example, a CpG methyltransferase from Spiroplasma sp. strain MQ1 (M.SssI), DNA- methyltransferase 1 (DNMT1), DNA-methyltransferase 3 alpha (DNMT3A), DNA- methyltransferase 3 beta (DNMT3B), or DNA adenine methyltransferase (Dam). The CxMTase may be a CpG methyltransferase from Mycoplasma penetrans (M.Mpel).

[0390] In one embodiment, the methyltransferase enzyme is a variant of M.Mpel having an N374R substitution or an N374K substitution.

[0391] Optionally, the conversion procedure further includes enzymatic protection of 5hmCs, such as by glucosylation of the 5hmCs (e.g., using PGT) or by carbamoylation of the 5hmCs (e.g., using 5-hydroxymethylcytosine carbamoyltransferase), in the DNA prior to the deamination of unprotected modified cytosines. In this method, 5hmC can be protected from conversion, for example throughAttorney Docket No.: GH0253WO glucosylation using P-glucosyl transferase (PGT), forming (5- glucosylhydroxymethylcytosine) 5ghmC, or through carbamoylation using 5- hydroxymethylcytosine carbamoyltransferase, forming 5cmC. This is described, for example, in Yu et al., Cell 2012; 149: 1368-80, and in Yang et al., Bio-protocol, 2023; 12(17): e4496. Glucosylation or carbamoylation of 5hmC can reduce or eliminate deamination of 5hmC by a deaminase such as APOBEC3A. Treatment with an MTase or CxMTase then adds a protecting group to unmodified (unmethylated) cytosines in the DNA. 5mC (but not protected, unmodified cytosine and not 5ghmC or 5cmC) is then deaminated (converted to T in the case of 5mC) by treatment with a deaminase, for example, an APOBEC enzyme (such as APOBEC3 A). Sequencing of the converted DNA identifies positions that are read as cytosine as being either 5hmC or unmodified C positions. Meanwhile, positions that are read as T are identified as being T or 5mC. Performing DM-seq conversion with glucosylation of 5hmC on a sample as described herein thus facilitates distinguishing positions containing unmodified C or 5hmC on the one hand from positions containing 5mC using the sequence reads obtained.

[0392] Also provided herein are methods in which alternative base conversion schemes can be used. For example, unmethylated cytosines can be left intact while methylated cytosines and hydroxymethylcytosines are converted to a base read as a thymine (e.g., uracil, thymine, or dihydrouracil).

[0393] In some embodiments, methylating a cytosine in at least one first complementary strand or second complementary strand comprises contacting the cytosine with a methyltransferase such as DNMT1 or DNMT5. In such embodiments, the step of oxidizing a 5-hydroxymethylated cytosine to 5-formylcytosine (such as by contacting the 5 -hydroxymethyl cytosine in a first strand and a second strand with KRuC ) can be optional.

[0394] In some embodiments, converting the modified cytosine in at least one first or second strand to a thymine or a base read as thymine comprises oxidizing a hydroxymethyl cytosine, e.g., the hydroxymethyl cytosine is oxidized to formylcytosine. In some embodiments, oxidizing the hydroxymethyl cytosine to formylcytosine comprises contacting the hydroxymethyl cytosine with a ruthenate, such as potassium ruthenate (KRuCU).Attorney Docket No.: GH0253WO

[0395] In some embodiments, the modified cytosine is converted to thymine, uracil, or dihydrouracil. In any such embodiments, amplification methods may comprise uracil- and / or dihydrouracil-tolerant amplification methods, such as PCR using a uracil- and / or dihydrouracil-tolerant DNA polymerase.

[0396] In some embodiments, the method comprises converting a formylcytosine and / or a methylcytosine to carboxylcytosine as part of converting the modified cytosine in at least one first or second strand to a thymine or a base read as thymine. For example, converting the formylcytosine and / or the methylcytosine to carboxylcytosine can comprise contacting the formylcytosine and / or the methylcytosine with a TET enzyme, such as TET1, TET2, or TET3. In some embodiments, the method comprises reducing the carboxylcytosine as part of converting the modified cytosine in at least one first or second strand to a thymine or a base read as thymine, and / or the carboxylcytosine is reduced to dihydrouracil. In some embodiments, reducing the carboxylcytosine comprises contacting the carboxylcytosine with a borane or borohydride reducing agent.

[0397] In some embodiments, the borane or borohydride reducing agent comprises pyridine borane, 2-picoline borane, borane, tert-butylamine borane, ammonia borane, sodium borohydride, sodium cyanoborohydride (NaBHsCN), lithium borohydride (LiBEU), ethylenediamine borane, dimethylamine borane, sodium triacetoxyborohydride, morpholine borane, 4-methylmorpholine borane, trimethylamine borane, dicyclohexylamine borane, or a salt thereof. In other embodiments, the reducing agent comprises lithium aluminum hydride, sodium amalgam, amalgam, sulfur dioxide, dithionate, thiosulfate, iodide, hydrogen peroxide, hydrazine, diisobutylaluminum hydride, oxalic acid, carbon monoxide, cyanide, ascorbic acid, formic acid, dithiothreitol, beta-mercaptoethanol, or any combination thereof.

[0398] As discussed above, in some embodiments, a TET protein can be used to convert 5mC and optionally 5hmC (but not unmodified C) into substrates (e.g., 5caC) that cannot be deaminated by a deaminase, and then a deaminase (e.g., APOBEC3 A) can be used to deaminate unmodified cytosines, converting them to uracils. Various TET enzymes may be used in the disclosed methods as appropriate. In some embodiments, the one or more TET enzymes comprise TETv. TETv is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 1 therein. In some embodiments, theAttorney Docket No.: GH0253WO one or more TET enzymes comprise TETcd. TETcd is described in US Patent 10,260,088 and its sequence is SEQ ID NO: 3 therein. In some embodiments, the one or more TET enzymes comprise TET1. In some embodiments, the one or more TET enzymes comprise TET2. TET2 may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker as described, e.g., in US Patent 10,961,525. In some embodiments, the one or more TET enzymes comprise TET1 and TET2. In some embodiments, the one or more TET enzymes comprise a T1372 TET mutant, such as T1372S. In some embodiments, the one or more TET enzymes comprise a VI 900 TET mutant, such as a V1900A, V1900C, V1900G, VI 9001, or V1900P TET mutant. In some embodiments, the one or more TET enzymes comprise a VI 900 TET2 mutant, such as a V1900A, V1900C, V1900G, VI 9001, or V1900P TET2 mutant. It can be beneficial to use a TET enzyme that maximizes formation of 5-carboxylcytosine (5-caC) relative to less oxidized modified cytosines, particularly 5 -formylcytosine, because 5-caC is not a substrate for enzymatic deamination, e.g., by APOBEC enzymes such as APOBEC3 A. Maximizing formation of 5-caC thus reduces the risk of false calls in which a base is identified as unmethylated because it underwent deamination even though it was methylated (or hydroxymethylated) in the original sample. Accordingly, in some embodiments, the TET enzyme comprises a mutation that increases formation of 5-caC. Exemplary mutations are set forth above. “A mutation that increases formation of 5-caC” means that the TET enzyme having the mutation produces more 5-caC than a TET enzyme that lacks the mutation but is otherwise identical. 5-caC production can be measured as described, e.g., in Liu et al., Nat Chem Biol 13: 181-187 (2017) (see Online Methods section, TET reactions in vitro subsection, “driving” conditions). Any variants and / or mutants described in Liu et al. (2017) can be used in the disclosed methods as appropriate.

[0399] In some embodiments, the one or more TET enzymes comprise a TET2 enzyme comprising a T1372S mutation, such as TET2-CS-T1372S and TET2-CD- T1372S. A TET2 comprising a T1372S mutation is described in US Patent 10,961,525 and may be expressed and used as a fragment comprising TET2 residues 1129-1480 joined to TET2 residues 1844-1936 by a linker. Position 1372 of TET2 corresponds to position 258 of SEQ ID NO: 21 (wild type TET2 catalytic domain) of US PatentAttorney Docket No.: GH0253WO10,961,525. Thus, the sequence of a T1372S TET2 catalytic domain may be obtained by changing the threonine at position 258 of SEQ ID NO: 21 of US Patent 10,961,525 to serine. TET2 comprising a T1372S mutation is also described in Liu et al., Nat Chem Biol. 2017 February; 13(2): 181-187. As demonstrated in Liu et al., TET2 comprising a T1372S mutation can more efficiently oxidize 5mC to produce 5-carboxylcytosine (5- caC) than other versions of TET2 such as TET2 lacking a T1372S mutation.

[0400] In some embodiments, the deaminase is thermally inactivated after contacting nucleic acids (e.g., DNA such as cfDNA) with the deaminase. In some embodiments, the thermal inactivation comprises heating or cooling of the deaminase to a temperature at which the deaminase has reduced or inhibited activity relative to a deaminase that has not been subjected to heating or cooling. In some embodiments, the thermal inactivation completely inhibits the activity of the deaminase or reduces the activity of the deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, about 99%, or 100% relative to a deaminase that has not been subjected to heating or cooling.G. Detection of somatic mutations

[0401] In some embodiments, the present disclosure provides methods comprising detecting somatic mutations of nucleic acids. In some embodiments, the second assay detects somatic mutations within the nucleic acid population. Somatic mutations can include single nucleotide variants (SNVs); insertions / deletions (indels); copy number variations (CNVs); structural rearrangements, such as gene fusions. In some embodiments, the somatic mutations comprise SNVs. In some embodiments, the nucleic acid population undergoes amplification prior to or subsequent to the partitioning step to produce progeny nucleic acids. Sequence reads of the resulting progeny nucleic acids within the second partition can be grouped according to their shared parent nucleic acid, and a consensus sequence for the parent nucleic acid can be determined. The consensus sequence of the parent nucleic acids can be used to identify somatic mutations of the nucleic acids, e.g. by alignment to a reference sequence.

[0402] Parent nucleic acids of the sample may be tagged with sample indexes, partition tags and / or molecular barcodes (referred to generally as “tags”). Tags can form part of an adapter. In some embodiments, the addition of tags (e.g. sample indexes,Attorney Docket No.: GH0253WO partition and / or sub-partition tags and / or molecular barcodes) to parent nucleic acids can be done through amplification, wherein the tags are comprised in primers used for amplification. In these embodiments, tags, such as molecular barcodes, are generally attached to individual nucleic acids such that the combination of the tag and endogenous sequence information of the sample nucleic acid that it is attached to creates a unique sequence that may be used for grouping the sequence reads into families, wherein a family corresponds to sequence reads derived from the same parent nucleic acid. Sequence reads of a family can be used for the determination of a consensus sequence of the parent nucleic acid and identification of somatic mutations within the parent nucleic acids.

[0403] In some embodiments, the nucleic acids are ligated to adapters comprising molecular barcodes. These molecular barcodes (optionally in combination with endogenous sequence information) can then be used for grouping the sequence reads into families, wherein a family corresponds to sequence reads derived from the same parent nucleic acid. The grouped sequence reads can be analyzed, for example, to identify somatic mutations within the sequence of parent nucleic acids.

[0404] Endogenous sequence information which can be used for grouping the sequence reads into families includes the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the parent nucleic acid in the sample, start and stop genomic positions corresponding to the sequence of the parent nucleic acid in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the parent nucleic acids in the sample. In some embodiments, the beginning region comprises the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used for grouping the sequence reads into families, wherein a family corresponds to sequenceAttorney Docket No.: GH0253WO reads derived from the same parent nucleic acid. The grouped sequence reads can be analyzed, for example, to identify somatic mutations within the sequence of parent nucleic acids.

[0405] In some embodiments, each nucleic acid of a sample or subsample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation) to individual nucleic acids such that the combination of the molecular barcode and the sequence of the sample nucleic acid that it is attached to creates a unique sequence that may be used for grouping the sequence reads into families, wherein a family corresponds to sequence reads derived from the same parent nucleic acid. Detection of non-unique molecular barcodes in combination with endogenous sequence information typically allows for the assignment of a unique identity to a particular molecule.

[0406] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos. 2001 / 0053519, 2003 / 0152490, and 2011 / 0160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992. Alternatively, in some embodiments, grouping of sequence reads into families can be performed using only endogenous sequence information (e.g., start and / or stop positions, sub-sequences of one or both ends of a sequence, and / or lengths). The addition of tags (e.g. sample indexes, partition and / or sub-partition tags and / or molecular barcodes) to parent nucleic acids can be done through amplification, wherein the tags are comprised in primers used for amplification.

[0407] In some embodiments, the second assay can comprise targeted amplification of amplicons containing genomic regions of interest. This step can occur prior to or subsequent to the partitioning steps. Targeted amplification can occur in combination with the addition of tags to parent nucleic acids wherein the tags are comprised in primers used for amplification. In some embodiments, the targeted amplification can occur in a separate amplification step. The resulting families correspond to sequenceAttorney Docket No.: GH0253WO reads derived from parent nucleic acids that contain the targeted genomic regions of interest.

[0408] Additionally, or alternatively, the second assay can comprise hybrid capture of nucleic acids comprising genomic regions of interest. Capture may be performed using any suitable approach known in the art. Target capture can involve use of a bait set comprising oligonucleotide baits labeled with a capture group, such as the examples noted below. The probes can have sequences selected to tile across a panel of regions, such as genes. Such bait sets are combined with a sample under conditions that allow hybridization of the target molecules with the baits. Then, captured molecules are isolated using the capture group. For example, a biotin capture group can be captured by bead-based streptavidin. Such methods are further described herein and in, for example, U.S. 9,850,523. The resulting families correspond to sequence reads derived from parent nucleic acids that contain the targeted genomic regions of interest.

[0409] In some embodiments, the second assay can detect somatic mutations of nucleic acids in the second partition wherein the assay can be used to detect disease, for example the second assay can be used for the detection of cancer. The assay can be used for the detection of known somatic mutations associated with disease, particularly those associated with cancer.H. Molecular Tagging

[0410] In some embodiments, the nucleic acid molecules (e.g., DNA such as cfDNA) of the sample may be tagged with sample indexes and / or molecular barcodes (referred to generally as “tags”). In some embodiments, the nucleic acid molecules (e.g., DNA such as cfDNA) of the sample comprise barcodes.

[0411] Tags can be molecules, such as nucleic acids, containing information that indicates a feature of the molecule with which the tag is associated. For example, molecules can bear a sample tag or sample index (which distinguishes molecules in one sample from those in a different sample), a partition tag (which distinguishes molecules in one partition from those in a different partition) and / or a molecular barcode (which distinguishes different molecules from one another (in both unique and non-unique tagging scenarios). In certain embodiments, a tag can comprise one or a combination of barcodes.Attorney Docket No.: GH0253WO

[0412] Tagging strategies can be divided into unique tagging and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample bear a different tag, so that reads can be assigned to original molecules based on tag information alone. Tags used in such methods are sometimes referred to as “unique tags”. In non-unique tagging, different molecules in the same sample can bear the same tag, so that other information in addition to tag information is used to assign a sequence read to an original molecule. Such information may include start and stop coordinate, coordinate to which the molecule maps, start or stop coordinate alone, etc. Tags used in such methods are sometimes referred to as “non-unique tags”. Accordingly, it is not necessary to uniquely tag every molecule in a sample. It suffices to uniquely tag molecules falling within an identifiable class within a sample. Thus, molecules in different identifiable families can bear the same tag without loss of information about the identity of the tagged molecule.

[0413] Optionally, adapters may contain a partition-specific barcode and / or a molecular barcode. As used herein, the term “barcode” refers to a nucleic acid molecule having a particular nucleotide sequence, or to the nucleotide sequence, itself, depending on context. A barcode can have, for example, between 10 and 100 nucleotides. A collection of barcodes can have degenerate sequences or can have sequences having a certain Hamming distance, as desired for the specific purpose. So, for example, a molecular barcode can be comprised of one barcode or a combination of two barcodes, each attached to different ends of a molecule. Additionally, or alternatively, for different partitions, different sets of molecular barcodes can be used such that the barcodes serve as a molecular tag through their individual sequences and also serve to identify the partition to which they correspond based the set of which they are a member. For example, barcodes can be used to allow the origin of the DNA (e.g., the subject, biological sample (e.g., samples collected at various time points), enriched DNA sample (e.g., enriched DNA comprising an epigenetic target region set or enriched DNA comprising a sequence-variable target region set), partition, or similar) to be identified, e.g., following pooling of a plurality of samples for parallel sequencing. Tags comprising barcodes can be incorporated into or otherwise joined to adapters. Tags can be incorporated by ligation, overlap extension PCR among other methods. Tags can be used to label the individual polynucleotide population partitions so as to correlate theAttorney Docket No.: GH0253WO tag (or tags) with a specific partition. Alternatively, tags can be used in embodiments of the disclosure that do not employ a partitioning step. In some embodiments, a single tag can be used to label a specific partition. In some embodiments, multiple different tags can be used to label a specific partition. In embodiments employing multiple different tags to label a specific partition, the set of tags used to label one partition can be readily differentiated for the set of tags used to label other partitions. In some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations, for example as in Kinde et al., Proc Nat’l Acad Sci USA 108: 9530-9535 (2011), Kou et al., PLoS ONE,11 : e0146638 (2016)) or used as non-unique molecule identifiers, for example as described in US Pat. No. 9,598,731. Similarly, in some embodiments, the tags may have additional functions, for example the tags can be used to index sample sources or used as non-unique molecular identifiers (which can be used to improve the quality of sequencing data by differentiating sequencing errors from mutations).

[0414] Tags may be incorporated into or otherwise joined to adapters by chemical synthesis, ligation (e.g., as described above, e.g. by blunt-end ligation or sticky-end ligation), or overlap extension polymerase chain reaction (PCR), among other methods. Such adapters are ultimately joined to the parent nucleic acids. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce sample indexes to a nucleic acid using conventional nucleic acid amplification methods. The amplifications may be conducted in one or more reaction mixtures (e.g., a plurality of microwells in an array). Molecular barcodes, partition tags and / or sample indexes may be introduced simultaneously, or in any sequential order. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after a partitioning procedure. In some embodiments, molecular barcodes and / or sample indexes are introduced prior to and / or after sequence capturing steps, if present, are performed. In some embodiments, only the molecular barcodes are introduced prior to probe capturing and the sample indexes are introduced after sequence capturing steps are performed. In some embodiments, both the molecular barcodes and the sample indexes are introduced prior to performing probe-based sequence capturing steps, if present. In some embodiments, the sample indexes are introduced after sequenceAttorney Docket No.: GH0253WO capturing steps are performed, if present. In some embodiments, sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).

[0415] In some embodiments, the tags may be located at one end or at both ends of the sample nucleic acids. In some embodiments, tags are predetermined or random or semi-random sequences. In some embodiments, the tag(s) may together be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, or 5 nucleotides in length. Typically, tags are about 5 to 20 or 6 to 15 nucleotides in length. The tags may be linked to sample nucleic acids randomly or non-randomly.

[0416] In some embodiments, each sample is distinctly tagged with a sample index or a combination of sample indexes. In some examples, when multiple partitions are subsequently processed after the partitioning step, each partition can be distinctly tagged with a partition tag or a combination of partition tags. In some embodiments, each nucleic acid of a sample or subsample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, a plurality of molecular barcodes may be used such that molecular barcodes are not necessarily unique to one another in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are generally attached (e.g., by ligation) to individual nucleic acids such that the combination of the molecular barcode and the sequence of the sample nucleic acid that it is attached to creates a unique sequence that may be used for grouping the sequence reads into families, wherein a family corresponds to sequence reads derived from the same parent nucleic acid. Detection of non-unique molecular barcodes in combination with endogenous sequence information (e.g., the beginning (start) and / or end (stop) genomic location / position corresponding to the sequence of the original nucleic acid molecule in the sample, start and stop genomic positions corresponding to the sequence of the original nucleic acid molecule in the sample, the beginning (start) and / or end (stop) genomic location / position of the sequence read that is mapped to the reference sequence, start and stop genomic positions of the sequence read that is mapped to the reference sequence, sub-sequences of sequence reads at one or both ends, length of sequence reads, and / or length of the original nucleic acid molecule in the sample) typically allows for the assignment of a unique identity to a particular molecule. In some embodiments, beginning region comprises the first 1, first 2, the first 5, the first 10, the first 15, the first 20, the first 25, the first 30 or at least theAttorney Docket No.: GH0253WO first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the end region comprises the last 1, last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30 or at least the last 30 base positions at the 3' end of the sequencing read that align to the reference sequence. The length, or number of base pairs, of an individual sequence read are also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand, and / or a complementary strand.

[0417] In certain embodiments of non-unique tagging, the number of different tags used can be sufficient that there is a very high likelihood (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999% that all DNA molecules of a particular group bear a different tag. It is to be noted that when barcodes are used as tags, and when barcodes are attached, e.g., randomly, to both ends of a molecule, the combination of barcodes, together, can constitute a tag. This number, in term, is a function of the number of molecules falling into the calls. For example, the class may be all molecules mapping to the same start-stop position on a reference genome. The class may be all molecules mapping across a particular genetic locus, e.g., a particular base or a particular region (e.g., up to 100 bases or a gene or an exon of a gene).

[0418] In certain embodiments, the number of different tags used to uniquely identify a number of molecules, z, in a class can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, 9*z, 10*z, 11 *z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z or 100*z (e.g., lower limit) and any of 100,000*z, 10,000*z, 1000*z or 100*z (e.g., upper limit). In some embodiments, molecular barcodes are introduced at an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. One example format uses from about 2 to about 1,000,000 different molecular barcode sequences, or from about 5 to about 150 different molecular barcode sequences, or from about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule. Alternatively, from about 25 to about 1,000,000 different molecular barcode sequences may be used. For example, 20- 50 x 20-50 molecular barcode sequences (i.e., one of the 20-50 different molecular barcode sequences can be attached to each end of the parent nucleic acids) can be used.Attorney Docket No.: GH0253WOSuch numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of molecules have the same combinations of molecular barcodes. For example, in a sample of about 5 ng to 30 ng of cell free DNA, one expects around 3000 molecules to map to a particular nucleotide coordinate, and between about 3 and 10 molecules having any start coordinate to share the same stop coordinate. Accordingly, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can suffice to uniquely tag all such molecules. To uniquely tag all 3000 molecules mapping across a nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0419] In some embodiments, the assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is hereby incorporated by reference in its entirety. Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only endogenous sequence information (e.g., start and / or stop positions, sub-sequences of one or both ends of a sequence, and / or lengths). Tags can be linked to sample nucleic acids randomly or non-randomly.

[0420] In some embodiments, the assignment of unique molecular barcodes in reactions is performed using methods and systems described in Lim et al., Communications Biology. (2025)8: 1098, e.g., SPIDER-seq. In some such embodiments, amplicons are tagged with a pair of two unique molecular barcodes using primers that contain a barcode. Successive daughter strands synthesized through each round of PCR amplification are grouped into clusters (e.g., peer-to peer networks, as illustrated in Fig. 1c of Lim et al.) based on a chain of common unique barcodes between immediate parent and daughter strands. That is, strand synthesis events (with a synthesized strand as a template) involve copying one barcode from the template and include one new barcode from the primer, so each daughter strand shares a unique barcode with its parent. By clustering strands in this way, a consensus can be generated that reduces errors.Attorney Docket No.: GH0253WO

[0421] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, the unique tags may be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the unique tags may be loaded so that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags are loaded per genome sample. In some cases, the average number of unique tags loaded per sample genome is less than, or greater than, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000 or 1,000,000,000 unique tags per genome sample.

[0422] In some embodiments a format uses 20-50 different tags (e.g., barcodes) ligated to both ends of target nucleic acids. For example, 35 different tags (e.g., barcodes) ligated to both ends of target molecules creating 35 x 35 permutations, which equals 1225 for 35 tags. Such numbers of tags are sufficient so that different molecules having the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different combinations of tags. Other barcode combinations include any number between 10 and 500, e.g., about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0423] In some cases, unique tags may be predetermined or random or semirandom sequence oligonucleotides. In other cases, a plurality of barcodes may be used such that barcodes are not necessarily unique to one another in the plurality. In this example, barcodes may be ligated to individual molecules such that the combination of the barcode and the sequence it may be ligated to creates a unique sequence that may be individually tracked. As described herein, detection of non-unique barcodes in combination with sequence data of beginning (start) and end (stop) portions of sequence reads may allow assignment of a unique identity to a particular molecule. The length or number of base pairs, of an individual sequence read may also be used to assign a unique identity to such a molecule. As described herein, fragments from a single strandAttorney Docket No.: GH0253WO of nucleic acid having been assigned a unique identity, may thereby permit subsequent identification of fragments from the parent strand.

[0424] In some embodiments, the method includes adding one or more internal control DNAs and forward and reverse primers for amplifying the internal control DNAs. The internal control DNAs may be added before amplification using the primers that anneal upstream and downstream of the rearrangement breakpoints. The forward and reverse primers for amplifying the internal control DNAs may be included with, or added at the same time as, the primers that anneal upstream and downstream of the rearrangement breakpoints. The internal control DNAs may comprise or consist of sequences that do not occur in the genome of the subject, or that do not occur in the genome of the species of which the subject is a member (e.g., the human genome). The forward and / or reverse primers for amplifying the internal control DNAs may comprise sequences that are not complementary to any sequence in the genome of the subject, e.g., the human genome. The internal control DNAs may be used to ensure that the amplification process proceeded as designed. As such, the method may comprise detecting (e.g., sequencing) molecules amplified from and / or captured by the one or more internal control DNAs. The method can comprise comparing an amount of internal control DNAs (e.g., number of molecules or reads detected that correspond to an internal control DNA sequence) to a predetermined threshold, and either rejecting sequencing results if the predetermined threshold is not met or accepting sequencing results if the predetermined threshold is met. The predetermined threshold may be established, e.g., based on historical data or by testing the method on samples of DNA from test subjects, such as healthy volunteers. For example, amplification and detection of the one or more internal control DNAs provides confirmation that the amplification process proceeded properly, thus reducing the likelihood of a false negative.I. Sample and Subjects

[0425] The present disclosure relates to methods of analyzing a nucleic acid population comprising nucleic acids of different lengths, e.g. DNA in a sample such as cfDNA. In some cases, the nucleic acid is obtained or has been obtained from a subject. In some embodiments, the nucleic acid sample may comprise or consist of nucleic acids (e.g. DNA) from a biological sample obtained from a subject. The subject may be aAttorney Docket No.: GH0253WO human, a mammal, an animal, a primate, rodent (including mice and rats), or other common laboratory, domestic, companion, service or agricultural animal, for example a rabbit, dog, cat, horse, cow, sheep, goat or pig. The subject may in some cases have or be suspected of having a cancer, tumor or neoplasm. In other cases, the subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission, e.g. from a tumor, cancer, or neoplasia (e.g., following treatment such as chemotherapy, surgical resection, radiation, or a combination thereof). The subject may or may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutations / disorders. In some embodiments, the sample is a polynucleotide sample obtained from a tumor tissue biopsy. The cancer, tumor or neoplasm may generally be of any type, for example a cancer tumor or neoplasm of the lung, colon, rectal (or colorectal), kidney, breast, prostate, or liver, or other type of cancer as described herein. In any of the foregoing embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia may be of the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the lung. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre- cancer, cancer, tumor, or neoplasia is of the colon or rectum. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the breast. In some embodiments, the pre-cancer, cancer, tumor, or neoplasia or suspected pre-cancer, cancer, tumor, or neoplasia is of the prostate. In any of the foregoing embodiments, the subject may be a human subject. In some embodiments, the sample is obtained from a subject having a stage I cancer, stage II cancer, stage III cancer or stage IV cancer.

[0426] In some embodiments, the subject may have an infection, a transplant rejection, or other disease or disorder related to changes in the immune system. The subject may not have cancer or a detectable cancer symptom. The subject may have been treated with one or more cancer therapy, e.g., any one or more of chemotherapies, antibodies, vaccines or biologies. The subject may be in remission. The subject may orAttorney Docket No.: GH0253WO may not be diagnosed as being susceptible to cancer or any cancer-associated genetic mutati ons / di sorder s .

[0427] The sample can be any biological sample isolated from a subject. The sample can be a bodily sample. Samples can include body tissues, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine. Samples are preferably body fluids, particularly blood and fractions thereof, and urine. A sample can be in the form originally isolated from a subject or can have been subjected to further processing to remove or add components, such as cells, or enrich for one component relative to another.

[0428] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis.

[0429] In some embodiments, a population of nucleic acids is obtained from a serum, plasma or blood sample from a subject having or suspected of having neoplasia, a tumor, precancer, or cancer or previously diagnosed with neoplasia, a tumor, precancer, or cancer. The population includes nucleic acids having varying levels of sequence variation and / or epigenetic variation, such as post-translation modifications (PTMs) of chromatin and / or nucleobase modifications, e.g., modifications of cytosine, particularly at the 5-position of the nucleobase, e.g., 5 -methylcytosine, 5- hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

[0430] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis.

[0431] In a particular embodiment, the DNA sample comprises cell-free DNA. In another particular embodiment the DNA sample is a DNA sample from a formalin fixed paraffin embedded (FFPE) sample.Attorney Docket No.: GH0253WO

[0432] The sample may be plasma. The volume of plasma can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For example, the volume can be 0.5 ml, 1 ml, 5 ml, 10 ml, 20 ml, 30 ml, or 40 ml. A volume of sampled plasma may be 5 to 20 ml. In some embodiments, the sample volume is 3-5 mL of plasma, such as 4 mL of plasma, per 10 mL whole blood.

[0433] In some embodiments, the sample comprises whole blood. Exemplary volumes of sampled whole blood are 0.4-40 mL, 5-20 mL, 10-20 mL, 1-6 mL, 1-3 mL, and 3-5 mL. For example, the volume can be 0.5 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled whole blood may be 5 to 20 mL. In some embodiments, the sample volume is 1-5 mL of whole blood, such as 2.5 mL of whole blood.

[0434] In some embodiments, the sample comprises buffy coat separated from whole blood. Exemplary volumes of sampled buffy coat are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled buffy coat may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of buffy coat, such as 0.3 mL of buffy coat, per 10 mL whole blood.

[0435] In some embodiments, the sample comprises PBMCs separated from whole blood. Exemplary volumes of sampled PBMCs are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2- 0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL 10 mL, or 20 mL. A volume of sampled PBMCs may be 1 to 10 mL. In some embodiments, the sample volume is 0.1-0.5 mL of PBMCs, such as 0.3 mL of PBMCs, per 10 mL whole blood.

[0436] In some embodiments, the sample comprises leukocytes separated from subject blood using leukapheresis. Exemplary volumes of sampled leukocytes from leukapheresis are 0.1-20 mL, 1-10 mL, 1-5 mL, 0.2-0.6 mL, and 0.3-0.5 mL. For example, the volume can be 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, or 20 mL. A volume of sampled leukocytes from leukapheresis may be 1 to 10 mL. In some embodiments, theAttorney Docket No.: GH0253WO sample volume is 0.1-0.6 mL of leukocytes from leukapheresis, such as 0.4 mL of leukocytes, per 10 mL whole blood.

[0437] A sample can comprise various amount of nucleic acid that contains genome equivalents. For example, a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cell free DNA (cfDNA), about 200 billion (2xlOn) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion (6 x 1011) individual molecules.

[0438] A sample can comprise nucleic acids from different sources, e.g., cellular DNA and cell-free DNA of the same subject, or cellular DNA and cell-free DNA of different subjects. A sample can comprise nucleic acids carrying mutations. For example, a sample can comprise DNA carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations existing in germline DNA of a subject. Somatic mutations refer to mutations originating in somatic cells of a subject, e.g., cancer cells. A sample can comprise DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). A sample can comprise an epigenetic variant (i.e. a chemical or protein modification), wherein the epigenetic variant associated with the presence of a genetic variant such as a cancer-associated mutation. In some embodiments, the sample comprises an epigenetic variant associated with the presence of a genetic variant, wherein the sample does not comprise the genetic variant.

[0439] The sample may comprise cell free nucleic acids, such as cfDNA. The cfDNA may be obtained from a test subject, for example as described above. For example, the sample for analysis may be plasma or serum containing cell-free nucleic acids. “Cell-free DNA” “cfDNA molecules,” or “cfDNA”, for example, include DNA molecules that naturally occur in a subject in extracellular form (e.g., in blood, serum, plasma, or other bodily fluids such as lymph, cerebrospinal fluid, urine, or sputum). While the cfDNA originally existed in a cell or cells in a large complex biological organism, e.g., a mammal, it has undergone release from the cell(s) in vivo into a fluid found in the organism, and may be obtained by obtaining a sample of the fluid without the need to perform an in vitro cell lysis step. In other words, cell-free nucleic acids or cfDNA are nucleic acids or cfDNA not contained within or otherwise bound to a cell. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, including cfDNAAttorney Docket No.: GH0253WO derived from genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or a hybrid thereof. A cell-free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluid from cancer cells e.g., circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell free nucleic acids are produced by tumor cells. In some embodiments, cell free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.

[0440] Exemplary amounts of cell-free nucleic acids in a sample before amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acids. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acids. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acids. The method can comprise obtaining 1 femtogram (fg) to 200 ng cell-free nucleic acids from samples.

[0441] Cell-free nucleic acids have an exemplary size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides.

[0442] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. Fractionation may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together.Generally, after addition of buffers and wash steps, nucleic acids can be precipitatedAttorney Docket No.: GH0253WO with an alcohol. Further clean up steps may be used such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, such as Cl DNA, DNA or protein for hybridization may be added throughout the reaction to optimize certain aspects of the procedure such as yield.

[0443] After such processing, samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA and single stranded RNA. In some embodiments, single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps.

[0444] In some embodiments, the nucleic acid sample is obtained using a sample preparation method optimized for extracting longer nucleic acids. Off the shelf kits for the extraction of nucleic acids from a sample often favor shorter nucleic acids. To maximize the nucleic acids in the partitions comprising longer nucleic acids, and hence maximize the information such partitions can provide, extraction methods need to be optimized to increase the average length of nucleic acid extracted.

[0445] Methods for optimizing sample preparation for extracting longer nucleic acids are well known in the art. Commercially available DNA extraction kits can be modified to provide a workflow that results in an increase in average nucleic acid length. Known methods for extracting longer nucleic acids include, but are not limited to, phenol-chloroform nucleic acid extraction, gravity-flow based filtration methods and solid-phase reversible immobilization methods (SPRI). Developments in long read sequencing methods has led to an increase in commercially available kits for the extraction of high molecular weight (HMW) nucleic acids. Examples of commercial kit for the extraction of HMW nucleic acids include, but are not limited to, Quick-DNA HMW MagBead (Zymo Research), ZymoBiomics DNA Miniprep (Zymo Research) or DNAexpress (Claremont Bio).

[0446] The methods disclosed herein are also particularly suited for the analysis of DNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. While the formalin fixation process adequately preserves the ultrastructure of the tissues, it results in various types of damage to the DNA within the tissues, such as nicks in the DNA. As explained elsewhere herein, these nicks can lead to synthesis of regions of the DNA molecule in the end repair process. The methods disclosed herein allow for these regions to be identified and the sequence data to be interpreted accordingly.Attorney Docket No.: GH0253WO

[0447] Reference or control molecules can be added to or spiked into a sample as a control or normalization standard. For example, a certain amount of modified DNA from a species other than the species of the subject from which the sample was obtained or synthetic nucleic acids comprising certain modifications may be added to the sample. In some embodiments, the reference or control molecules are distinguishable from the molecules originally present in the sample. In some embodiments, the detected DNA sequences are normalized to the reference or control molecules.J. End repair and A-tailing

[0448] In some embodiments, the disclosed methods comprise subjecting nucleic acids in a sample (e.g., DNA such as cell-free DNA (cfDNA)) to end repair to generate end-repaired nucleic acid molecules (e.g., end-repaired DNA molecules). In some embodiments, the methods comprise subjecting the nucleic acids in a sample (e.g., DNA such as cell-free DNA (cfDNA)) to end repair to generate end-repaired nucleic acid molecules (e.g., end-repaired DNA such as end-repaired cfDNA). In some embodiments, the end repair is performed prior to separating (e.g., partitioning) the nucleic acids in a sample (e.g., DNA such as cell-free DNA (cfDNA)) into a plurality of subsamples. In some embodiments, the end repair is performed prior to enriching the nucleic acids (e.g., DNA such as cell-free DNA (cfDNA)) for long DNA (e.g., at least 180 bp in length) and / or for short DNA (e.g., less than 180 bp). In some embodiments, the end repair is performed after separating (e.g., partitioning) the nucleic acids (e.g., DNA such as cfDNA) into a plurality of subsamples, such as the first partition and the second partition. In some embodiments, the end repair is performed prior to sequencing at least a portion of the nucleic acids (e.g., DNA such as cfDNA).

[0449] End repair refers to methods for repairing DNA by the conversion of nonblunt ended DNA into blunt ended DNA. Sequencing workflows typically use end repair to make ends of DNA molecules compatible with adapters, which are subsequently ligated onto the DNA. Fragmented and / or damaged DNA (e.g. cfDNA or DNA from FFPE samples) often contain non-blunt ends, which contain 3 ’overhangs and / or 5 ’overhangs. A 3 ’overhang refers to the 3’ end of a DNA strand which extends beyond the 5 ’end of the paired strand, resulting in one or more unpaired nucleotides at the 3 ’end of the DNA strand. Conversely, a 5 ’overhang refers to the 5’ end of a DNAAttorney Docket No.: GH0253WO strand which extends beyond the 3 ’end of the paired strand, resulting in one or more unpaired nucleotides at the 5 ’end of the DNA strand.

[0450] The process of end repair involves the conversion of double-stranded DNA with 3 ’overhangs and / or 5 ’overhangs to double-stranded DNA without overhangs. This can be done using one or more enzymes such as T4 DNA polymerase and / or Klenow fragment. The 3’ to 5’ exonuclease activity of these enzymes removes the 3 ’ends at 3 ’overhangs and the 5’ to 3’ polymerase activity of these enzymes extends the 3’ ends at 5’ overhangs to remove the 5’ overhang, thereby generating a blunt-ended DNA molecule. In order to fill in these 5’ overhangs, end repair is conducted in the presence of dATP, dCTP, dGTP and dTTP. In order to fill in these 5’ overhangs, end repair is conducted in the presence of dNTPs. End repair can also include a second step, which involves the addition of a phosphate group to the 5' ends of DNA, by an enzyme such as polynucleotide kinase. This makes the 5 ’ends of the end-repaired DNA molecules compatible with the subsequent action of DNA polymerases and DNA ligases.

[0451] As used herein, the term “A-tailing” refers to the addition of a single deoxyadenosine residue to the end of a blunt-ended double-stranded DNA fragment to form a 3' deoxyadenosine single-base overhang. Such A tailing reactions are conducted with polymerases which have the ability to add a non-templated A to the 3' end of a blunt, double-stranded DNA molecule. Polymerases capable of A-tailing typically do not possess 3’-5’ exonuclease activity. When A-tailing is performed as a separate reaction to end repair, it is typically conducted in the presence of dATP, but the absence of dCTP, dTTP and dGTP. A-tailed fragments are not compatible for self-ligation (i.e., self-circularizatian and concantenation of the DNA), but they are compatible with 3' T- overhangs, which can be used on adapters. Methods comprising end repair, A-tailing and ligation to adapters with 3' T-overhangs can result in higher efficiency ligation, compared to blunt ended ligation, as blunt ligation can lead to self-ligation of the adapters and / or DNA molecules.

[0452] In some embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by blunt end ligation of adapters. In other embodiments, the methods disclosed herein comprise end repair of the DNA molecules followed by A- tailing and sticky-end ligation of T-tailed adapters. When the methods disclosed herein comprise an A-tailing step, it may be performed separately from the end repair with anAttorney Docket No.: GH0253WO intervening reaction clean-up step or it may be performed in the same reaction as the end repair (e.g. using NEBNext® Ultra™ II End Repair / dA-Tailing Module (E7546)). In some embodiments, the reaction clean-up step removes unincorporated dNTPs. In instances wherein the A-tailing reaction is performed in the same reaction as end repair, the sticky-end ligation may be performed with a mixture of T-tailed adapters and C- tailed adapters.

[0453] End repair and A tailing reactions can have varying impacts on the composition of the DNA molecule, dependent on the exact workflow and reaction components used. These reactions can lead to the synthesis of regions at the 3 ’ends of DNA strands, but also the synthesis of internal regions through nick translation and through gap filling followed by ligation.

[0454] In some embodiments, end repair can lead to 3 ’fill in with unmethylated cytosines, which may not reflect the true methylation status of that position in the DNA molecule prior to the generation of the 5 ’overhang. In nicked DNA, polymerases which contain 5’ to 3’ exonuclease activity and / or strand displacement activity can lead to the synthesis of internal regions of the DNA molecule through nick translation. If the end repair reaction is conducted with non-methylated deoxy cytidine triphosphate (dCTP), the synthesized regions will incorporate the non-methylated dCTP, potentially at positions which initially comprised methylated cytosines. In gapped DNA, both the DNA polymerases used in end repair and A tailing can lead to the generation of synthesized regions. The gaps can be filled in with DNA polymerases used in the end repair reaction, regardless of whether they possess 5’ to 3’ exonuclease activity or strand displacement activity. After this gap filling, a nick will still exist between the synthesized region and the region of the original DNA molecule 3’ of the gap. The A- tailing enzymes may then introduce further synthesized regions through nick translation, as described for the nicked DNA. This synthesized region may extend to the 3 ’end of the DNA molecule.

[0455] In some embodiments, the end-repair and the A-tailing reactions are performed in a single tube. In such cases, the A tailing reaction can be performed at a higher temperature than the end repair. Optionally, end repair is performed at ambient temperature (e.g. 15-35°C) and A tailing is performed at a temperature over 60°C, including e.g., about 60°C-75°C. The A tailing reaction can be performed using aAttorney Docket No.: GH0253WO thermostable polymerase (e.g. Taq DNA polymerase, Tfl DNA polymerase, Bst DNA Polymerase, Large Fragment or Tth DNA polymerase) and the method further comprises increasing temperature of the sample after the end repair to inactivate the polymerase used in end repair (e.g. T4 DNA polymerase or Klenow fragment). In some embodiments the A-tailing is performed using a DNA polymerase that: (i) does not possess 5 ’-3’ exonuclease activity; and / or (ii) is not a strand displacing DNA polymerase. These properties reduce the ability of the DNA polymerase to extend from nick. This reduces the level of synthesis which may occur during the end repair and A- tailing reactions thus reducing the proportion of sequencing data that may be filtered out as potentially containing artifactual data. Accordingly, in some embodiments, the A- tailing is performed using a DNA polymerase that cannot extend from a nick in the DNA such as HemoKlen Taq. In other embodiments, the A-tailing is performed using Taq DNA polymerase. In other embodiments, the A-tailing is performed using Tfl polymerase, Bst DNA Polymerase, Large Fragment or Tth polymerase.

[0456] . In order to reduce the level of synthesized regions, the end repair reaction can be performed using DNA polymerases can be used which lack 5 ’to 3’ exonuclease activity and / or strand displacement activity (e.g. T4 DNA polymerase or Klenow fragment).

[0457] In nicked DNA and gapped DNA, nick translation is reduced in end repair through the use of polymerases which lack 5’to 3’ exonuclease activity and / or strand displacement activity. The separation of the end repair and A tailing reaction by a reaction clean-up means that only dATP (not dCTP, dTTP or dGTP) is present in the A tailing reaction. This means that efficie...

Claims

1. Attorney Docket No.: GH0253WOWhat is claimed is:

1. A method of analyzing a nucleic acid population comprising nucleic acids of different lengths, wherein the method comprises:(a) separating the nucleic acid population into at least two partitions according to nucleic acid length to provide at least a first partition and a second partition, wherein the nucleic acids in the first partition have a higher average length relative to the nucleic acids in the second partition;(b) performing a first assay on the first partition, wherein:(i) the first assay determines active chromatin regions of nucleic acids in the first partition; and / or(ii) the first assay determines the fragmentation profile of nucleic acids in the first partition; and(c) performing a second assay on the second partition, wherein:(i) the second assay determines the modification status of nucleic acids in the second partition; and / or(ii) the second assay detects somatic mutations of nucleic acids in the second partition.

2. The method of the immediately preceding claim, wherein the nucleic acid population is obtained using a sample preparation method optimized for extracting longer nucleic acids.

3. The method of claim 1 or claim 2, wherein the nucleic acid population is partitioned using in vitro size selection.

4. The method of any one of the preceding claims, wherein the nucleic acids in the first partition are at least 180 bp in length.

5. The method of any one of the preceding claims, wherein the nucleic acids in the second partition are less than 180 bp in length.Attorney Docket No.: GH0253WO6. The method of any one of the preceding claims, wherein the separating the nucleic acid population into at least two partitions comprises binding nucleic acids with a higher average length with one or more beads.

7. The method of the immediately preceding claim, wherein the one or more beads comprise a coating of silica and / or carboxyl groups.

8. The method of claim 6 or claim 7, wherein the one or more beads comprise one or more paramagnetic beads.

9. The method of any one of claims 6-8, wherein the one or more beads are in a solution comprising polyethylene glycol and a salt.

10. The method of any one of claims 6-9, wherein the separating the nucleic acid population into at least two partitions comprises solid-phase reversible immobilization (SPRI).

11. The method of any one of claims 1-5, wherein the separating the nucleic acid population into at least two partitions comprises column separation.

12. The method of the immediately preceding claim, wherein the column separation comprises using a silica column.

13. The method of claim 11 or 12, wherein the column separation comprises mixing the nucleic acid population with a buffer.

14. The method of any one of claims 1-5, wherein the separating the nucleic acid population into at least two partitions comprises gel electrophoresis.

15. The method of any one of the preceding claims, wherein the first assay and / or the second assay are untargeted.Attorney Docket No.: GH0253WO16. The method of any one of claims 1-14, wherein the first assay and / or the second assay are targeted such that the first partition and / or the second partition are enriched for a plurality of target genomic regions.

17. The method of the immediately preceding claim, wherein enrichment for target genomic regions is performed using hybrid capture or targeted amplification.

18. The method of any one of the preceding claims, wherein the first assay determines active chromatin regions of nucleic acids in the first partition.

19. The method of the immediately preceding claim, wherein the first assay comprises identifying genomic features within the nucleic acids of the first partition.

20. The method of the immediately preceding claim, wherein the genomic features comprise promoters and / or exons.

21. The method of claim 19 or claim 20, further comprising identifying regions of active transcription using the identified genomic features.

22. The method of any one of claims 19-21, further comprising identifying patterns of gene expression using the identified genomic features.

23. The method of any one of the preceding claims, wherein the first assay comprises sequencing, optionally next-generation sequencing (NGS).

24. The method of any one of claims 1-22, wherein the first assay comprises quantitative PCR (qPCR).

25. The method of any one of the preceding claims, wherein the first assay determines the fragmentation profile of the nucleic acids in the first partition.Attorney Docket No.: GH0253WO26. The method of the immediately preceding claim, wherein the fragmentation profile is generated using the length distribution of nucleic acids in the first partition, optionally wherein the first assay comprises a next generation sequencing assay and the fragmentation profile is generated using data from the next generation sequencing assay.

27. The method of any one of the preceding claims, wherein the second assay determines the modification status of nucleic acids in the second partition.

28. The method of claim 27, wherein the second assay comprises a baseconversion based sequencing method.

29. The method of the immediately preceding claim, wherein the base-conversion based sequencing method comprises bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, enzymatic methyl-seq (EM-seq) conversion, Tet-assisted pyridine borane sequencing (TAPS), APOBEC-coupled epigenetic (ACE) conversion, and / or direct methylation sequencing (DM-seq).

30. The method of the immediately preceding claim, wherein the Tet-assisted conversion further comprises a substituted borane reducing agent, optionally wherein the substituted borane reducing agent comprises 2-picoline borane, borane pyridine, tert-butylamine borane, and / or ammonia borane.

31. The method of claim 29 or claim 30, wherein the base-conversion based sequencing method comprises contacting the nucleic acids with a CpG-specific DNA methyltransferase (MTase) or a CpG-specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a deaminase.

32. The method of the immediately preceding claim, wherein the deaminase is a dsDNA deaminase.

33. The method of claim 31 or 32, wherein the deaminase is a methyl insensitive deaminase.Attorney Docket No.: GH0253WO34. The method of the immediately preceding claim, wherein the methyl insensitive deaminase is an APOBEC enzyme, optionally wherein the APOBEC enzyme is AP0BEC3A.

35. The method of any one of claims 31 or 32, wherein the deaminase is a methyl sensitive deaminase.

36. The method of the immediately preceding claim, wherein the methyl sensitive deaminase is modification-sensitive DNA deaminase A (MsddA) or a modificationsensitive DNA deaminase A (MsddA)-like deaminase.

37. The method of claim 27, wherein the second assay comprises generating subpartitions of nucleic acids according to the modification status of the nucleic acids, wherein at least one of the sub-partitions is enriched for nucleic acids with a modification of interest, wherein generating sub-partitions comprises providing a binding agent that preferentially binds to nucleic acids within the second partition that contain the modification of interest.

38. The method of the immediately preceding claim, further comprising separating the nucleic acids bound to the binding agent from the unbound nucleic acids to produce an unbound sub-partition.

39. The method of claim 37 or claim 38, further comprising eluting nucleic acids bound to the binding agent to provide a sub-partition containing nucleic acids comprising the modification.

40. The method of any one of claims 37-39, wherein bound nucleic acids are subjected to a plurality of conditions which increasingly favor elution of the nucleic acids bound to the binding agent to provide multiple sub-partitions wherein the nucleic acids eluted in the first of the plurality of conditions have a lower extent of the modification compared to the nucleic acids eluted in the last of the plurality of conditions.Attorney Docket No.: GH0253WO41. The method of any one of claims 37-40, wherein the binding agent comprises: (i) a methyl-binding domain (MBD) protein; and / or (ii) an anti-5mC antibody or an antigen-binding fragment thereof.

42. The method of any one of claims 27-41, further comprises quantifying the level of modifications, optionally using quantitative PCR (qPCR).

43. The method of the immediately preceding claim, wherein the quantification is used to provide a modification score.

44. The method of any one of claims 27-43, wherein the modification is a methylated nucleotide.

45. The method of any one of claims 27-44, wherein the modification is 5- methylcytosine (5mC).

46. The method of any one of the preceding claims, wherein the second assay detects somatic mutations within the nucleic acid population.

47. The method of the immediately preceding claim, wherein the second assay comprises hybrid capture of nucleic acids comprising genomic regions of interest.

48. The method of claim 46, wherein the second assay comprises targeted amplification of amplicons containing genomic regions of interest.

49. The method of any one of claims 46-48, wherein the second assay comprises sequencing, optionally next generation sequencing (NGS), or qPCR.

50. The method of any one of the preceding claims, further comprising preparing one or more sequencing libraries from at least a portion of the nucleic acids in the firstAttorney Docket No.: GH0253WO partition and / or the second partition, further comprising sequencing the one or more sequencing libraries.

51. The method of any one of claims 23-50, wherein the NGS comprises pyrosequencing, sequencing-by-synthesis, semiconductor sequencing, sequencing-by- ligation, or sequencing-by-hybridization.

52. The method of any one of claims 23-50, wherein the sequencing comprises nanopore-based sequencing or single-molecule real time (SMRT) sequencing.

53. The method of any one of claims 23-50, wherein the sequencing comprises long-read sequencing.

54. The method of any one of claims 23-50, wherein the sequencing comprises nanopore-based sequencing.

55. The method of any one of claims 23-50, wherein the sequencing comprises 5- letter or 6-letter sequencing.

56. The method of any one of claims 23-50 and 54, wherein the sequencing comprises nanopore-based sequencing and the method further comprises subjecting the nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

57. The method of any one of claims 23-50 and 52, wherein the sequencing comprises single-molecule real time (SMRT) sequencing and the method further comprises subjecting the nucleic acids in the sample to end repair to generate end- repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, and the at least one type of dNTP comprising aAttorney Docket No.: GH0253WO modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

58. The method of claim 56 or claim 57, wherein the modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, dUTP, a dNTP comprising fluorodeoxyuridine (FldU), a dNTP comprising 5-iododeoxyuridine (IdU), a dNTP comprising 5-ethynyldeoxyuridine (EdU), and / or a dNTP comprising 8oxoG.

59. The method of any one of claims 56-58, further comprising subjecting the nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is other than 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

60. The method of any one of claims 56-58, further comprising subjecting nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, and the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

61. The method of any one of claims 56-58, further comprising subjecting the nucleic acids in the sample to end repair to generate end-repaired DNA molecules, wherein the end repair is performed using at least one type of dNTP which comprises a modified base, wherein the modified base is a methylated cytosine, optionally wherein the methylated base is 5mC or 5hmC, wherein the at least one type of dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations, and the repaired region is defined as:Attorney Docket No.: GH0253WO(i) the sequence between two non-methylated cytosines which span one or more methylated CpH cytosines; and / or(ii) the sequence between a methylated CpH cytosine and an end of a sequence read, wherein the methylated CpH cytosine is the CpH cytosine most distant from the end of the sequence read, or a subsequence thereof comprising one or more methylated CpH cytosines.

62. The method of any one of claims 56-61, further comprising analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations present in the nucleic acids in the sample.

63. The method of any one of claims 56-62, wherein the method further comprises detecting the methylation status of cytosines in the nucleic acids in the sample, and wherein the analyzing the sequence data further comprises filtering out the one or more repaired regions of the end-repaired DNA molecules such that the one or more repaired regions are not used to determine the methylation status of cytosines in the nucleic acids in the sample.

64. The method of any one of claims 56-63, wherein the method is for detecting the single nucleotide variants (SNVs) in the nucleic acids in the sample, and wherein the analyzing the sequence data further comprises classifying all base calls within the one or more repaired regions as not having double stranded support.

65. The method of any one of claims 56-64, further comprising analyzing the sequence data to determine a level of measured artifacts in the nucleic acids.

66. The method of any one of the preceding claims, further comprising amplifying at least a portion of the nucleic acids using a DNA polymerase.

67. The method of any one of claims 1-55 and 65-66, further comprising subjecting the nucleic acids in the sample to end repair to generate end-repaired nucleicAttorney Docket No.: GH0253WO acid molecules, wherein the end repair is performed using deoxynucleotide triphosphates (dNTPs).

68. The method of the immediately preceding claim, wherein at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into a repaired region of the end-repaired DNA molecules at one or more locations.

69. The method of claim 67 or claim 68, wherein the end repair is performed using a DNA polymerase that does not have 5 ’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase.

70. The method of claim 67 or claim 68, wherein the end repair is performed using a DNA polymerase that has 5’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

71. The method of any one of claims 67-70, wherein the at least one type of dNTP which comprises a modified base, wherein the modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6- methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG).

72. The method of any one of claims 67-71, wherein the end repair occurs prior to step a).

73. The method of any one of claims 67-71, wherein the end repair occurs after step a).

74. The method of any one of the preceding claims, further comprising performing an A-tailing reaction, optionally after a step of subjecting the nucleic acids in the sample to end repair.Attomey Docket No.: GH0253WO75. The method of the immediately preceding claim, wherein the end-repair and the A-tailing reaction are performed in the same reaction mixture, optionally wherein the end-repair and the A-tailing reaction are performed a single tube and / or optionally wherein the end-repair and the A-tailing reaction are performed without an intervening clean-up step.

76. The method of claim 74 or claim 75, wherein the A-tailing is performed using a DNA polymerase that does not possess 5’-3’ exonuclease activity and / or is not a strand displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

77. The method of any one of claims 74-76, wherein the A-tailing is performed using a thermostable DNA polymerase.

78. The method of any one of the preceding claims, wherein one or more adapters are ligated to the end-repaired DNA molecules or one or more adapters are ligated to the nucleic acids in the sample.

79. The method of the immediately preceding claim, wherein the one or more adapters comprise molecular barcodes.

80. The method of claim 78 or claim 79, wherein at least one cytosine in the one or more adapters is a modification resistant cytosine, optionally wherein each cytosine in the one or more adapters is a modification resistant cytosine.

81. The method of the immediately preceding claim, wherein the modification resistant cytosine is a deaminase resistant cytosine.

82. The method of the immediately preceding claim, wherein the deaminase resistant cytosine is 5-propynylC (5pyC), 5-pyrrolo-dC (5pyrC), 5-Attorney Docket No.: GH0253WO hydroxymethylcytosine (5hmC), glucosylated5-hydroxymethylcytosine (5ghmC), cytosine 5-methylenesulfonate (CMS), or N4-modified cytosine.

83. The method of any one of claims 78-82, wherein the one or more adapters are Y-shaped adapters.

84. The method of any one of the preceding claims, wherein the method comprises ligating one or more adapters to the nucleic acids after the step of separating the nucleic acid population into at least two partitions.

85. The method of any one of claims 1-83, wherein the method comprises ligating one or more adapters to the nucleic acids prior to the step of separating the nucleic acid population into at least two partitions.

86. The method of any one of the preceding claims, wherein the nucleic acids in the sample comprises barcodes.

87. The method of any one of the preceding claims, further comprising enriching the nucleic acids in the sample for a plurality of target regions.

88. The method of the immediately preceding claim, wherein the enriching the nucleic for a plurality of target regions occurs: i) after partitioning the nucleic in the sample into a plurality of subsamples; ii) prior to a step of sequencing the nucleic acids in the sample; iii) after amplifying the nucleic acids; and / or iv) prior to amplifying the nucleic acids.

89. The method of claim 87 or claim 88, wherein the plurality of target regions comprises epigenetic target regions.Attorney Docket No.: GH0253WO90. The method of the immediately preceding claim, wherein the epigenetic target regions comprise hypermethylation variable target regions.

91. The method of the immediately preceding claim, wherein the hypermethylation variable region comprises regions having a higher degree of methylation in at least one type of tissue or in blood than the degree of methylation in nucleic acids from a healthy subject.

92. The method of any one of claims 89-91, wherein the epigenetic target regions comprise hypomethylation variable target regions.

93. The method of the immediately preceding claim, wherein the hypomethylation variable region comprises regions having a lower degree of methylation in at least one type of tissue or in blood than the degree of methylation in nucleic acids from a healthy subject.

94. The method of any one of claims 89-93, wherein the epigenetic target regions comprise a methylation control target region set.

95. The method of any one of claims 89-94, wherein the epigenetic target region set comprise a fragmentation variable target region set.

96. The method of the immediately preceding claim, wherein the fragmentation variable target region set comprises transcription start site regions.

97. The method of claim 95 or claim 96, wherein the fragmentation variable target region set comprises CTCF binding regions.

98. The method of any one of claims 87-97, wherein the plurality of target regions comprise sequence-variable target regions.Attorney Docket No.: GH0253WO99. The method of any one of the preceding claims, further comprising performing a methylation-preserving amplification of the nucleic acids of the sample.

100. The method of the immediately preceding claim, wherein the methylationpreserving amplification is a linear, methylation-preserving amplification.

101. The method of claim 99 or claim 100, wherein the methylation-preserving amplification comprises contacting the nucleic acids with a methyltransferase.

102. The method of any one of claims 99-101, wherein the methylation-preserving amplification comprises one or more of polymerase chain reaction, linear amplification, rolling circle amplification, ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.

103. The method of any one of claims 99-101, wherein the methylation-preserving amplification comprises thermocycled amplification.

104. The method of any one of claims 99-101, wherein the methylation-preserving amplification comprises isothermal amplification.

105. The method of any one of claims 99-104, wherein the methylation-preserving amplification occurs prior to step a).

106. The method of any one of the preceding claims, wherein the nucleic acid population comprises cell-free DNA (cfDNA), optionally wherein the cfDNA is from a bodily fluid, such as blood, serum, or plasma.

107. The method of any one of the preceding claims, wherein the sample is a tissue sample.

108. The method of any one of the preceding claims, wherein the sample is a blood sample.Attorney Docket No.: GH0253WO109. The method of the immediately preceding claim, wherein the blood sample is a whole blood sample, a plasma sample, a buffy coat sample, a leukapheresis sample, or a peripheral blood mononuclear cell (PBMC) sample.

110. The method of any one of the preceding claims, wherein the sample is from a subject.

111. The method of any one of the preceding claims, wherein the sample is from a subject and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.

112. The method of any one of claims 110 or 111, wherein the subject is an animal.

113. The method of the immediately preceding claim, wherein the subject is a human.

114. The method of any one of claims 110-113, wherein the subject has or is at risk of having a cancer.

115. The method of any one of claims 110-114, further comprising determining the presence or status of a cancer in the subject.

116. The method of any one of claims 110-115, further comprising determining a likelihood that the subject has cancer.

117. The method of claims 23-116, wherein the sequencing comprises generating a plurality of sequencing reads; and the method further comprises mapping the plurality of sequence reads to one or more reference sequences to generate mapped sequence reads, and processing the mapped sequence reads to determine the likelihood that the subject has cancer.

118. The method of the immediately preceding claim, further comprising detecting a presence or absence of nucleic acids originating or derived from a tumor cell using the mapped sequence reads.Attorney Docket No.: GH0253WO119. The method of the immediately preceding claim, further comprising determining a cancer recurrence score that is indicative of the presence or absence of the nucleic acids originating or derived from the tumor cell for the test subject, optionally further comprising determining a cancer recurrence status based on the cancer recurrence score, wherein the cancer recurrence status of the test subject is determined to be at risk for cancer recurrence when a cancer recurrence score is determined to be at or above a predetermined threshold or the cancer recurrence status of the test subject is determined to be at lower risk for cancer recurrence when the cancer recurrence score is below the predetermined threshold.

120. The method of the immediately preceding claim, further comprising comparing the cancer recurrence score of the test subject with a predetermined cancer recurrence threshold, wherein the test subject is classified as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold or not a candidate for a subsequent cancer treatment when the cancer recurrence score is below the cancer recurrence threshold.

Images