Determining mutational profiles in cell-free nucleic acids

Abstract

The disclosure relates to methods for analyzing cell free nucleic acids obtained from a biological sample of a subject. In some embodiments, the method comprises partitioning the cell free nucleic acids based on the methylation status of the cell free nucleic acids, wherein the partitioning provides a hypermethylated partition and / or a hypomethylated partition; sequencing cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition, or derivatives thereof, to provide sequence reads; and analyzing the sequence reads to obtain a mutational profile of the cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition.

Description

Atty. Docket No. GH0264WODETERMINING MUTATIONAL PROFILES IN CELL-FREE NUCLEIC ACIDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 755,579, filed February 7, 2025 and PCT Patent Application No. PCT / US2025 / 060742, filed December 19, 2025, which are incorporated by reference herein in its entirety for all purposes.BACKGROUND

[0002] Nucleic base modifications can occur de novo, or can be inherited. Modified bases, such as methylation, and abnormal patterns of these modifications in the genome can be indicative of aberrant biological processes. Methylation and methylation patterns across the genome have been highlighted as key biomarkers for disease, particularly regarding cancer. Due to the enrichment of mutations in these highly methylated regions, focusing analysis on methylated nucleic acids allows for a reduction in background noise, facilitating an increased accuracy in, for example, cancer diagnosis and prognosis. Increased sensitivity can be achieved by the inclusion of regions with lower levels of methylation, however this comes at the cost of increased background noise.

[0003] While individual mutations are known to be key indications of cancer, single mutations are often not a sole cause or result of cancer development. Rather, cancer is often governed by a network of mutations. Additionally, mutational patterns in the genome can act as cancer signatures, indicating cancer presence and / or development. Hence, integrating analysis workflows to probe combinations of mutations within specific genomic regions, or throughout the genome, can provide a more informed and accurate insight into disease detection and tracking.

[0004] There is, therefore, a need for improved integrated workflows that are able to identify and analyze mutational profiles in methylated genomic regions while limiting background noise and assay costs.Atty. Docket No. GH0264WOSUMMARY

[0005] The present invention provides a method of analyzing the mutational profiles in nucleic acid populations from hypermethylated partitions and / or a hypomethylated partitions.

[0006] In an aspect, the present disclosure provides a method that allows for the analysis of the mutational profile of a nucleic acid population containing differentially methylated nucleic acids by enriching for nucleic acids of a particular methylation state. In some embodiments, this is achieved through partitioning the nucleic acid sample into at least a hypomethylated and hypermethylated partition. The nucleic acids within a partition can be amplified and sequenced. Subsequently, the sequence reads are grouped according to their parent nucleic acid or parent strand of nucleic acids. Groups can be paired according to whether the sequence reads represent opposing strands of the same parent nucleic acid, allowing the identification of mutations within the sequence reads of the group that have double stranded support. Such mutations can then be used to produce a mutational profile for a given genomic region which can be further analyzed, such as by using supervised machine learning algorithms trained on reference data sets, to provide information of disease (e.g. cancer) detection, progression and / or prognosis. Where biological noise increases due to an increase in background methylation of genomic regions, error correction assays or analysis methods can be used to minimise technical noise such as by minimising sequencing or amplification errors. Preferred error correction assays are those which do not require an increased sequencing depth.

[0007] In certain aspects, the present methods utilize a “hyperSNV” approach in an minimal residual disease (MRD) assay, such as plasma-only, plasma+buffy (both tumor-naive) or plasma+buffy+tissue (tumor-informed assay). In a tumor-informed assay, the method can generate a high cancer signal - abundant # of hyper SNVs, with tumor indexing protecting against false positives (FPs) from all non-cancer hyper SNVs in plasma and buffy subtracting of (de novo) germline FPs. While for tumor-naive, there is additional need for low non-cancer hyperSNV noise from non-blood cell contributors to cfDNA - buffy subtraction possible, mitigates only germline and CHIP, the latter which are harder to catch.Atty. Docket No. GH0264WO

[0008] Accordingly, in the first aspect, the present disclosure provides a method of analyzing cell free nucleic acids obtained from a biological sample of a subject, wherein the method comprises: (a) partitioning the cell free nucleic acids based on the methylation status of the cell free nucleic acids, wherein the partitioning provides a hypermethylated partition and / or a hypomethylated partition; (b) sequencing cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition, or derivatives thereof, to provide sequence reads; (c) analyzing the sequence reads to obtain a mutational profile of the cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition.

[0009] In some embodiments, adaptors are ligated to the cell free nucleic acids either before or after partitioning. In some embodiments, the adaptors comprise molecular barcodes.

[0010] In some embodiments, the sequence reads are grouped, wherein a group contains sequence reads derived from the same cell free nucleic acid in the biological sample.

[0011] In some embodiments, the sequence reads are grouped, wherein a group contains sequence reads derived from the same strand of a cell free nucleic acid in the biological sample. In some embodiments, the groups are paired such that the two groups in a pair represent different strands of the same cell free nucleic acid. In some embodiments, a mutation is classified as having double strand support when it is present in both strands of the same cell free nucleic acid. In some embodiments, the mutational profile of the cell free nucleic acids is determined using only mutations with double strand support.

[0012] In some embodiments, the method comprises hybrid capture of nucleic acids derived from target genomic regions. In some embodiments, the target genomic regions comprise differentially methylated regions.

[0013] In some embodiments, the method comprises sequencing cell free nucleic acids from the hypermethylated partition, or derivatives thereof. In some embodiments, the method comprises sequencing cell free nucleic acids from the hypomethylated partition, or derivatives thereof.

[0014] In some embodiments, the mutational profile comprises the frequency of mutations observed in the sequence reads relative to a reference sequence.Atty. Docket No. GH0264WO

[0015] In some embodiments, the mutational profile comprises the frequency of nucleotide transitions, including A— G, G— A, T— C, and / or C— T. In some embodiments, the mutational profile comprises the frequency of nucleotide transversions, including A— T, G— C, T— A, and / or C— G. In some embodiments, the mutational profile comprises the frequency of mutations within exons, introns, regulatory regions, and / or intergenic regions.

[0016] In some embodiments, the mutational profile comprises the frequency of copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), fusions, and / or structural variants. In some embodiments, the mutational profile comprises the frequency of SNVs.

[0017] In some embodiments, the mutational profile comprises the frequency of mutations wherein the mutation is one of at least two mutations within a genomic region. In some embodiments, the genomic region is between 50 and 400 nucleotides in length.

[0018] In some embodiments, the mutational profile is computationally defined using a scoring system that assigns weights to mutations based on their frequency, type, and / or genomic context. In some embodiments, the hypermethylated partition is subjected to treatment with a methylation sensitive restriction enzyme (MSRE). In some embodiments, the hypomethylated partition is subjected to treatment with a methylation dependent restriction enzyme (MDRE).

[0019] In some embodiments, the sequencing is whole genome or whole exome sequencing.

[0020] In some embodiments, analyzing the mutational profile comprises comparing the mutational profile to a reference dataset and classifying the subject based on the comparison. In some embodiments, the method further comprises classifying the subject as having cancer or a type of cancer by applying a supervised machine learning algorithm trained on a reference dataset containing mutational profiles of known cancer types. In some embodiments, the classification process involves the integration of clinical metadata with the mutational profile, and using a machine learning algorithm trained on combined datasets.

[0021] In some embodiments, the mutational profile is analyzed using a tumour-informed minimal residual disease (MRD) model to classify whether the subject hasAtty. Docket No. GH0264WOMRD. In some embodiments, the mutational profile is analyzed for tumour-naive minimal residual disease (MRD) detection, to classify whether the subject has MRD.

[0022] In some embodiments, analyzing the mutational profile comprises determining the somatic mutational profile by comparing the mutational profile to a germline sample from the same subject.

[0023] In some embodiments, the cell free nucleic acids from the sample are enriched for methylated molecules using a chemical or enzymatic process, such as bisulfite sequencing or EM-Seq techniques. In some embodiments, the cell free nucleic acids are enriched using a methylation-sensitive restriction enzyme (MSRE) and / or a methylation-dependent restriction enzyme (MORE). In some embodiments, a first portion of the sample obtained from the subject is enriched for methylated molecules, and optionally, a second portion is of the sample is used for somatic detection. In some embodiments, the enriched methylated molecules and / or unmethylated molecules are analyzed according to the bioinformatic workflows described herein to generate the mutational profile.

[0024] In some embodiments, methylated molecules can be determined via a sequencing analysis process without undergoing a conversion (e.g., by bisulfite or EM-Seq), such as by nanopore sequencing.

[0025] In some embodiments, the method comprises analyzing sequence reads derived from target genomic regions with a high cancer-to-normal methylation ratio. In some embodiments, the method comprises enrichment of nucleic acids derived from target genomic regions with a high cancer-to-normal methylation ratio.

[0026] In some embodiments, the method comprises analyzing sequence reads derived from target genomic regions with a mid cancer-to-normal methylation ratio and wherein the method comprises an additional step to reduce errors in the sequence reads. In some embodiments, the method comprises enrichment of nucleic acids derived from target genomic regions with a mid cancer-to-normal methylation ratio and wherein the method comprises an additional step to reduce errors in the sequence reads.

[0027] 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 true methylation status of cytosines or variants, as obtained by the methods disclosed herein, or information derived therefrom, can be displayed directly in such aAtty. Docket No. GH0264WOreport. 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.

[0028] 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.

[0029] 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

[0030] FIG. l is a schematic diagram of an example of a system suitable for use with some embodiments of the disclosure.DETAILED DESCRIPTION

[0031] 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.

[0032] 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 clearly dictates otherwise. Thus, for example, reference to “a nucleic acid” includes a plurality of nucleic acids.

[0033] 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.Atty. Docket No. GH0264WO

[0034] 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.

[0035] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way. In the event that any document or other material incorporated by reference contradicts any explicit content of this specification, including definitions, this specification controls.

[0036] 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.Definitions

[0037] As used herein the “plus” and “minus” strand represent the complementary strands of an original double stranded DNA duplex, for any given region. The two DNA strands may also be referred to as “positive” and “negative” strands, or “Watson” and “Crick” strands. For coding regions, the plus strand may be a sense strand and the minus may be an antisense strand or vice versa.

[0038] As used herein, “double stranded support” for a base call refers to the same nucleotide base being present in a given position based on the sequence reads corresponding to both the plus strand and the minus strand of an original sequence (e.g., an A is present in the plus strand and a T is present in the minus strand).

[0039] As used herein, “high stringency detection” and “high stringency identification” refer to variant detection and identification procedures that have a more stringent threshold than base or variant calling typically used for sequencing of bulk DNA or unmethylated DNA, e.g., a greater stringency than requiring two independent instances of the variant. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting somatic variants present in at least 3 molecules represented in the plurality of sequencing reads generated from sequencing DNA from a sample. In some embodiments, candidate variants that are present in fewerAtty. Docket No. GH0264WOthan 3 molecules represented in the plurality of sequencing reads are discarded. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting somatic variants present in at least 4 molecules represented in the plurality of sequencing reads generated from sequencing DNA from a sample. In some embodiments, candidate variants that are present in fewer than 4 molecules represented in the plurality of sequencing reads are discarded. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting somatic variants present in at least 5 molecules, at least 6 molecules, at least 7 molecules, at least 8 molecules, at least 9 molecules, or at least 10 molecules represented in the plurality of sequencing reads. In some embodiments, candidate variants that are present in fewer than 5 molecules, fewer than 6 molecules, fewer than 7 molecules, fewer than 8 molecules, fewer than 9 molecules, or fewer than 10 molecules represented in the plurality of sequencing reads are discarded. In some embodiments, the identifying or detecting with high stringency comprises excluding candidate somatic variants present in CpG dinucleotides. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting one or more somatic variants present in CpG dinucleotides that are present in at least 3 molecules represented in the plurality of sequencing reads. In some embodiments, candidate variants that are present in CpG dinucleotides that are present in fewer than 3 molecules represented in the plurality of sequencing reads are discarded. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting one or more somatic variants present in CpG dinucleotides that are present in at least 4 molecules represented in the plurality of sequencing reads. In some embodiments, candidate variants that are present in CpG dinucleotides that are present in fewer than 4 molecules represented in the plurality of sequencing reads are discarded. In some embodiments, the identifying or detecting with high stringency comprises identifying or detecting one or more somatic variants present in CpG dinucleotides that are present in at least 5 molecules, at least 6 molecules, at least 7 molecules, at least 8 molecules, at least 9 molecules, or at least 10 molecules represented in the plurality of sequencing reads. In some embodiments, candidate variants that are present in CpG dinucleotides that are present in fewer than 5 molecules, fewer than 6 molecules, fewer than 7 molecules, fewer than 8 molecules,Atty. Docket No. GH0264WOfewer than 9 molecules, or fewer than 10 molecules represented in the plurality of sequencing reads are discarded.

[0040] As used herein, “minimum residual disease”, “minimal residual disease”, “measurable residual disease”, or “MRD” refers to a small number of diseased cells (e.g., cancer cells) that remain in the body after treatment (e.g., after chemotherapeutic or immunotherapeutic treatment). Detecting MRD may require highly sensitive diagnostic testing such as next generation sequencing and / or methods provided herein.

[0041] As used herein, “clonal hematopoiesis of indeterminate potential (CHIP)” is a clonally expanded hematopoietic stem cell with any one or more mutations (e.g., variants) that is detectable in a blood sample from a subject (e.g., a buffy coat or plasma sample). See e.g., Mamell et al., J. Mol Cell Cardiol (2021 doi:10.1016 / j.yjmcc.2021.07.004. CHIP variants can be detected in the absence of disease (e.g., in a subject that does not have cancer). In some embodiments, the methods provided herein can be used to detect the presence of CHIP variants in a subject. In some embodiments, CHIP variants are not used to determine the presence or absence of cancer, precancer, or MRD.

[0042] “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 previously 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. cfDNA molecules may occur as DNA fragments.

[0043] 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.

[0044] As used herein, “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 andAtty. Docket No. GH0264WOplatelets of the sample. The huffy 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.

[0045] “Enriching” or “capturing” one or more target nucleic acids or one or more nucleic acids comprising at least one target region refers to preferentially isolating or separating the one or more target nucleic acids or one or more nucleic acids comprising at least one target region from non-target nucleic acids or from nucleic acids that do not comprise at least one target region.

[0046] An “enriched set” or “captured set” of nucleic acids or “enriched” or “captured” nucleic acids refers to nucleic acids that have undergone capture.

[0047] As used herein, a “capture moiety” is a molecule that allows affinity separation of molecules, such as nucleic acids, 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.

[0048] As used herein, a “cell type” is a set of cells having a shared characteristic. For example, cell types can include cells of different origins, differentiation types, different activation types, or any combination of different origins, different differentiation types, and different activation types. Indeed, differentiation status and activation status can overlap and often change together in a given cell, such as an immune cell or a cancer cell. For example, activation of an immune cell may induce differentiation of the cell. In some embodiments, cell types may be distinguished based on characteristics such as one or more cell surface markers, a genetic signature (such as expression (or expression level) of a particular gene or set of genes), and / or an epigenetic signature, such as regions of DNA hypermethylation or hypomethylation.

[0049] As used herein, a “cell cluster” or “cluster” is a plurality of related cell types, e.g., immune cell types, tissue-specific cell types, and / or cancer cell types. In someAtty. Docket No. GH0264WOembodiments, the cell types within a cluster have similar DNA methylation profiles, e.g., in a plurality of hypermethylation variable target regions and / or hypomethylation variable target regions.

[0050] A “converted nucleobase” is a nucleobase having an altered base pairing specificity, wherein the original base pairing specificity of the nucleobase 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. As used herein, a “converted sample” is a sample comprising DNA comprising at least one converted nucleobase.

[0051] As used herein, an “oligonucleotide probe” refers to a single-stranded nucleic acid molecule (such as a DNA molecule or an RNA molecule) that can hybridize to a complementary nucleic acid sequence (e.g., a target sequence) within a target nucleic acid. An oligonucleotide probe may be 10 to 50 bases in length and may be synthesized as a specified base sequence.

[0052] “Specifically binds” in the context of a primer, a probe, or other oligonucleotide and a target sequence (e.g., a nucleic acid comprising a sequence that is partially or completely complementary to the primer, probe, or other oligonucleotide) means that under appropriate hybridization conditions, the primer, probe, or other oligonucleotide hybridizes to its target sequence, or replicates thereof, to form a stable hybrid, while at the same time formation of stable non-target hybrids is minimized. Thus, a primer, probe, or other oligonucleotide hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a non-target sequence, to ultimately enable enrichment 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, 2nd ed. (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). An oligonucleotide probe can “preferentially form a substrate for extension with,” e.g., a target nucleic acidAtty. Docket No. GH0264WOcomprising a wild-type sequence relative to a target nucleic acid comprising a variant sequence, a target nucleic acid that comprises a converted nucleotide relative to a target nucleic acid that does not comprise the converted nucleotide, or a target nucleic acid that does not comprise a converted nucleotide relative to a target nucleic acid that comprises the converted nucleotide. For example, an oligonucleotide probe that preferentially forms a substrate for extension with a target nucleic acid comprising a wild-type sequence relative to a target nucleic acid comprising a variant sequence may be at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times more likely to bind the target nucleic acid comprising the wild-type sequence relative to the target nucleic acid comprising the variant sequence.

[0053] A “target region” refers to a genomic locus targeted for identification and / or capture, for example, by using probes (e.g., through sequence complementarity). A “target region” or “set of target regions” refers to a plurality of genomic loci targeted for identification and / or capture, for example, by using a set of probes (e.g., through sequence complementarity). A “target region” can comprise regions that share at least one common feature. In some embodiments, a target region is identified by the at least one common feature. For example, a hypermethylation variable target region comprises regions of DNA that are hypermethylated.

[0054] “Sequence-variable target regions” refer to 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) relative to normal cells. A “sequence-variable target region” refers to a set of sequence-variable target regions. In some embodiments, the sequencevariable target regions are target regions that may exhibit changes that affect less than or equal to 50 contiguous nucleotides, e.g., less than or equal to 40, 30, 20, 10, 5, 4, 3, 2, or 1 nucleotides.

[0055] “Epigenetic target regions” refers to target regions that may show sequenceindependent differences in different cell or tissue types (e.g., different types of immune cells) or in abnormal cells, such as neoplastic cells (e.g., tumor cells and cancer cells), relative to normal cells; or that may show sequence-independent differences (i.e., in which there is no change to the nucleotide sequence, e.g., differences in methylation,Atty. Docket No. GH0264WOnucleosome distribution, or other epigenetic features) in DNA, e.g., from different cell types or from subjects having cancer relative to DNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, changes in methylation (increases or decreases), nucleosome distribution, fragmentation patterns, CCCTC-binding factor (“CTCF”) binding, transcription start sites (e.g., with respect to any one of more of binding of RNA polymerase components, binding of regulatory proteins, fragmentation characteristics, and nucleosomal distribution), and regulatory protein binding regions. Epigenetic target regions thus include, but are not limited to, hypermethylation variable target regions, hypomethylation variable target regions, and fragmentation variable target regions, such as CTCF binding sites and transcription start sites. For present purposes, loci susceptible to neoplasia-, tumor-, or cancer-associated focal amplifications and / or gene fusions may also be included in an epigenetic target region 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. An “epigenetic target region” is a set of epigenetic target regions.

[0056] 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 differentially methylated region has a detectably higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type, such as at least one immune cell type, relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type, such as other immune cell types, or from the same cell or tissue type from a healthy subject. In some embodiments, a differentially methylated region has a detectably lower degree of methylation (e.g., a hypom ethylated region) in at least oneAtty. Docket No. GH0264WOcell or tissue type, such as at least one immune cell type, relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type, such as other immune cell types, or from the same cell or tissue type from a healthy subject. A “corresponding DMR” refers to a DMR from the same locus or covering the same sequence as a first (e.g., variant) DMR.

[0057] A nucleic acid is “produced by a tumor” if it originated from a tumor cell. 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). As used herein, “precancer” or a “precancerous condition” is an abnormality that has the potential to become cancer, wherein the potential to become cancer is greater than the potential if the abnormality was not present, i.e., was normal. Examples of precancer include but are not limited to adenomas, hyperplasias, metaplasias, dysplasias, benign neoplasias (benign tumors), premalignant carcinoma in situ, and polyps. It should be noted that certain types of carcinoma in situ are recognized in the field as cancerous, e.g., Stage 0 cancer, as opposed to premalignant.

[0058] The term “methylation” or “DNA methylation” refers to addition of a methyl group to a nucleobase 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. In some embodiments, DNA methylation is 5-methylation (modification of the 5th carbon of the 6-carbon ring of cytosine). 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 3rd carbon of the 6-carbon ring of cytosine). 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. DNAAtty. Docket No. GH0264WOmethylation 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.

[0059] The term “hypermethylation” refers to an increased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules comprising the same genetic information 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.

[0060] The term “hypomethylation” refers to a decreased level or degree of methylation of nucleic acid molecule(s) relative to the other nucleic acid molecules comprising the same genetic information 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.

[0061] 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. MethylAtty. Docket No. GH0264WObinding reagents include but are not limited to methyl binding domains (MBDs) and methyl binding proteins (MBPs) and antibodies specific for methyl cytosine. In some embodiments, such antibodies bind to 5-methyl cytosine in DNA. In some such embodiments, the DNA may be single-stranded or double-stranded. Suitable agents include agents that recognize modified nucleotides in double-stranded DNA, singlestranded DNA, and both double-stranded and single-stranded DNA.

[0062] The term “epigenetic status” refers to a certain level or extent of a sequenceindependent variable that may be present in a DNA sequence. In some embodiments, the epigenetic status of a DNA sequence refers to the extent or level of methylation, nucleosome distribution, cfDNA fragmentation pattern, CCCTC-binding factor (“CTCF”) binding, transcription start site, or regulatory protein binding region of the sequence. Epigenetic statuses thus include, but are not limited to, hypermethylation, hypomethylation, and the presence of absence of CTCF binding sites or transcription start sites. The epigenetic status of a sequence may be a “reference epigenetic status” that can be used for comparison to the epigenetic status of the corresponding sequence in other DNA molecules. An example of a reference epigenetic status is a status that is prevalent in samples obtained from healthy subjects and is not associated with cancer.

[0063] As used herein, “methylation status” refers to the presence or absence of a methyl group on a DNA nucleobase (e.g. cytosine) at a particular genomic position in a nucleic acid, the degree of methylation of a nucleic acid (e.g., high, low, intermediate, or unmethylated), or the number of nucleotides methylated in a particular nucleic acid molecule. A nucleic acid “in methylated form” means that it comprises a sequence containing a methylated DNA nucleobase, e.g., a methylated cytosine in a CpG dinucleotide.

[0064] 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 a referred to as a cancer or a cancerous tumor.

[0065] 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 10 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.,Atty. Docket No. GH0264WOrepresenting 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 two molecules may have the same endogenous sequence information (e.g., start and / or stopAtty. Docket No. GH0264WOpositions, subsequences of one or both ends of a sequence, and / or lengths) and also have the same molecular barcode.

[0066] As used herein, “partitioning” refers to physically separating, sorting, and / or fractionating a mixture of nucleic acid molecules in a sample into a plurality of subsamples or subpopulations of nucleic acids based on a characteristic of the nucleic acid molecules. A sample or population may be partitioned into one or more partitioned subsamples or subpopulations based on a characteristic that is indicative of a genetic or epigenetic change or a disease state. 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. Stated differently, partitioning may include physically partitioning nucleic acid molecules based on the presence or absence of one or more methylated nucleobases. 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. Partitioning can involve 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.

[0067] 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 binding 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 orAtty. Docket No. GH0264WOdegree 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 partition, or residual partitioned set). 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). For example, if the partitioning involves 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.

[0068] As used herein, a “restriction endonuclease” refers to an enzyme that can cleave a double stranded nucleic acid molecule (such as a double stranded DNA or cDNA) at a specific recognition site (a particular nucleotide sequence recognized by the restriction endonuclease, also referred to herein as a “cleavage site”) in the double stranded DNA or cDNA molecule. Exemplary restriction endonucleases of use herein include, but are not limited to, BspQI, Afllll, BsiHKAI, Btrl, Maell, and Sdul. In some embodiments, the restriction endonuclease is BspQI and claves a double-stranded nucleic acid at a 5’-GCTCTTCN-3’ recognition site.

[0069] As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0070] 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, panelAtty. Docket No. GH0264WOsequencing, 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 at 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, long-read 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.

[0071] 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. Example 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.

[0072] 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” areAtty. Docket No. GH0264WOintended 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.

[0073] As used herein, the phrase “non-complementary restriction endonuclease cleavage site” refers to a restriction endonuclease cleavage site in a first strand of a double-stranded nucleic acid in which the second strand comprises at least one nucleotide that is not complementary to the corresponding nucleotide of the first strand. Non-complementary restriction endonuclease cleavage sites can comprise 1, 2, 3, 4, 5, 6, or more than 6 nucleotides that are not complementary to one another in this fashion. For example, in some embodiments of a double-stranded nucleic acid comprising 5’ and 3’ adapters on both strands, a 5’ adapter and a 3’ adapter comprise “non-complementary restriction endonuclease cleavage sites” when the cleavage site in the 5’ adapter comprises at least one nucleotide that is not complementary to the paired nucleotide of the 3’ adapter when the nucleic acids are aligned antiparallel to one another. Restriction endonuclease cleavage sites of use herein include cleavage sites that are recognized by a restriction endonuclease that cleaves a double-stranded nucleic acid comprising the cleavage site. Thus, in some embodiments, non-complementary restriction endonuclease cleavage sites, such as non-complementary restriction cleavage sites in otherwise complementary sense and antisense strands of a nucleic acid, may comprise cleavage sites that are recognized by different restriction endonucleases. Non-complementary restriction endonuclease cleavage sites can thus comprise a sense strand cleavage site that is longer or shorter than the cleavage site in the antisense strand.

[0074] As used herein, “substantially” refers to a range of numerical values (e.g., values within 5-10% of the specified vale) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). For example, “substantially” can include values greater than or equal to 90% of a specified value. Substantially does not require 100% (such as exactly 100% of the target nucleic acids of a population of target nucleic acids), but can include an amount greater than orAtty. Docket No. GH0264WOequal to 90%, such as an amount greater than or equal to any one of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% (such as 90%-100% of the target nucleic acids of a population of target nucleic acids).

[0075] “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.

[0076] “ Germline mutation” and “germline variation” are used interchangeably and refer to an inherited mutation (i.e., not one arising post-conception). Germline mutations may be the only mutations that can be passed on to the offspring and may be present in every somatic cell and germline cell in the offspring. As used herein, the term “germline sequence” refers to the genetic sequences from the germline (the haploid gametes and those diploid cells from which they are formed).

[0077] “ Somatic mutation” and “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.

[0078] As used herein, a “passenger mutation” or “hitchhiker mutation” refers to a mutation which does not alter fitness but occurred in a cell that coincidentally or subsequently acquired a driver mutation, and are therefore found in every cell with that driver mutation. Conversely, a “driver mutation” refers to a mutation that confer a fitness advantage to somatic cells in their microenvironment, thereby driving the cell lineage to cancer.

[0079] 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. A reference sequence can align with a single contiguous sequence of a genome or chromosome or chromosome arm or can include non-contiguous segments that alignAtty. Docket No. GH0264WOwith different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as, hgl9 and hg38.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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 leastAtty. Docket No. GH0264WOone 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. The 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.

[0086] 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.

[0087] The term “sequence-specific nuclease” means a nuclease that preferentially cleaves nucleic acid sequences that comprise a particular sequence or consensus sequence. In some embodiments, sequence-specific nucleases bind to a guide RNA that hybridizes to or near to the sequence to be cleaved by the nuclease. Examples of sequence-specific nucleases include, but are not limited to, CRISPR (e.g., a Cas nuclease such as Cas9), Argonaute, TALEN, and zinc finger nucleases. Unless otherwise indicated, a sequence-specific nuclease may be a variant sequence-specific nuclease that comprises at least one modification to at least one amino acid compared to the sequence-specific nuclease from which it was derived and has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 98%, or 99% sequence identity) to the sequencespecific nuclease from which it was derived and has retains at least some or enhanced nuclease function. The modification may be an amino acid substitution, deletion, or insertion.

[0088] 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.

[0089] As used herein, “peripheral blood mononuclear cells” or “PBMCs” refers to immune cells having a single, round nucleus that originate in bone marrow and areAtty. Docket No. GH0264WOfound 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.

[0090] 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. 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 is irrelevant because uracil nonetheless most preferentially pairs with A among the four standard DNA bases.

[0091] A “XlnnnX2 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 human polypeptide unless indicated otherwise. The polypeptide comprising the XlnnnX2 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.

[0092] “ Or” is used in the inclusive sense, i.e., equivalent to “and / or,” unless the context requires otherwise.Atty. Docket No. GH0264WOPartitioning

[0093] The present disclosure provides a method for analyzing a nucleic acid population by determining a mutational profile for the population wherein the method comprises partitioning the nucleic acid population.

[0094] 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. In some embodiments, the population of nucleic acids are subjected to a methylation-based partitioning assay, wherein the methylation-based partitioning assay partitions nucleic acids using methyl-binding domain (MBD). In such methods, the methylation-based partitioning assay can form a hypermethylated partition and / or a hypomethylated partition. After partitioning, one or more of the resulting partitions can be analyzed by the methods disclosed herein to determine a mutational profile. In some embodiments, the hypermethylated partition is further analyzed to determine a mutational profile of nucleic acids within the partition. In some embodiments, the hypomethylated partition is further analyzed to determine a mutational profile of nucleic acids within the partition.

[0095] Partitioning may include physically partitioning nucleic acids into partitions based on the presence or absence of one or more methylated nucleobases. A sample may be partitioned into partitions based on a characteristic that is indicative of differential gene expression or a disease state. A sample may be partitioned based on a characteristic that provides a difference in signal between a normal and diseased state during analysis of nucleic acids, e.g., cell free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0096] In some instances, a nucleic acid sample is partitioned into two or more partitions (e g., at least 3, 4, 5, 6 or 7 partitions). The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is aAtty. Docket No. GH0264WOmodified cytosine, such as a methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. 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 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 partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5-carboxylcytosine (5caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs), including proteins such as MeCP2.

[0097] Additional, non-limiting examples of 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.

[0098] In some embodiments, partitioning can comprise both binary partitioning and partitioning based on degree / level of modifications. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from unmethylated fragments using methyl binding domain proteins (e.g., Methyl Miner Methylated DNA Enrichment Kit (ThermoFisher Scientific)). Subsequently, additional partitioning 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.

[0099] Various levels of methylation can be partitioned using sequential elutions. For example, a hypomethylated partition (no methylation) can be separated from a methylated 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 methylatedAtty. Docket No. GH0264WOnucleic 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 level of methylated nucleic acids from those with lower level of methylation. The elution and magnetic separation steps can be repeated to create various partitions such as a hypomethylated partition (enriched in nucleic acids comprising no methylation), a methylated partition (enriched in nucleic acids comprising low levels of methylation), and a hypermethylated partition (enriched in nucleic acids comprising high levels of methylation). Any one or more partitions can then be analyzed using the methods disclosed herein.

[0100] In some methods, nucleic acids bound to an agent used for affinity separationbased 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).

[0101] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0102] In some embodiments, the nucleic acids can be partitioned into different partitions based on the nucleic acids that are bound to a specific protein or a fragment thereof and those that are not bound to that specific protein or fragment thereof.

[0103] Nucleic acids 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, protein A and protein G. Any suitable method can be used to partition the nucleic acids based on protein bound regions. Examples of methods used to partition nucleic acids based onAtty. Docket No. GH0264WOprotein bound regions include, but are not limited to, SDS-PAGE, chromatin-immunoprecipitation (ChIP), heparin chromatography, and asymmetrical field flow fractionation (AF4).

[0104] In some embodiments, the partitioning 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.

[0105] 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.

[0106] 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, (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 preferentially bind to 5-hydroxymethyl-cytosine over unmodified cytosine, (c) FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind to 5 -formyl -cytosine over unmodified cytosine (lurlaro et al., Genome Biol. 14: R119 (2013)), and (d) antibodies specific to one or more methylated or modified nucleobases or conversion products thereof, such as 5mC, 5caC, or DHU.

[0107] 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 aAtty. Docket No. GH0264WOpopulation 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 (a residual 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.

[0108] In some embodiments, the partitioned nucleic acids can be contacted with a methylation sensitive restriction enzyme (MSRE) and / or a methylation dependent restriction enzyme (MORE). In one embodiment, a partition which is enriched for methylated nucleic acids (e.g. a hypermethylated partition and / or a residual partition) is treated with an MSRE such that unmethylated nucleic acids within the partition are digested. This can reduce the number of incorrectly partitioned nucleic acids in the partition enriched for methylated nucleic acids. In one embodiment, a partition which is unenriched for methylated nucleic acids (e.g. the hypomethylated partition) can be treated with an MDRE such that methylated nucleic acids within the partition are digested. This can reduce the number of incorrectly partitioned nucleic acids in the partition enriched for unmethylated nucleic acids.

[0109] In some embodiments, the partitioning 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.

[0110] In some embodiments, DNA from one or more samples (e.g., the first, second, and / or third sample) or a subsample thereof (e.g., a subsample prepared by partitioning a sample as described herein, such as on the basis of a level of a cytosine modification, such as methylation, e.g., 5-methylation, e.g., a hypermethylated or hypomethylatedAtty. Docket No. GH0264WOsubsample) is contacted with a methylation-dependent nuclease or methylation-sensitive nuclease. The contacting can be performed using a sample that has been divided into a plurality of subsamples as disclosed herein, and / or using a sample that has been partitioned into a plurality of subsamples as disclosed herein. Alternatively, the contacting can be performed using a sample that has not been divided into a plurality of subsamples and / or has not been partitioned into a plurality of subsamples. Unless otherwise indicated, where partitioning is performed on the basis of a cytosine modification, the first subsample is the subsample with a higher level of the modification (e.g., a hypermethylated subsample); the second subsample is the subsample with a lower level of the modification (e.g., a hypomethylated subsample); and, when present, the third subsample has a level of the modification intermediate between the first and second subsamples.[OHl] In some embodiments, methods herein comprise contacting DNA with a methylation-sensitive nuclease, thereby degrading DNA comprising unmethylated sequences or sequences having low levels of methylation. In some such embodiments, the methylation-sensitive nuclease is a methylation-sensitive restriction enzyme (MSRE), thereby degrading DNA comprising an unmethylated recognition site of the MSRE. Methylation-sensitive nucleases can thus be used in methods herein comprising one or more steps that deplete unmodified or unmethylated sequences, such as those that are prevalent in cfDNA from a subject.

[0112] In some embodiments, methods herein comprise contacting DNA with a methylation-dependent nuclease, thereby degrading DNA comprising methylated sequences or sequences having high levels of methylation. In some such embodiments, the methylation-dependent nuclease is a methylation-dependent restriction enzyme (MDRE), thereby degrading DNA comprising a methylated recognition site of the MDRE. Methylation-dependent nucleases can thus be used in methods herein comprising one or more steps that deplete modified or methylated sequences, such as those that are prevalent in cfDNA from a subject.

[0113] In some embodiments, the contacting the DNA with the MSRE occurs after ligating one or more adapters to the end-repaired DNA molecules, after contacting the CpG-binding protein with the DNA in the sample, after separating the CpG proteinbound DNA from unbound DNA, after removing the CpG-binding protein from theAtty. Docket No. GH0264WOCpG protein-bound DNA, and / or after contacting the CpG-dense DNA with the methylsensitive deaminase. In some embodiments, the one or more adapters is resistant to digestion by the MSRE. In some embodiments, the one or more adapters that is resistant to digestion by MSRE comprises one or more methylated nucleotides, one or more nucleotide analogs resistant to methylation sensitive restriction enzymes, or does not comprise a nucleotide sequence recognized by the MSRE. In some embodiments, the methylated nucleotides in the one or more adapters that is resistant to digestion by MSRE comprise 5-methylcytosine and / or 5-hydroxymethylcytosine.

[0114] In some embodiments, the contacting the DNA with the MDRE occurs after ligating one or more adapters to the end-repaired DNA molecules, after contacting the CpG-binding protein with the DNA in the sample, after separating the CpG proteinbound DNA from unbound DNA, after removing the CpG-binding protein from the CpG protein-bound DNA, and / or after contacting the CpG-dense DNA with the methylsensitive deaminase. In some embodiments, the one or more adapters is resistant to digestion by the MDRE. In some embodiments, the one or more adapters that is resistant to digestion by MDRE comprises one or more unmethylated nucleotides, one or more nucleotide analogs resistant to methylation dependent restriction enzymes, or does not comprise a nucleotide sequence recognized by the MDRE.

[0115] In some embodiments, CpG protein-bound DNA may be contacted with an MSRE. In some embodiments, DNA unbound by a CpG-binding protein may be contacted with an MDRE.

[0116] Partitioning procedures may result in imperfect sorting of DNA molecules among the subsamples. The choice of a methylation-dependent nuclease or methylationsensitive nuclease can be made so as to degrade nonspecifically partitioned DNA. For example, the second (e.g., hypomethylated) subsample can be contacted with a methylation-dependent nuclease, such as a methylation-dependent restriction enzyme. This can degrade nonspecifically partitioned DNA in the second subsample (e.g., methylated DNA) to produce a treated second subsample. Alternatively or in addition, the first (e.g., hypermethylated) subsample can be contacted with a methylationsensitive endonuclease, such as a methylation-sensitive restriction enzyme, thereby degrading nonspecifically partitioned DNA in the first subsample to produce a treated first subsample. Degradation of nonspecifically partitioned DNA in either or both of theAtty. Docket No. GH0264WOfirst or second subsamples is proposed as an improvement to the performance of methods that rely on accurate partitioning of DNA on the basis of a cytosine modification, e.g., to detect the presence of aberrantly modified DNA in a sample, to determine the tissue of origin of DNA, and / or to determine whether a subject has cancer. For example, such degradation may provide improved sensitivity and / or simplify downstream analyses. In general, where nonspecifically partitioned DNA would be hypermethylated, such as in a hypomethylated partition, a methylationdependent nuclease, such as a methylation-dependent restriction enzyme, should be used. Conversely, where nonspecifically partitioned DNA would be hypomethylated, such as in a hypermethylated partition, a methylation-sensitive nuclease, such as a methylation-sensitive restriction enzyme, should be used. Methylation-dependent nucleases, such as methylation-dependent restriction enzymes, preferentially cut methylated DNA relative to unmethylated DNA, while methylation-sensitive nucleases, such as methylation-sensitive restriction enzymes, preferentially cut unmethylated DNA relative to methylated DNA.

[0117] In contacting a subsample with a nuclease, one or more nucleases can be used. In some embodiments, a subsample is contacted with a plurality of nucleases. The subsample may be contacted with the nucleases sequentially or simultaneously.Simultaneous use of nucleases may be advantageous when the nucleases are active under similar conditions (e.g., buffer composition) to avoid unnecessary sample manipulation. Contacting the second subsample with more than one methylationdependent restriction enzyme can more completely degrade nonspecifically partitioned hypermethylated DNA. Similarly, contacting the first subsample with more than one methylation-sensitive restriction enzyme can more completely degrade nonspecifically partitioned hypomethylated and / or unmethylated DNA.

[0118] In some embodiments, a methylation-dependent nuclease comprises one or more of MspJI, LpnPI, FspEI, or McrBC. In some embodiments, at least two methylation-dependent nucleases are used. In some embodiments, at least three methylation-dependent nucleases are used. In some embodiments, the methylationdependent nuclease comprises FspEI. In some embodiments, the methylation-dependent nuclease comprises FspEI and MspJI, e.g., used sequentially.Atty. Docket No. GH0264WO

[0119] In some embodiments, a methylation-sensitive nuclease comprises one or more of Aatll, AccII, Acil, Aorl3HI, Aorl5HI, BspT104I, BssHII, BstUI, CfrlOI, Clal, Cpol, Eco52I, Haell, HapII, Hhal, Hin6I, Hpall, HpyCH4IV, Mlul, MspI, Nael, Notl, Nrul, Nsbl, PmaCI, Pspl406I, Pvul, SacII, Sall, Smal, and SnaBI. In some embodiments, at least two methylation-sensitive nucleases are used. In some embodiments, at least three methylation-sensitive nucleases are used. In some embodiments, the methylationsensitive nucleases comprise BstUI and Hpall. In some embodiments, the two methylation-sensitive nucleases comprise Hhal and AccII. In some embodiments, the methylation-sensitive nucleases comprise BstUI, Hpall and Hin6I.

[0120] In some embodiments, FspEI is used for digesting the nucleic acid molecules in at least one subsample (e.g., a hypomethylated partition). In some embodiments, BstUI, Hpall and Hin6I are used for digesting the nucleic acid molecules in at least one subsample (e.g., a hypermethylated partition) and FspEI is used for digesting the nucleic acid molecules in at least one other subsample (e.g., a hypomethylated partition). In embodiments involving an intermediately methylated partition, the nucleic acid molecules therein may be digested with a methylation-sensitive nuclease or a methylation-dependent nuclease. In some embodiments, the nucleic acid molecules in an intermediately methylated partition are digested with the same nuclease(s) as the hypermethylated partition. For example, the intermediately methylated partition may be pooled with the hypermethylated partition and then the pooled partitions may be subjected to digestion. In some embodiments, the nucleic acid molecules in an intermediately methylated partition are digested with the same nuclease(s) as the hypomethylated partition. For example, the intermediately methylated partition may be pooled with the hypomethylated partition and then the pooled partitions may be subjected to digestion.

[0121] In some embodiments, a subsample is contacted with a nuclease as described above after a step of tagging or attaching adapters to both ends of the DNA. The tags or adapters can be resistant to cleavage by the nuclease using any of the approaches described above. In this approach, cleavage can prevent the nonspecifically partitioned molecule from being carried through the analysis because the cleavage products lack tags or adapters at both ends.Atty. Docket No. GH0264WO

[0122] Alternatively, a step of tagging or attaching adapters can be performed after cleavage with a nuclease as described above. Cleaved molecules can be then identified in sequence reads based on having an end (point of attachment to tag or adapter) corresponding to a nuclease recognition site. Processing the molecules in this way can also allow the acquisition of information from the cleaved molecule, e.g., observation of somatic mutations. When tagging or attaching adapters after contacting the subsample with a nuclease, and low molecular weight DNA such as cfDNA is being analyzed, it may be desirable to remove high molecular weight DNA (such as contaminating genomic DNA) from the sample before the contacting step. It may also be desirable to use nucleases that can be heat-inactivated at a relatively low temperature (e.g., 65°C or less, or 60°C or less) to avoid denaturing DNA, in that denaturation may interfere with subsequent ligation steps.

[0123] Where a sample is partitioned into three subsamples, including a third subsample containing intermediately methylated molecules, the third subsample is in some embodiments contacted with a methylation-sensitive nuclease. Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above. In some embodiments, the first and third subsamples are combined before being contacted with a methylation-sensitive nuclease. Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above. In some embodiments, the first and third subsamples are differentially tagged before being combined.

[0124] Alternatively, where a sample is partitioned into three subsamples, including a third subsample containing intermediately methylated molecules, the third subsample is in some embodiments contacted with a methylation-dependent nuclease. Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above. In some embodiments, the second and third subsamples are combined before being contacted with a methylation-dependent nuclease. Such a step may have any of the features described elsewhere herein with respect to contacting steps, and may be performed before or after a step of tagging or attaching adapters as discussed above.Atty. Docket No. GH0264WOIn some embodiments, the second and third subsamples are differentially tagged before being combined.

[0125] In some embodiments, the DNA is purified after being contacted with the nuclease, e.g., using SPRI beads. Such purification may occur after heat inactivation of the nuclease. Alternatively, purification can be omitted; thus, for example, a subsequent step such as amplification can be performed on the subsample containing heat-inactivated nuclease. In another embodiment, the contacting step can occur in the presence of a purification reagent such as SPRI beads, e.g., to minimize losses associated with tube transfers. After cleavage and heat inactivation, the SPRI beads can be re-used for cleanup by adding molecular crowding reagents (e.g., PEG) and salt.

[0126] 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.

[0127] In some embodiments, the nucleic acids of the nucleic acid sample may be exposed to methylation-sensitive restriction enzymes (MRSEs). Such restriction enzymes do not cleave methylated residues, leaving only the methylated nucleic acids of the nucleic acid sample intact. Exposure to such restriction enzymes would result in a sample of only methylated nucleic acids, which could then be analyzed by the disclosed methods. Exposure to differential concentrations of MRSEs would result in subsamples that contain nucleic acids increasingly enriched for hypermethylated nucleic acids.

[0128] In some instances, the DNA from one or more samples (e.g., the first, second, and / or third 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 DNA isAtty. Docket No. GH0264WOpartitioned based on the presence or absence of 5-methylcytosine (5mC) or5-hydroxymethylcytosine (5hmC) nucleic acid bases in the nucleic acids.

[0129] 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, 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 one or more base modifications and without the one or more base 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 singlestranded 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).

[0130] 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.

[0131] In some embodiments, partition tagging comprises tagging molecules in each partition with a partition tag. After re-combining partitions (e.g., to reduce the numberAtty. Docket No. GH0264WOof sequencing runs needed and avoid unnecessary cost) 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 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.Atty. Docket No. GH0264WODifferentially tagged partitions can be separately sequenced or sequenced together concurrently, e.g., in the same flow cell of an Illumina sequencer.

[0136] 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 genomic region while lower coverage can correlate with lower nucleosome occupancy or a nucleosome depleted region (NDR).

[0137] In some embodiments, methods disclosed herein 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. 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.

[0138] Methylation profiling 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.

[0139] In some embodiments, the partitioning comprises contacting the DNA with an agent that recognizes a modification associated with (e.g., in) the DNA. In some embodiments, the agent that recognizes the modification is an antibody. In someAtty. Docket No. GH0264WOembodiments, the agent is immobilized on a solid support. In some embodiments, the partitioning comprises immunoprecipitation, e.g., using a binding agent agent, such as an antibody, immobilized on solid support.

[0140] 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, an acetylated protein, such as a methylated or acetylated histone. In some embodiments, the binding reagent specifically binds an unmethylated or unacetylated protein epitope.

[0141] 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).

[0142] Where immunoprecipitation is used and involves an antibody that recognizes single-stranded DNA, the DNA may be converted to double-stranded form byAtty. Docket No. GH0264WOcomplementary strand synthesis before a subsequent step. Such synthesis may use an adapter as a primer binding site, or can use random priming.

[0143] 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 hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning a sample into hypermethylated and hypomethylated nucleic acid molecules. 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., cell free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell free nucleic acids (cfNA).

[0144] 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.

[0145] In some instances, heterogeneous DNA in a 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. 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.

[0146] In some embodiments, sequence reads from differentially tagged and pooled DNA are obtained and analyzed in silico. After sequencing, analysis of reads can beAtty. Docket No. GH0264WOperformed 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).

[0147] The agents used to partition populations of nucleic acids within a sample can be affinity agents, such as antibodies with the desired specificity, natural binding partners or variants thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or artificial peptides selected e.g., by phage display to have specificity to a given target. In some embodiments, the agent used in the partitioning 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 recognized by the agent is a product of a procedure that affects the first nucleobase in the DNA differently from the second nucleobase in the DNA of the sample. 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 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 someAtty. Docket No. GH0264WOembodiments, the partitioning agent is an antibody that recognizes a modified cytosine other than 5-methylcytosine, such as 5-carboxylcytosine (5-caC). Alternative partitioning agents include methyl binding domain (MBDs) and methyl binding proteins (MBPs) as described herein, 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 singlestranded 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 hemi-methylated 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.

[0148] Additional, non-limiting examples of 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.

[0149] In some embodiments, partitioning can comprise both binary partitioning and partitioning based on degree / level of modifications. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from unmethylated fragments using methyl binding domain proteins (e.g., MethylMiner Methylated DNA Enrichment Kit (ThermoFisher Scientific). Subsequently, additional partitioning 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.

[0150] Analyzing DNA may comprise detecting or quantifying DNA of interest. Analyzing DNA can comprise detecting genetic variants and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation). In some embodiments, the DNA of interest is one or more differentially methylated regions of the DNA. In some embodiments, the detecting or quantifying the DNA of interest comprises quantifying and / or detecting a level of methylation at one or more differentially methylated regions of the DNA. In some embodiments, quantifying and / or detecting the level ofAtty. Docket No. GH0264WOmethylation at one or more differentially methylated regions of the DNA comprises sequencing at least a portion of the amplified DNA or quantitative PCR (qPCR).

[0151] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversion such as 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.

[0152] 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 affinity 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.

[0153] When using MeDIP or MethylMiner®Methylated DNA Enrichment Kit (ThermoFisher Scientific) various levels of methylation can be partitioned using sequential elutions. For example, a hypomethylated partition (no methylation) can be separated from a methylated partition by contacting the nucleic acid population with the MBD from the kit, which is attached to magnetic beads. The beads are used to separate out the methylated nucleic acids from the non- methylated nucleic acids. Subsequently,Atty. Docket No. GH0264WOone 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 is once again used to separate higher level of methylated nucleic acids from those with lower level of methylation. The elution and magnetic separation steps can be repeated to create various partitions such as a hypomethylated partition (enriched in nucleic acids comprising no methylation), a methylated partition (enriched in nucleic acids comprising low levels of methylation), and a hyper methylated partition (enriched in nucleic acids comprising high levels of methylation).

[0154] In some methods, nucleic acids bound to an agent used for affinity separation based 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).

[0155] 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.

[0156] For further details regarding portioning nucleic acid samples based on characteristics such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0157] In some embodiments, the nucleic acid molecules can be partitioned into different partitions based on the nucleic acid molecules that are bound to a specificAtty. Docket No. GH0264WOprotein or a fragment thereof and those that are not bound to that specific protein or fragment thereof.

[0158] 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, 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).

[0159] In some embodiments, the partitioning 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.

[0160] 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.

[0161] In some embodiments, the partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.

[0162] Sequences that comprise aberrantly high copy numbers may tend to be hypermethylated. Accordingly, in some embodiments, the DNA contacted with captureAtty. Docket No. GH0264WOprobes specific for members of an epigenetic target region 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.

[0163] 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.

[0164] In some embodiments, methods disclosed 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. 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.

[0165] Methylation profiling 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 otherAtty. Docket No. GH0264WOregions, 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.Base Conversion

[0166] In some embodiments, the nucleic acids of the sample undergo base conversion methods. Such conversion methods include bisulfite conversion, oxidative bisulfite conversion, Tet-assisted bisulfite conversion, EM-seq, TAPS conversion, ACE-seq and direct-methylation sequencing (DM-seq). This allows for the determination of the modification status of nucleic acids within the sample at a single base resolution. The modification status of the nucleic acids within the sample can be used to determine a modification profile for genomic regions or interest or for the sample as a whole. In some embodiments, the sequence of base converted nucleic acids can be compared to sequences reads derived from the opposing strand of the same nucleic acid, which can be used to distinguish between base conversions and mutations present in the original nucleic acid, as described elsewhere. In other words, this comparison facilitates distinguishing mutations from base conversion by requiring mutations to have double stranded support, wherein the mutation is present on both stands of the same nucleic acid. The mutations identified to have double stranded support can be used to determine a mutational profile. On the contrary, sequence differences resulting from base conversions would not be expected to have double stranded support.

[0167] In some embodiments, the nucleic acids comprising a particular modification, identified by the base conversion methods, are quantified. Quantification of the modification in the sample can be determined, for example, using quantitative PCR (q-PCR). Q-PCR can be employed to determine the modification status of nucleic acids by utilizing 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 quantifiedAtty. Docket No. GH0264WOvalue of the modifications in the sample can be used to provide a mutational profile for the nucleic acid sample.

[0168] Such conversion procedures can comprise subjecting the DNA to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA. In some embodiments, methods disclosed herein comprise a step of subjecting DNA, or a subsample thereof, to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA, 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, DNA is subjected to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase in the DNA before library preparation using the DNA, before a first amplification of the DNA and / or before the ligation of adapters. In certain embodiments, the DNA is subjected to the procedure before or after contacting the DNA with a methylation-sensitive nuclease.

[0169] 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 cytosine (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 orAtty. Docket No. GH0264WOhmC 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.

[0170] 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 bi sulfite-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.

[0171] 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, P-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. 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.Atty. Docket No. GH0264WOSequencing 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.

[0172] 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.

[0173] 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 useAtty. Docket No. GH0264WOin 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, tertbutylamine 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 a DNA 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.

[0174] 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.

[0175] 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. bioRxiv. 2019; DOI:Atty. Docket No. GH0264WO10.1101 / 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.

[0176] 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 double-stranded 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 APOBEC3A-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.

[0177] 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 labelingAtty. Docket No. GH0264WOprocedure 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 a 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.

[0178] In some embodiments, the method provided herein further comprise subjecting the DNA or one or more subsamples thereof to a procedure that affects a first nucleobase in the DNA differently from a second nucleobase, 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 that affects a first nucleobase in the DNA differently from a second nucleobase comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base, depending on the modification status of the base. In some embodiments, the first nucleobase is an unmodified cytosine and the second nucleobase is a modified cytosine, optionally wherein the modified cytosine is 5-methylcytosine or 5-hydroxymethylcytosine. 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. In some embodiments, the methylation-sensitive conversion is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymaticAtty. Docket No. GH0264WOmethyl-seq (EM-seq) conversion, single-enzyme 5-methylcytosine sequencing (SEM-seq) conversion, or direct methylation sequencing (DM-seq). In some embodiments, the Tet-assisted conversion further comprises 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 some embodiments, the conversion procedure comprises contacting the DNA with a CpG-specific DNA methyltransferase (MTase) or a CpG-specific carboxymethyltransferase (CxMTase), a methyl donor or a carboxymethyl donor, and a cytosine deaminase. In some embodiments, the cytosine deaminase is an APOBEC enzyme, optionally wherein the APOBEC enzyme is APOBEC3A.

[0179] In some embodiments, the level of methylation is measured using single stranded methylation conversion (SSM). In some embodiments, the level of methylation is determined based on partitioning of the DNA via methyl-specific enrichment probes. In some embodiments, the level of methylation comprises a count of molecules including the one or more variant DMRs. In some embodiments, cancer is detected based on a count of molecules including the one or more variant DMRs. In some embodiments, cancer is detected when a count of molecules including the one or more variant DMRs is one or more in the sample from the subject or a subsample thereof, e.g., in a first subsample that comprises methylated DNA, e.g., to a greater extent than a second subsample. Such first and, optionally, second subsamples may be obtained by partitioning as described elsewhere herein.

[0180] In some embodiments, methods disclosed herein can comprise converting DNA, e.g., contacting DNA from one or more samples (e.g., the first, second, and / or third sample) or a subsample thereof (e.g., a hypermethylated and / or hypomethylated subsample thereof) with a deaminase, such as a methyl-sensitive deaminase, thereby providing a converted sample in which unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, conversion is performed prior to the sequencing. In some embodiments, conversion is performed prior to a capturing step. In some embodiments, conversion is performed after a capturing step and prior to the sequencing. In some embodiments, conversion occurs after one or more of contacting DNA in a sample with a CpG-binding protein, eluting CpG-dense DNA from the CpG-binding protein, an end-Atty. Docket No. GH0264WOrepair step, and / or a tagging (e.g., adapter ligation step) and may also occur before one or more of an amplification step, and a sequencing step.

[0181] In some embodiments, the methyl-sensitive deaminase is a dsDNA deaminase and / or a ssDNA deaminase. In some embodiments, the dsDNA deaminase has methylsensitive deaminase activity on ssDNA. The step of contacting the DNA with a methylsensitive 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 methyl-sensitive deaminase, see, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. DNA in the converted sample is then amplified, separated, partitioned, and / or enriched. In some embodiments, the converted DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of endrepair prior to the sequencing, prior to contacting the DNA with a methyl-sensitive deaminase, and / or prior to contacting the DNA in the sample with a CpG-binding protein.

[0182] In some embodiments, the contacting the DNA with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, all unmethylated CpGs in the sample can be converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of unmethylated CpGs are not converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of methylated CpGs can be converted to UpGs using the methyl-sensitive deaminase.

[0183] 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.

[0184] In some embodiments, the conversion procedure 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) or does not change the base pairing specificity of any modified nucleoside (e.g. modified cytosine,Atty. Docket No. GH0264WOadenosine, guanosine and thymidine (or uracil)). In methods that require denaturation for conversion, failure to denature a DNA molecule will result in non-conversion 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) nonconversion events can appear as false negatives (non-methylated regions). Random nonconversion 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 is used.

[0185] 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.

[0186] The methods described herein could in principle use any suitable methylsensitive enzymatic conversion procedure that changes the base-pairing specificity of the unmodified cytosine and thereby allows the modified base to be distinguished from the corresponding unmodified cytosine and / or other types of modification when sequenced. For example, any enzymatic conversion procedure could be used allowing 5-methylcytosine (m5c or 5-mC or 5mC) to be distinguished from unmodified cytosine. In particular embodiments, the conversion procedure converts unmodified (e.g., unmethylated) cytosines to uracils using a methyl-sensitive deaminase.

[0187] In some embodiments, the conversion procedure comprises enzymatic conversion of a nucleobase using a non-specific, modification-sensitive double-stranded DNA deaminase, e.g., as in SEM-seq. See, e.g., Vaisvila et al. Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA. Mol Cell. 2024 Mar 7;84(5):854-866.e7. SEM-Seq employs a non-specific, modification-sensitive double-stranded DNA deaminase (e.g., modification-sensitive DNA deaminase A (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 ofAtty. Docket No. GH0264WOuse, e.g., in AP0BEC3A-based protocols. Additionally, MsddA does not deaminate 5-formylated cytosines (5fC) or 5-carboxylated cytosines (5-caC). 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 conversion procedure comprises enzymatic conversion of unmodified cytosine 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 can 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 theAtty. Docket No. GH0264WONEBNext 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.

[0188] In some embodiments, a methyl-sensitive deaminase used in a method described herein is a dsDNA deaminase and / or a ssDNA deaminase. In some embodiments, the dsDNA deaminase has methyl-sensitive deaminase activity on ssDNA. The step of contacting the DNA with a methyl-sensitive 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 methyl-sensitive deaminase, see, e.g., Vaisvila et al., Mol Cell. 2024 Mar 7;84(5):854-866.e7. DNA in the converted sample is then amplified, separated, partitioned, and / or enriched. In some embodiments, the converted DNA is sequenced, and a level of methylation at one or more differentially methylated regions of the DNA is quantified. Such embodiments may also comprise a step of end-repair prior to the sequencing, prior to contacting the DNA with a methyl-sensitive deaminase, and / or prior to contacting the DNA in the sample with a CpG-binding protein.

[0189] In some embodiments, the contacting the DNA with a methyl-sensitive deaminase provides a converted sample in which at least a portion of unmethylated CpGs in the DNA are converted to UpGs. In some embodiments, all unmethylated CpGs in the sample can be converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of unmethylated CpGs are not converted to UpGs using the methyl-sensitive deaminase. In some embodiments, at least a portion of methylated CpGs can be converted to UpGs using the methyl-sensitive deaminase.

[0190] 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 canAtty. Docket No. GH0264WObe protected from conversion, for example through 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 MsddA or an MsddA-like deaminase.

[0191] 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., MsddA or an MsddA-like deaminase) 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, the 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 methyl-sensitive deaminase enzymes such as MsddA or an MsddA-like deaminase. 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.Atty. Docket No. GH0264WOAccordingly, 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.

[0192] 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 Patent 10,961,525. 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. In some embodiments, the TET2 enzyme is a human TET2 enzyme comprising a T1372S mutation.

[0193] In some embodiments, the methyl-sensitive deaminase is thermally inactivated after contacting the mCpG-dense DNA with the methyl-sensitive deaminase. In some embodiments, the thermal inactivation comprises heating or cooling of the methylsensitive deaminase to a temperature at which the methyl-sensitive deaminase has reduced or inhibited activity relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling. In some embodiments, the thermal inactivation completely inhibits the activity of the methyl-sensitive deaminase or reduces the activity of the methyl-sensitive deaminase by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 90%, about 95%, about 98%, aboutAtty. Docket No. GH0264WO99%, or 100% relative to a methyl-sensitive deaminase that has not been subjected to heating or cooling.Enrichment using capture probes

[0194] In some embodiments, the method may comprise a hybrid capture step to enrich nucleic acids of interest within the sample. The capturing step can form a separate fraction of nucleic acids that are enriched for nucleic acids comprising sequences of genomic regions of interest. Genomic regions of interest may include genomic regions of know mutations associated with disease, such as cancer.

[0195] Genomic regions of interest can comprise differentially methylated regions. Genomic regions of interest can also comprise differentially methylated regions (DMRs), which are specific genomic loci exhibiting abnormal DNA methylation patterns in cancerous cells compared to normal tissues. In the context of cancer, DMRs can be classified into hypermethylated and hypomethylated regions. Hypermethylated regions may comprise promoter regions of tumor suppressor genes. Several types of cancer have well-characterized DMRs that serve as biomarkers for diagnosis, prognosis, and therapeutic response prediction. For example, in colorectal cancer aberrant hypermethylation of the SEPT9 gene promoter can be used for early CRC detection. Additionally, hypermethylation of MLH1 can be used to detect microsatellite instability in colorectal tumors. For example, hypermethylation of RASSF1A, CDKN2A (p 16), and / or MGMT promoters can be used to detect non-small cell lung cancer (NSCLC).

[0196] In some embodiments, the genomic regions of interest may have no or minimal methylation in non-diseased cells, but are methylated in diseased cells. In some embodiments, the diseased cells can be cancer cells. Where a subject is suspected of having a particular type of cancer, the genomic regions of interest may be methylated in cancer cells of the particular cancer. Further, the genomic regions of interest may be methylated in cancer cells at a specific stage of cancer. Such regions may have a high cancer-to-normal methylation ratio. A high cancer-to-normal methylation ratio can be defined as at least 5:1, at least 10:1, at least 20:1, at least 50:1, at least 100:1, or at least 1000:1.

[0197] The cancer-to-normal methylation ratio can be determined by quantifying the DNA methylation levels at the genomic region of interest in both known cancerous andAtty. Docket No. GH0264WOknown normal tissues and then computing the methylation ratio between these values. DNA methylation levels can be measured using various techniques, including bisulfite sequencing, methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP), pyrosequencing, or methylated DNA immunoprecipitation sequencing (MeDIP-seq).

[0198] In some cases, a threshold methylation ratio can be established based on empirical data to distinguish cancerous from non-cancerous tissues. For example, a genomic region that exhibits negligible methylation in normal tissue but shows consistent hypermethylation in cancerous tissue may have a cancer-to-normal methylation ratio exceeding a predefined diagnostic threshold, thereby enabling its use as a biomarker for cancer detection. A high cancer-to-normal methylation ratio can be defined as at least 5:1, at least 10:1, at least 20:1, at least 50:1 at least 100:1, or at least 1000:1. A genomic region of interest may comprise no detectable level of methylation in non-diseased tissue, but does exhibit methylation in cancer tissue.

[0199] The use of regions with a high cancer-to-normal methylation ratio minimizes biological noise in the target genomic regions. In some embodiments, the genomic regions of interest are ranked by their cancer-to-normal methylation ratio and this ranking is used to select genomic regions that are targeted for enrichment by capture probes. Capture probes can be used to target genomic regions with the highest cancer-to-normal methylation ratio, thereby minimizing biological noise in the target genomic regions.

[0200] In some embodiments, the top 10%, the top 5%, the top 1%, the top 0.1%, the top 0.01%, the top 0.001% or the top 0.0001% of ranked genomic regions are targeted for enrichment by capture probes. In some embodiments, genomic regions with a cancer-to-normal methylation ratio are targeted for enrichment by capture probes wherein the cancer-to-normal methylation ratio is above a set threshold. In some embodiments, the threshold can be defined as at least 5:1, at least 10:1, at least 20:1, at least 50:1, at least 100:1, or at least 1000:1. The cancer-to-normal methylation ratio can be determined for a specific cancer type, or a specific cancer stage. For example, if a subject is suspected of having a particular type of cancer, the genomic regions can be selected such that they have a high cancer-to-normal methylation ratio for that particular type of cancer.

[0201] In some embodiments, genomic regions with mid cancer-to-normal methylation ratio are used. Genomic regions with mid cancer-to-normal methylation ratioAtty. Docket No. GH0264WOcan be targeted in combination with genomic regions with a high cancer-to-normal methylation ratio. The additional targeting of genomic regions with mid cancer-to-normal methylation ratio increases the number of informative regions that can be analyzed, but the genomic regions with mid cancer-to-normal methylation ratio are associated with higher biological noise. This increased noise can be mitigated, at least in part, by using error correction methods, as described elsewhere herein.

[0202] In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are those not classified as having a high cancer-to-normal methylation ratio but are within the top 10%, the top 5%, the top 1%, the top 0.1% or the top 0.01% of ranked genomic regions. In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are those not classified as having a high cancer-to-normal methylation ratio but have a cancer-to-normal methylation ratio of at least 2: 1, at least 5:1, at least 10:1, at least 20:1, at least 50:1, or at least 100:1. In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are targeted for capture enrichment. In genomic regions with a mid cancer-to-normal methylation ratio, there will be an increase in biological noise compared to genomic regions with a high cancer-to-normal methylation ratio. Increased biological noise may be a result of increased background methylation in non-diseased cells. As such, error correction methods can be used to reduce the background noise in samples derived from workflows that have enriched for genomic regions with a mid cancer-to-normal methylation ratio. Error correction methods can reduce the overall background noise by reducing technical noise in the method, such as by filtering for amplification or sequencing errors or damaged bases. In some embodiments, error correction can be performed subsequent to enrichment. In some embodiments, error correction can be performed subsequent to enrichment using capture probes.

[0203] By utilizing hybrid capture techniques, it is possible to selectively enrich for nucleic acids from these regions, and ultimately enrich for cancer-derived nucleic acids. The mutational status of the nucleic acids in the enriched fraction can be assessed and a mutational profile for the targeted sequence can be determined.

[0204] 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 sequencesAtty. Docket No. GH0264WOselected 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.

[0205] Capture groups include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, a hapten recognized by an antibody, and magnetically attractable particles. The capture group can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture group that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. The capture group can be any type of molecule that allows affinity separation of nucleic acids bearing the capture group from nucleic acids lacking the capture group. An exemplary capture group are 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.

[0206] In some embodiments, the methods herein comprise capturing nucleic acids comprising epigenetic and / or sequence-variable target regions. In some embodiments, the methods herein comprise capturing nucleic acids comprising epigenetic target regions, such as differentially methylated regions. In some embodiments, the capturing step comprises enriching for methylated nucleic acids. Cancer is known to be associated with hypermethylation across the genome. In some embodiments, the capturing step enriches for genomic regions known to be hypermethylated in cancer. In some embodiments, the methods herein comprise capturing nucleic acids from genomic regions that are known to be hypermethylated in particular types of cancer. In some embodiments, the methods herein comprise capturing nucleic acids from genomic regions that are known to be hypermethylated in particular stages of cancer. Genomic regions known to be hypermethylated in cancer can include, but are not limited to, promoter regions, enhancer regions, or CpG islands. In some embodiments, reference databases may be used to identify genomic regions which are known to be hypermethylated in cancer. Such identified regions can be enriched for in the capturingAtty. Docket No. GH0264WOstep. The identified genomic regions may be associated with a specific type of cancer and / or specific stage of cancer development.

[0207] Such regions may be captured from a sample (e.g., a subsample) that has undergone attachment of adapters, derivatization, partitioning, and / or amplification. Enriching for or capturing DNA comprising epigenetic and / or sequence-variable target regions may comprise contacting the DNA with a set of target-specific probes. When the method comprises a partitioning step, capturing may be performed prior and subsequent to the sequencing step. Capturing may be performed on one or more partitions. When capturing is performed on multiple partitions, the capture probes used for each partition may be different. In some embodiments, DNA is captured from the first partition and / or the second partition.

[0208] The capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In some embodiments, complexes of target-specific probes and DNA are formed.

[0209] In some embodiments, the capturing step may comprise capturing a plurality of sets of target regions. The target regions may comprise intronic regions, VDJ regions that may comprise rearrangements or other genomic features. The target regions may comprise epigenetic target regions, which may show differences in methylation levels depending on whether they originated from a tumor or from healthy cells. The target regions may comprise sequence-variable regions, which may show differences in sequence, other than rearrangements, depending on whether they originated from a tumor or from healthy cells. The target regions may comprise both epigenetic target regions and sequence-variable regions. The capturing step produces a captured set of DNA molecules. In some embodiments, the DNA molecules corresponding to the sequence-variable target region set are captured at a greater capture yield in the captured set of DNA molecules than DNA molecules corresponding to the epigenetic target region set. In some embodiments, a method described herein comprises contacting DNA with a set of target-specific probes, wherein the set of target-specific probes is configured to capture cfDNA corresponding to the sequence-variable target region set atAtty. Docket No. GH0264WOa greater capture yield than DNA corresponding to the epigenetic target region set. For additional discussion of capturing steps, capture yields, and related aspects, see W02020 / 160414, incorporated herein by reference.

[0210] It can be beneficial to capture DNA corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set because a greater depth of sequencing may be necessary to analyze the sequence-variable target regions with sufficient confidence or accuracy than may be necessary to analyze the epigenetic target regions. The volume of data needed to determine fragmentation patterns (e.g., to test for perturbation of transcription start sites or CTCF binding sites) or methylation status is generally less than the volume of data needed to determine the presence or absence of genetic variants, such as cancer-related sequence mutations. Capturing the target region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and / or in the same sequencing cell).

[0211] In some embodiments, partitioning is performed before the capturing step. In some embodiments, amplification is performed before the capturing step. In some embodiments, amplification is performed after the capturing step. In some embodiments, an amplification step is performed before and after the capturing step. In some embodiments, the methods further comprise sequencing the captured DNA to different degrees of sequencing depth for the epigenetic and sequence-variable target region sets and for rearrangements, consistent with the discussion herein.

[0212] In some embodiments, a capturing step is performed with probes for a sequence-variable target region set and probes for an epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition. This approach provides a relatively streamlined workflow. In some embodiments, the concentration of the probes for the sequence-variable target region set is greater than the concentration of the probes for the epigenetic target region set.

[0213] Alternatively, a capturing step is performed with a sequence-variable target region probe set in a first vessel and with an epigenetic target region probe set in a second vessel, or a contacting step is performed with a sequence-variable target region probe set at a first time and a first vessel and an epigenetic target region probe set at aAtty. Docket No. GH0264WOsecond time before or after the first time. This approach allows for preparation of separate first and second compositions comprising captured DNA corresponding to a sequence-variable target region set and captured DNA corresponding to an epigenetic target region set. The compositions can be processed separately as desired. These can then be pooled in appropriate proportions to provide material for further processing and analysis such as sequencing.

[0214] In some embodiments, a capturing step is performed to generate a fraction enriched for nucleic acids comprising sequences of a genomic region of interest. In some embodiments, the capturing step can be performed on the hypermethylated partition. In some embodiments, the capturing step can be performed on the hypomethylated partition. The capturing step can be used to decrease background noise by enriching for nucleic acids that comprise sequences from genomic regions known to be associated with diseases, such as cancer.

[0215] In some embodiments, the capturing step can be used to form a fraction enriched for nucleic acids comprising sequences of a genomic region of interest. In some embodiments, the capturing step can be performed on a sample of nucleic acids that have undergone a base conversion method. The capturing step can use probes that target the methylated nucleic acids containing the sequence of interest. Methylated nucleic acids which comprise the sequence of interest can undergo base conversion process, as detailed elsewhere. Only nucleic acids that are have a specific modification status will undergo base conversion. Nucleic acids that contain the sequence of interest but do not have the specific modification status will not undergo conversion. Probes specific to the base converted sequence of interest can be used to discriminate between differentially methylated nucleic acids that contain the same original sequence. The capture probes may be specific to nucleic acids which have undergone a base conversion method and have a sequence which represents a specific modification status of the pre-converted nucleic acid. Base conversion methods include bisulfite conversion, oxidative bisulfite conversion, Tet-assisted bisulfite conversion, EM-seq, TAPS conversion, ACE-seq and direct-methylation sequencing (DM-seq). As described elsewhere, each of these methods result in different base conversions on the target nucleic acids. For example, bisulfite conversion converts unmodified cytosine and certain modified cytosine nucleotides (e.g., 5-formyl cytosine (fC) or 5-Atty. Docket No. GH0264WOcarboxylcytosine (caC)) to uracil whereas other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted. Whereas 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. Therefore, hybrid capture probes that target base converted samples must be designed with the knowledge of the resulting sequence once the target sequence, including its mutations, have undergone the particular base conversion sequence being utilized.

[0216] Grouping Sequence Reads

[0217] In some embodiments, sequence reads of the partitions are grouped, wherein a group contains sequence reads derived from the same nucleic acid in the original sample. In some embodiments, the grouping of sequencing reads can be achieved through the use of molecular barcodes, which indicate the parent nucleic acid of the given sequence reads in the sample. In further embodiments, the sequence reads are grouped, wherein a group contains sequence reads derived from the same strand of a nucleic acid in the original sample. In some embodiments, groups can be paired such that the two groups in a pair represent opposing strands of the same nucleic acid from the original sample. Pairing of the group allows the identification of mutations with double strand support, wherein the mutation is classified as having double strand support when it is present in both strands of the same nucleic acids.

[0218] Molecular barcodes are a type of tag. 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.

[0219] 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. AAtty. Docket No. GH0264WOcollection 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.

[0220] 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, adaptors are ligated to nucleic acids in the sample before or after the partitioning step. In some embodiments, sample indexes are incorporated through overlap extension polymerase chain reaction (PCR).

[0221] 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 semirandom 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.

[0222] 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,Atty. Docket No. GH0264WOeach 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 than 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.

[0223] 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. Such numbers of identifiers are typically sufficient for different molecules having the same start and stop points to have a high probability of receiving different combinations of identifiers.

[0224] In some embodiments, DNA from one or more samples (e.g., the first, second, and / or third sample) or a subsample thereof (e.g., a hypermethylated or hypomethylated subsample thereof) can be contacted with a plurality of capture probes (which can be “capture,” “enriching,” or “enrichment”), in which molecules having target sequences are captured for subsequent analysis or depleted from a sample.Atty. Docket No. GH0264WO

[0225] In some embodiments, contacting is performed prior to a step of amplifying DNA and prior to a step of sequencing the DNA, and optionally after contacting the DNA with a deaminase.

[0226] In some embodiments, the contacting step is performed prior to a step of amplifying DNA, prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methyl-sensitive deaminase, and after separating the CpG proteinbound DNA from unbound DNA. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methyl-sensitive deaminase, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, prior to a step of sequencing the DNA, after separating the CpG protein-bound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of amplifying methylation-separated DNA, after contacting the CpG-dense DNA with the methyl-sensitive deaminase, after separating the CpG proteinbound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples. In some embodiments, the capture step is performed prior to a step of sequencing the DNA, after contacting the CpG-dense DNA with the methylsensitive deaminase, after separating the CpG protein-bound DNA from unbound DNA, and after partitioning the DNA in the sample into a plurality of subsamples.

[0227] In some embodiments, methods disclosed herein comprise capturing (i.e., enriching) one or more sets of target regions of DNA, such as cfDNA. In some embodiments, capture is performed prior to a step of amplifying DNA, prior to a step of sequencing the DNA, after contacting the DNA with a deaminase, and / or after partitioning the DNA in the sample into a plurality of subsamples.

[0228] Capture may be performed using any suitable approach using a plurality of capture probes. In some embodiments, the capture probes comprise a target-hybridizing sequence of about 80 to about 140 nucleotides in length. In some embodiments, the capture probes comprise a target-hybridizing sequence of about 90 to about 130 nucleotides in length. In some embodiments, the capture probes comprise a targethybridizing sequence of about 100 to about 120 nucleotides in length. In someAtty. Docket No. GH0264WOembodiments the capture probes comprise a target-hybridizing sequence of 100 nucleotides in length. In some embodiments, the capture probes comprise a targethybridizing sequence of 120 nucleotides in length. In some embodiments, the capture probes are about 80 to about 140 nucleotides in length. In some embodiments, the capture probes are about 90 to about 130 nucleotides in length. In some embodiments, the capture probes are about 100 to about 120 nucleotides in length. In some embodiments the capture probes are 100 nucleotides in length. In some embodiments, the capture probes are 120 nucleotides in length. In some embodiments, the capture probes are more than 120 nucleotides in length. In some embodiments, the capture probes are less than 120 nucleotides in length. Target capture can involve use of a bait set comprising oligonucleotide baits (a type of probe useful herein) labeled with a capture moiety, such as biotin or the other 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 moiety. For example, a biotin capture moiety by bead-based streptavidin. Such methods are further described in, for example, U.S. patent 9,850,523, issuing December 26, 2017, which is incorporated herein by reference.

[0229] Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, digoxygenin, a histidine tag, an affinity tag, an immunoglobulin constant domain, a hapten recognized by an antibody, and magnetically attractable particles. In some embodiments, the immunoglobulin constant domain may be bound using protein A, protein G, or a secondary antibody. In some embodiments, the secondary antibody comprises an antimouse secondary antibody. In some embodiments, the anti-mouse secondary antibody is a goat anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, or a donkey anti-mouse secondary antibody. In some embodiments, a CpG-binding protein comprises a capture moiety. The extraction moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. The capture moiety can be any type of moleculeAtty. Docket No. GH0264WOthat allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are 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.

[0230] A panel of regions targeted for enrichment can be selected such that they do not contain regions known to include the base modification used in the end repair reaction. When the end repair is performed with dNTPs comprising 5mC or 5hmC, a panel of regions targeted for enrichment may be selected such that they do not contain CpH dinucleotides which are known to be naturally methylated in the subject (e.g. humans). Such CpH dinucleotides can be identified through the use of publicly available resources (e.g. MethBank3.0: a database ofDNA methylomes across a variety of species Nucleic Acids Res 2018). Such an approach has the advantage that any detected methylated CpH dinucleotides can unambiguously be attributed to regions synthesized in the end repair.

[0231] In some embodiments, capturing comprises contacting the DNA to be captured with a set of target-specific probes. The set of target-specific probes may have any of the features described herein for sets of target-specific probes, including but not limited to in the embodiments set forth above and the sections relating to probes below.Capturing may be performed on one or more subsamples (e.g., one or more hypomethylated subsamples) of each of the first, second, and / or third samples prepared during methods disclosed herein.

[0232] The capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In some embodiments, complexes of target-specific probes and DNA are formed.

[0233] In some embodiments, a method described herein comprises capturing DNA obtained from a subject (e.g., DNA from the first, second, and / or third samples or a subsample thereof (e.g., a hypomethylated subsample thereof)) for a plurality of sets ofAtty. Docket No. GH0264WOtarget regions. In some embodiments, the DNA obtained from a subject is cell free DNA (cfDNA).

[0234] In some embodiments, the target regions comprise at least a portion of the plurality of somatic variants identified by comparing the sequencing data from the first (e.g., tumor) sample to the sequencing data from the second (e.g., buffy) sample. In some such embodiments, the target regions comprise somatic variant target regions. In some embodiments, the plurality of somatic variants comprises one or more SNV.

[0235] In some embodiments, the target regions comprise epigenetic target regions, which may show differences in methylation levels and / or fragmentation patterns depending on whether they originated from a tumor or from healthy cells. In some embodiments, the target regions comprise sequence-variable target regions, which may show differences in sequence depending on whether they originated from a tumor or from healthy cells. The capturing step produces a captured set of DNA molecules, and the DNA molecules corresponding to the sequence-variable target region set are captured at a greater capture yield in the captured set of DNA molecules than DNA molecules corresponding to the epigenetic target region set. For additional discussion of capturing steps, capture yields, and related aspects, see W02020 / 160414, which is incorporated herein by reference for all purposes.

[0236] In some embodiments, a method described herein comprises contacting DNA (e.g., cfDNA) obtained from a subject with a set of target-specific probes, wherein the set of target-specific probes is configured to capture DNA corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set.

[0237] It can be beneficial to capture DNA (e.g., cfDNA) corresponding to the sequence-variable target region set at a greater capture yield than DNA corresponding to the epigenetic target region set because a greater depth of sequencing may be necessary to analyze the sequence-variable target regions with sufficient confidence or accuracy than may be necessary to analyze the epigenetic target regions. The volume of data needed to determine fragmentation patterns (e.g., to test for perturbation of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is generally less than the volume of data needed to determine the presence or absence of cancer-related sequence mutations. Capturing theAtty. Docket No. GH0264WOtarget region sets at different yields can facilitate sequencing the target regions to different depths of sequencing in the same sequencing run (e.g., using a pooled mixture and / or in the same sequencing cell).

[0238] In various embodiments, the methods further comprise sequencing the captured DNA e.g., to different degrees of sequencing depth for the epigenetic and sequencevariable target region sets, consistent with the discussion herein.

[0239] In some embodiments, complexes of target-specific probes and DNA are separated from DNA not bound to target-specific probes. For example, where targetspecific probes are bound covalently or noncovalently to a solid support, a washing or aspiration step can be used to separate unbound material. Alternatively, where the complexes have chromatographic properties distinct from unbound material (e.g., where the probes comprise a ligand that binds a chromatographic resin), chromatography can be used.

[0240] As discussed in detail elsewhere herein, the set of target-specific probes may comprise a plurality of sets such as probes for a sequence-variable target region set and probes for an epigenetic target region set. In some such embodiments, the capturing step is performed with the probes for the sequence-variable target region set and the probes for the epigenetic target region set in the same vessel at the same time, e.g., the probes for the sequence-variable and epigenetic target region sets are in the same composition. This approach provides a relatively streamlined workflow. In some embodiments, the concentration of the probes for the sequence-variable target region set is greater than the concentration of the probes for the epigenetic target region set.

[0241] Alternatively, the capturing step is performed with the sequence-variable target region probe set in a first vessel and with the epigenetic target region probe set in a second vessel, or the contacting step is performed with the sequence-variable target region probe set at a first time and a first vessel and the epigenetic target region probe set at a second time before or after the first time. This approach allows for preparation of separate first and second compositions comprising captured DNA corresponding to the sequence-variable target region set and captured DNA corresponding to the epigenetic target region set. The compositions can be processed separately as desired (e.g., to fractionate based on methylation as described elsewhere herein) andAtty. Docket No. GH0264WOrecombined in appropriate proportions to provide material for further processing and analysis such as sequencing.

[0242] In some embodiments, a captured set of DNA (e.g., cfDNA) is provided. With respect to the disclosed methods, the captured set of DNA may be provided, e.g., by performing a capturing step prior to a sequencing step as described herein. The captured set may comprise DNA corresponding to a sequence-variable target region set, an epigenetic target region set, or a combination thereof. In some embodiments, a capture step is performed prior to a conversion step or after a conversion step.

[0243] In some embodiments, a first target region set is captured (e.g., from one or more samples (e.g., from the first, second, and / or third sample) or a subsample thereof (e.g., a hypermethylated and / or hypomethylated subsample thereof)), comprising at least epigenetic target regions. The epigenetic target regions captured from the subsample may comprise hypermethylation variable target regions. In some embodiments, the hypermethylation variable target regions are CpG-containing regions that are unmethylated or have low methylation in DNA from healthy subjects (e.g., below-average methylation relative to bulk DNA). In some embodiments, the hypermethylation variable target regions are regions that show lower methylation in healthy DNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells may shed more DNA into the bloodstream than healthy cells of the same tissue type. As such, the distribution of tissue of origin of cfDNA may change upon carcinogenesis. Thus, an increase in the level of hypermethylation variable target regions in the first subsample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0244] In some embodiments, a second target region set is captured from the (e.g., from the first, second, and / or third sample or a subsample thereof) comprising at least epigenetic target regions. The epigenetic target regions may comprise hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are CpG-containing regions that are methylated or have high methylation in DNA from healthy subjects (e.g., above-average methylation relative to bulk DNA). In some embodiments, the hypomethylation variable target regions are regions that show higher methylation in healthy DNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells may shed more DNA into theAtty. Docket No. GH0264WObloodstream than healthy cells of the same tissue type. As such, the distribution of tissue of origin of cfDNA may change upon carcinogenesis. Thus, an increase in the level of hypomethylation variable target regions in the second subsample can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.

[0245] In some embodiments the quantity of captured sequence-variable target region DNA is greater than the quantity of the captured epigenetic target region DNA, when normalized for the difference in the size of the targeted regions (footprint size).

[0246] Alternatively, first and second captured sets may be provided, comprising, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set. The first and second captured sets may be combined to provide a combined captured set.

[0247] In some embodiments, a probe (e.g., a capture probe) is specific for members of an epigenetic target region set that includes only methylated DNA molecules. In some embodiments, the methylated DNA molecules comprise methylated cytosines. In some embodiments, the methylated cytosines are in CpG dinucleotides. In some embodiments, a probe (e.g., a capture probe) is specific for members of an epigenetic target region set that includes only unmethylated DNA molecules. In some embodiments, the unmethylated DNA molecules comprise uracils as a result of the conversion of unmethylated cytosines. In some embodiments, the unmethylated molecules comprise thymines as a result of the conversion of methylated cytosines. In some embodiments, the uracils are in UpG dinucleotides. In some embodiments, the thymines are in TpG dinucleotides. In some embodiments, a probe (e.g., a capture probe) captures both unmethylated and methylated DNA molecules.

[0248] In some embodiments in which a captured set comprising DNA corresponding to the sequence-variable target region set and the epigenetic target region set includes a combined captured set as discussed above, the DNA corresponding to the sequencevariable target region set may be present at a greater concentration than the DNA corresponding to the epigenetic target region set, e.g., a 1.1 to 1.2-fold greater concentration, a 1.2- to 1.4-fold greater concentration, a 1.4- to 1.6-fold greater concentration, a 1.6- to 1.8-fold greater concentration, a 1.8- to 2.0-fold greater concentration, a 2.0- to 2.2-fold greater concentration, a 2.2- to 2.4-fold greaterAtty. Docket No. GH0264WOconcentration a 2.4- to 2.6-fold greater concentration, a 2.6- to 2.8-fold greater concentration, a 2.8- to 3.0-fold greater concentration, a 3.0- to 3.5-fold greater concentration, a 3.5- to 4.0, a 4.0- to 4.5-fold greater concentration, a 4.5- to 5.0-fold greater concentration, a 5.0- to 5.5-fold greater concentration, a 5.5- to 6.0-fold greater concentration, a 6.0- to 6.5-fold greater concentration, a 6.5- to 7.0-fold greater, a 7.0-to 7.5-fold greater concentration, a 7.5- to 8.0-fold greater concentration, an 8.0- to 8.5-fold greater concentration, an 8.5- to 9.0-fold greater concentration, a 9.0- to 9.5-fold greater concentration, 9.5- to 10.0-fold greater concentration, a 10- to 11-fold greater concentration, an 11- to 12-fold greater concentration a 12- to 13 -fold greater concentration, a 13- to 14-fold greater concentration, a 14- to 15-fold greater concentration, a 15- to 16-fold greater concentration, a 16- to 17-fold greater concentration, a 17- to 18-fold greater concentration, an 18- to 19-fold greater concentration, a 19- to 20-fold greater concentration, a 20- to 30-fold greater concentration, a 30- to 40-fold greater concentration, a 40- to 50-fold greater concentration, a 50- to 60-fold greater concentration, a 60- to 70-fold greater concentration, a 70- to 80-fold greater concentration, a 80- to 90-fold greater concentration, or a 90- to 100-fold greater concentration. The degree of difference in concentrations accounts for normalization for the footprint sizes of the target regions.

[0249] In some embodiments, the DNA that is captured comprises intronic regions. In some embodiments, the intronic regions comprise one or more introns likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells, e.g., non-neoplastic circulating cells. For example, an intron comprising a rearrangement known to be present in some neoplastic cells and absent from healthy cells can be used to differentiate DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells. In some embodiments, the rearrangement is a translocation.

[0250] In some embodiments, captured intronic regions have a footprint of at least 30 bp, e.g., at least 100 bp, at least 200 bp, at least 500 bp, at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 50 kb, at least 200 kb, at least 300 kb, or at least 400 kb. In some embodiments, the intronic target region set has a footprint in the range of 30 bp-1000 kb, e.g., 30 bp-100 bp, 100 bp-200 bp, 200 bp-500 bp, 500 bp-Ikb, 1 kb-2 kb, 2 kb-5 kb, 5 kb-10 kb, 10 kb-20 kb, 20 kb-50 kb, 50 kb-100 kb, 100-200Atty. Docket No. GH0264WOkb, 200-300 kb, 300-400 kb, 400-500 kb, 500-600 kb, 600-700 kb, 700-800 kb, 800-900 kb, and 900-1,000 kb.

[0251] Exemplary rearrangements, such as intronic translocations that can be detected using the methods described herein include but are not limited to translocations wherein at least one of the two genes involved in the translocation is a receptor tyrosine kinase. Exemplary translocation products are the BCR-ABL fusion, and fusions comprising any of ALK, FGFR2, FGFR3, NTRK1, RET, or ROSE

[0252] In some embodiments, the DNA that is captured comprises target regions having a type-specific epigenetic variation. In some embodiments, an epigenetic target region set consists of target regions having a type-specific epigenetic variation. In some embodiments, the type-specific epigenetic variations, e.g., differential methylation or a type-specific fragmentation pattern, are likely to differentiate DNA from one or more related cell or tissue types cells from DNA from other cell or tissue types present in a sample or in a subject.

[0253] In some embodiments, nucleic acids captured or enriched using a method described herein comprise captured DNA, such as one or more captured sets of DNA. In some embodiments, the captured DNA comprise target regions that are differentially methylated in different immune cell types. In some embodiments, the immune cell types comprise rare or closely related immune cell types, such as activated and naive lymphocytes or myeloid cells at different stages of differentiation.In some embodiments, a captured epigenetic target region set captured from a sample (e.g., a first, second, and / or third sample or subsample thereof) comprises hypermethylation variable target regions. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one or more related cell or tissue types. In some embodiments, the hypermethylation variable target regions are differentially or exclusively hypermethylated in one cell type or in one immune cell type, or in one immune cell type within a cluster. In some embodiments, the hypermethylation variable target regions are hypermethylated to an extent that is distinguishably higher or exclusively present in one cell type or one immune cell type or one immune cell type within a cluster. Such hypermethylation variable target regions may be hypermethylated in other cell or tissue types but not to the extent observed in the one or more related cell or tissue types. In some embodiments, the hypermethylationAtty. Docket No. GH0264WOvariable target regions show lower methylation in healthy DNA (e.g., cfDNA) than in at least one other tissue type. In some embodiments, the hypermethylation variable target regions show even higher methylation in DNA (e.g., cfDNA) from a diseased cell of the one or more related cell or tissue types. In some embodiments, target regions comprise hypermethylated regions with aberrantly high copy number. In some such embodiments, the target regions are hypermethylated in healthy and diseased colon tissue and have aberrantly high copy number in pre-cancerous or cancerous colon tissue. Examples of such target regions are shown in Table 1 below. In some embodiments, a gene is considered to comprise a DMR when the DMR is located within an untranslated region (UTR), intron, or exon of the gene, or within 5000 nucleotides of either the 5’ end of the sense strand of the 5’ UTR or the 3’ end of the sense strand of the 3’ UTR. Table 1 : Hypermethylated target regions with aberrantly high copy number in colon cancer or pre-cancerAtty. Docket No. GH0264WOTable 2. Exemplary Hypermethylation Target Regions based on Lung Cancer studiesAtty. Docket No. GH0264WO

[0254] In some embodiments, a captured epigenetic target region set captured from a sample or subsample comprises hypomethylation variable target regions. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one or more related cell or tissue types. In some embodiments, the hypomethylation variable target regions are exclusively hypomethylated in one cell type or in one immune cell type or in one immune cell type within a cluster. In some embodiments, the hypomethylation variable target regions are hypomethylated to an extent that is exclusively present in one cell type or one immune cell type or in one immune cell type within a cluster. Such hypomethylation variable target regions may be hypomethylated in other cell or tissue types but not to the extent observed in the one or more cell or tissue types. In some embodiments, the hypomethylation variable target regions show higher methylation in healthy DNA (e.g., cfDNA) than in at least one other tissue type.

[0255] Without wishing to be bound by any particular theory, in an individual with cancer, proliferating or activated immune cells and / or dying cancer cells may shed more DNA into the bloodstream than immune cells in a healthy individual and / or healthy cells of the same tissue type, respectively. As such, the distribution of cell type and / or tissue of origin of cfDNA may change upon carcinogenesis. Thus, the presence and / or levels of cfDNA originating from certain cell or tissue types can be an indicator of disease. Variations in hypermethylation and / or hypomethylation can be an indicator of disease. For example, an increase in the level of hypermethylation variable target regions and / or hypomethylation variable target regions in a subsample following a partitioning step can be an indicator of the presence (or recurrence, depending on the history of the subject) of cancer.Atty. Docket No. GH0264WO

[0256] Exemplary hypermethylation variable target regions and hypomethylation variable target regions useful for distinguishing between various cell types, including but not limited to immune cell types, have been identified by analyzing DNA obtained from various cell types via whole genome bisulfite sequencing, as described, e.g, in Scott, C.A., Duryea, J.D., MacKay, H. et al., “Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data,” Genome Biol 21, 156 (2020) (doi.org / 10.1186 / sl3059-020-02065-5). Whole-genome bisulfite sequencing data is available from the Blueprint consortium, available on the internet at dcc.blueprint-epigenome.eu.

[0257] In some embodiments, first and second captured target region sets comprise, respectively, DNA corresponding to a sequence-variable target region set and DNA corresponding to an epigenetic target region set, for example, as described in WO 2020 / 160414. The first and second captured sets may be combined to provide a combined captured set. The sequence-variable target region set and epigenetic target region set may have any of the features described for such sets in WO 2020 / 160414, which is incorporated by reference herein in its entirety. In some embodiments, the epigenetic target region set comprises a hypermethylation variable target region set. In some embodiments, the epigenetic target region set comprises a hypomethylation variable target region set. In some embodiments, the epigenetic target region set comprises CTCF binding regions. In some embodiments, the epigenetic target region set comprises fragmentation variable target regions. In some embodiments, the epigenetic target region set comprises transcriptional start sites. In some embodiments, the epigenetic target region set comprises regions that may show focal amplifications in cancer, e.g, one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAFI. For example, in some embodiments, the epigenetic target region set comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets.

[0258] In some embodiments, the sequence-variable target region set comprises a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the sequence-variable target region set targets a plurality of different genes or genomic regions (“panel”) selected such that a determined proportion of subjects having a cancer exhibits a genetic variant or tumor marker in one or more different genes or genomicAtty. Docket No. GH0264WOregions in the panel. The panel may be selected to limit a region for sequencing to a fixed number of base pairs. The panel may be selected to sequence a desired amount of DNA, e.g., by adjusting the affinity and / or amount of the probes as described elsewhere herein. The panel may be further selected to achieve a desired sequence read depth. The panel may be selected to achieve a desired sequence read depth or sequence read coverage for an amount of sequenced base pairs. The panel may be selected to achieve a theoretical sensitivity, a theoretical specificity, and / or a theoretical accuracy for detecting one or more genetic variants in a sample.

[0259] Probes for detecting the panel of regions can include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be taken into account in designing probes, and / or probes can be designed to maximize the likelihood that particular sites (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on analysis of cfDNA coverage and fragment size variation impacted by nucleosome binding patterns and GC sequence composition. Regions used herein can also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0260] Examples of listings of genomic locations of interest may be found in Table 3 and Table 4 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the genes of Table 3 of WO 2020 / 160414. In some embodiments, a sequence-variable target region set used in the methods of the present disclosure comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4 of WO 2020 / 160414. Additionally or alternatively, suitable target region sets are available from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a panel of 35 cancer-related gene targets that can be used as part or all of a sequence-variable target region set. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT,Atty. Docket No. GH0264WOKRAS, MED 12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, andU2AFl.

[0261] In some embodiments, the sequence-variable target region set comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed above and in WO 2020 / 160414.

[0262] In some embodiments, a collection of capture probes is used in methods described herein, e.g., comprising capture probes prepared by any method disclosed herein for doing so. In some embodiments, the collection of capture probes further comprises target-binding probes specific for a sequence-variable target region set and / or target-binding probes specific for an epigenetic target region set. In some embodiments, the capture yield of the target-binding probes specific for the sequence- variable target region set is higher (e.g., at least 2-fold higher) than the capture yield of the targetbinding probes specific for the epigenetic target region set. In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set higher (e.g., at least 2-fold higher) than its capture yield specific for the epigenetic target region set.

[0263] In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the capture yield of the target-binding probes specific for the epigenetic target region set. In some embodiments, the capture yield of the target-binding probes specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than the capture yield of the target-binding probes specific for the epigenetic target region set.

[0264] In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than its capture yield for the epigenetic target region set. In some embodiments, the collection of capture probes is configured to have a capture yield specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5- to 1.75-, 1.75- toAtty. Docket No. GH0264WO2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than its capture yield specific for the epigenetic target region set.

[0265] The collection of probes can be configured to provide higher capture yields for the sequence-variable target region set in various ways, including concentration, different lengths and / or chemistries (e.g., that affect affinity), and combinations thereof. Affinity can be modulated by adjusting probe length and / or including nucleotide modifications as discussed below.

[0266] In some embodiments, the capture probes specific for the sequence-variable target region set are present at a higher concentration than the capture probes specific for the epigenetic target region set. In some embodiments, concentration of the targetbinding probes specific for the sequence-variable target region set is at least 1.25-, 1.5-, 1.75-, 2-, 2.25-, 2.5-, 2.75-, 3-, 3.5-, 4-, 4.5-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, or 15-fold higher than the concentration of the target-binding probes specific for the epigenetic target region set. In some embodiments, the concentration of the targetbinding probes specific for the sequence-variable target region set is 1.25- to 1.5-, 1.5-to 1.75-, 1.75- to 2-, 2- to 2.25-, 2.25- to 2.5-, 2.5- to 2.75-, 2.75- to 3-, 3- to 3.5-, 3.5- to 4-, 4- to 4.5-, 4.5- to 5-, 5- to 5.5-, 5.5- to 6-, 6- to 7-, 7- to 8-, 8- to 9-, 9- to 10-, 10- to 11-, 11- to 12-, 13- to 14-, or 14- to 15-fold higher than the concentration of the targetbinding probes specific for the epigenetic target region set. In such embodiments, concentration may refer to the average mass per volume concentration of individual probes in each set.

[0267] In some embodiments, the capture probes specific for the sequence-variable target region set have a higher affinity for their targets than the capture probes specific for the epigenetic target region set. Affinity can be modulated in any way known to those skilled in the art, including by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in certain sequence contexts), modifications that provide a heteroatom at the 2’ sugar position, and LNA nucleotides, can increase stability of double-stranded nucleic acids, indicating that oligonucleotides with such modifications have relatively higher affinity for their complementary sequences. See, e.g., Severin et al., Nucleic Acids Res. 39: 8740-8751Atty. Docket No. GH0264WO(2011); Freier et al., Nucleic Acids Res. 25: 4429-4443 (1997); US Patent No.9,738,894. Also, longer sequence lengths will generally provide increased affinity. Other nucleotide modifications, such as the substitution of the nucleobase hypoxanthine for guanine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, the capture probes specific for the sequence-variable target region set have modifications that increase their affinity for their targets. In some embodiments, alternatively or additionally, the capture probes specific for the epigenetic target region set have modifications that decrease their affinity for their targets. In some embodiments, the capture probes specific for the sequence-variable target region set have longer average lengths and / or higher average melting temperatures than the capture probes specific for the epigenetic target region set. These embodiments may be combined with each other and / or with differences in concentration as discussed above to achieve a desired fold difference in capture yield, such as any fold difference or range thereof described above.

[0268] In some embodiments, the capture probes comprise a capture moiety. The capture moiety may be any of the capture moi eties described herein, e.g., biotin. In some embodiments, the capture probes are linked to a solid support, e.g., covalently or non-covalently such as through the interaction of a binding pair of capture moieties. In some embodiments, the solid support is a bead, such as a magnetic bead.

[0269] In some embodiments, the capture probes specific for the sequence-variable target region set and / or the capture probes specific for the epigenetic target region set are a capture probe set as discussed above, e.g., probes comprising capture moieties and sequences selected to tile across a panel of regions, such as genes.

[0270] In some embodiments, the capture probes are provided in a single composition. The single composition may be a solution (liquid or frozen). Alternatively, it may be a lyophilizate.

[0271] Alternatively, the capture probes may be provided as a plurality of compositions, e.g., comprising a first composition comprising probes specific for the epigenetic target region set and a second composition comprising probes specific for the sequence-variable target region set. These probes may be mixed in appropriate proportions to provide a combined probe composition with any of the foregoing fold differences in concentration and / or capture yield. Alternatively, they may be used inAtty. Docket No. GH0264WOseparate capture procedures (e.g., with aliquots of a sample or sequentially with the same sample) to provide first and second compositions comprising captured epigenetic target regions and sequence-variable target regions, respectively.

[0272] In some embodiments, described herein are methods of identifying and / or detecting somatic variants in a subject, e.g., having or suspected of having a disease such as cancer. In some embodiments, the DNA of the first (e.g., tumor) sample collected from the subject contains a plurality of somatic variants that are not present in the DNA of the second (e.g., buffy) sample collected from the subject. The methods provided herein comprise obtaining a panel of capture probes configured to capture DNA comprising at least a portion of the plurality of somatic variants detected in DNA collected from the subject.

[0273] In some embodiments, the portion of the plurality of somatic variants is identified by comparing the sequence data (e.g., the plurality of sequencing reads) from the first (e.g., tumor) sample to the sequence data from the second (e.g., buffy) sample collected from the subject. In some embodiments, the portion of the plurality of somatic variants comprises variants present in the sequence data from the first (e.g., tumor) sample and not present in the sequence data from the second (e.g., buffy) sample.

[0274] In some embodiments, the portion of the plurality of somatic variants used to design and manufacture the somatic variant target capture probes does not comprise somatic variants detected in the sequence data from the second (e.g., buffy sample). In some embodiments, variants detectable in the sequence data from the second sample comprise CHIP variants.

[0275] In some embodiments, the panel of capture probes comprises at least 5, 7, 10, 15, 20, 30, or 50 capture probes. In some embodiments, the subset of the plurality of somatic variants comprises at least 5, 7, 10, 15, 20, 30, or 50 capture probes.

[0276] In some embodiments, the somatic variant target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb. In some embodiments, the somatic variant target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb. In some embodiments, the somatic variant target region probe set has a footprint of at least 50 kbp, e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or atAtty. Docket No. GH0264WOleast 400 kbp. In some embodiments, the somatic variant target region probe set has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp or 1.5-2 Mbp. In some embodiments, the somatic variant target region set has a footprint of at least 2 Mbp.

[0277] In some embodiments, obtaining the panel of capture probes comprises synthesizing or having synthesized a plurality of oligonucleotide capture probes, e.g., that are complementary to at least a portion of the plurality of somatic variants.

[0278] Probes specific for epigenetic target regions

[0279] Probes for an epigenetic target region set may comprise probes specific for one or more types of target regions likely to differentiate DNA from neoplastic (e.g., tumor or cancer) cells from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein, e.g., in the sections above concerning captured sets. The probes for the epigenetic target region set may also comprise probes for one or more control regions, e.g., as described herein.

[0280] In some embodiments, the probes for the epigenetic target region set have a footprint of at least 100 kbp, e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the epigenetic target region set has a footprint in the range of 100-20 Mbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-1,000 kbp, 1-1.5 Mbp, 1.5-2 Mbp, 2-3 Mbp, 3-4 Mbp, 4-5 Mbp, 5-6 Mbp, 6-7 Mbp, 7-8 Mbp, 8-9 Mbp, 9-10 Mbp, or 10-20 Mbp. In some embodiments, the epigenetic target region set has a footprint of at least 20 Mbp.

[0281] Hypermethylation variable target regions

[0282] In some embodiments, the probes for the epigenetic target region set comprise probes specific for one or more hypermethylation variable target regions.Hypermethylation variable target regions may also be referred to herein as hypermethylated DMRs (differentially methylated regions). The hypermethylation variable target regions may be any of those set forth above. For example, in some embodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1. In someAtty. Docket No. GH0264WOembodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 2. In some embodiments, the probes specific for hypermethylation variable target regions comprise probes specific for a plurality of loci listed in Table 1 or Table 2, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2. In some embodiments, for each locus included as a target region, there may be one or more probes with a hybridization site that binds between the transcription start site and the stop codon (the last stop codon for genes that are alternatively spliced) of the gene. In some embodiments, the one or more probes bind within 300 bp of the listed position, e.g., within 200 or 100 bp. In some embodiments, a probe has a hybridization site overlapping the position listed above. In some embodiments, the probes specific for the hypermethylation target regions include probes specific for one, two, three, four, or five subsets of hypermethylation target regions that collectively show hypermethylation in one, two, three, four, or five of breast, colon, kidney, liver, and lung cancers.

[0283] Hypomethylation variable target regions

[0284] In some embodiments, the probes for the epigenetic target region set comprise probes specific for one or more hypomethylation variable target regions.Hypomethylation variable target regions may also be referred to herein as hypomethylated DMRs (differentially methylated regions). The hypomethylation variable target regions may be any of those set forth above. For example, the probes specific for one or more hypomethylation variable target regions may include probes for regions such as repeated elements, e.g., LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and satellite DNA, and intergenic regions that are ordinarily methylated in healthy cells may show reduced methylation in tumor cells.

[0285] In some embodiments, probes specific for hypomethylation variable target regions include probes specific for repeated elements and / or intergenic regions. In some embodiments, probes specific for repeated elements include probes specific for one, two, three, four, or five of LINE 1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and / or satellite DNA.Atty. Docket No. GH0264WO

[0286] Exemplary probes specific for genomic regions that show cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1. In some embodiments, the probes specific for hypomethylation variable target regions include probes specific for regions overlapping or comprising nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1.

[0287] CTCF binding regions

[0288] In some embodiments, the probes for the epigenetic target region set include probes specific for CTCF binding regions. In some embodiments, the probes specific for CTCF binding regions comprise probes specific for at least 10, 20, 50, 100, 200, or 500 CTCF binding regions, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 CTCF binding regions, e.g., such as CTCF binding regions described above or in one or more of CTCFBSDB or the Cuddapah et al., Martin et al., or Rhee et al. articles cited above. In some embodiments, the probes for the epigenetic target region set comprise at least 100 bp, at least 200 bp at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream regions of the CTCF binding sites.

[0289] Transcription start sites

[0290] In some embodiments, the probes for the epigenetic target region set include probes specific for transcriptional start sites. In some embodiments, the probes specific for transcriptional start sites comprise probes specific for at least 10, 20, 50, 100, 200, or 500 transcriptional start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcriptional start sites, e.g., such as transcriptional start sites listed in DBTSS.In some embodiments, the probes for the epigenetic target region set comprise probes for sequences at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp upstream and downstream of the transcriptional start sites.

[0291] Focal amplifications

[0292] As noted above, although focal amplifications are somatic mutations, they can be detected by sequencing based on read frequency in a manner analogous to approaches for detecting certain epigenetic changes such as changes in methylation. As such, regions that may show focal amplifications in cancer can be included in theAtty. Docket No. GH0264WOepigenetic target region set, as discussed above. In some embodiments, the probes specific for the epigenetic target region set include probes specific for focal amplifications. In some embodiments, the probes specific for focal amplifications include probes specific for one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAFI. For example, in some embodiments, the probes specific for focal amplifications include probes specific for one or more of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the foregoing targets.

[0293] Control regions

[0294] It can be useful to include control regions to facilitate data validation. In some embodiments, the probes specific for the epigenetic target region set include probes specific for control methylated regions that are expected to be methylated in essentially all samples. In some embodiments, the probes specific for the epigenetic target region set include probes specific for control hypomethylated regions that are expected to be hypomethylated in essentially all samples.

[0295] Probes specific for sequence-variable target regions

[0296] Probes for a sequence- variable target region set may comprise probes specific for a plurality of regions known to undergo somatic mutations in cancer. The probes may be specific for any sequence-variable target region set described herein. Exemplary sequence-variable target region sets are discussed in detail herein, e.g., in the sections above concerning captured sets.

[0297] In some embodiments, the sequence-variable target region probe set has a footprint of at least 0.5 kb, e.g., at least 1 kb, at least 2 kb, at least 5 kb, at least 10 kb, at least 20 kb, at least 30 kb, or at least 40 kb. In some embodiments, the sequencevariable target region probe set has a footprint in the range of 0.5-100 kb, e.g., 0.5-2 kb, 2-10 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, and 90-100 kb. In some embodiments, the sequence-variable target region probe set has a footprint of at least 50 kbp, e.g., at least 100 kbp, at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the sequence-variable target region probe set has a footprint in the range of 100-2000 kbp, e.g., 100-200 kbp, 200-300 kbp, 300-400 kbp, 400-500 kbp, 500-600 kbp, 600-700 kbp, 700-800 kbp, 800-900 kbp, 900-Atty. Docket No. GH0264WO1,000 kbp, 1-1.5 Mbp or 1.5-2 Mbp. In some embodiments, the sequence-variable target region set has a footprint of at least 2 Mbp. In some embodiments, the sequencevariable target region probe set has a footprint in the range of 100-1000 Mbp, e.g., 100-200 Mbp, 200-300 Mbp, 300-400 Mbp, 400-500 Mbp, 500-600 Mbp, 600-700 Mbp, 700-800 Mbp, 800-900 Mbp, 900-1,000 Mbp. In some embodiments, the sequence variable target region set has a footprint of at least 100 Mbp.

[0298] In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 10, at least 20, at least 50, at least 75, at least 100, at least 200, at least 500, at least 750, at least 1,000, at least 2,000, at least 5,000, at least 7,500 or at least 10,000 variants (e.g., SNVs).

[0299] In some embodiments, the sequence-variable target regions are sequenced to a depth of at least 100 million reads. In some embodiments, the sequence-variable target regions are sequenced to a depth of about 100 million to about 500 million reads, e.g., about 100-200 million, about 200-300 million, about 300-400 million, or about 400-500 million reads; or about 100 million, about 200 million, about 300 million, about 400 million, or about 500 million reads.

[0300] In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or at 70 of the genes of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for the at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 of the SNVs of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 3. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, or 3 of the indels of Table 3. In some embodiments, probes specific for the sequencevariable target region set comprise probes specific for at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the genes of Table 4. In some embodiments, probes specific for the sequence-variable target region setAtty. Docket No. GH0264WOcomprise probes specific for at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 of the SNVs of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least 1, at least 2, at least 3, at least 4, at least 5, or 6 of the fusions of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or 18 of the indels of Table 4. In some embodiments, probes specific for the sequence-variable target region set comprise probes specific for at least a portion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 of the genes of Table 5.

[0301] Table 3

[0302] Table 4Atty. Docket No. GH0264WO

[0303] Table 5Atty. Docket No. GH0264WOAtty. Docket No. GH0264WO

[0304] In some embodiments, the probes specific for the sequence-variable target region set comprise probes specific for target regions from at least 10, 20, 30, or 35 cancer-related genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESRI, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED 12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, andU2AFl.

[0305] In some embodiments, the probes specific for the sequence-variable target region set comprise probes specific for mutations wherein the mutation is one of at least two mutations within a genomic region. In some embodiments, the probes specific for the sequence-variable target region set comprise probes specific genomic regions wherein there are at least two mutations within the genomic region.

[0306] In some embodiments, the genomic region may be between 50 and 400 nucleotides. In some embodiments, the genomic region that comprises at least twoAtty. Docket No. GH0264WOmutations may be between 100 and 300 nucleotides. In some embodiments, the genomic region that comprises at least two mutations may be at least 50, 75, 100, 125, 150, 175, 200, 225, or 250 nucleotides in length. In some embodiments, the genomic region that comprises at least two mutations may be no more than 400, 375, 350, 325, 300, 275, or 250 nucleotides in length.

[0307] In some embodiments, the at least two mutations may be identified on the same molecule. In some embodiments, the at least two mutations may be identified on the same cfDNA molecule. In some embodiments, the at least two mutations may be identified on different molecules. In some embodiments, the at least two mutations may be identified on different cfDNA molecules. In some embodiments, the at least two mutations may be SNVs. In some embodiments, the at least two mutations may be any of the SNVs disclosed in Tables 3 and 4.Mutational Profile

[0308] In some embodiments, sequence reads are analyzed to obtain a mutational profile of the nucleic acids. In some embodiments, the mutational profile is obtained from nucleic acids from a partition. In some embodiments, sequence reads from a hypermethylated partition may be analyzed to obtain a mutational profile. In some embodiments, sequence reads from a hypomethylated partition may be analyzed to obtain a mutational profile. In some embodiments, sequence reads are from a partition that has been enriched for a particular characteristic, such as a specific methylation state, may be analyzed to obtain a mutational profile. In some embodiments, the sequence reads are from a sample which has undergone base conversion. In some embodiments, the sequence reads are from a sample which has undergone base conversion and subsequently enriched for target sequence, such as through use of a capture probe. In some embodiments, the mutational profile of nucleic acids is determined using only mutations with double strand support, wherein a mutation is classified as having double strand support when it is present in both strands of the same nucleic acid.

[0309] In some embodiments, the mutational profile is generated from identified trackable mutations. Trackable mutations may be, for example, mutations which are associated with cancer. In some embodiments, trackable mutations can be obtained fromAtty. Docket No. GH0264WOreference datasets. In some embodiments, trackable mutations are obtained from data from the present subject, such as data from a tumor biopsy sample from a subject.

[0310] In some embodiments, the mutations used to determine a mutational profile are found in sequences comprised in nucleic acids originating from cancer cells. If a subject is suspected of having a particular type of cancer, mutations used to determine a mutational profile can be found in sequences comprised in nucleic acids originating from the particular type of cancer. In some embodiments, the mutations used to determine a mutational profile are genomic regions of interest which are differentially methylated regions, for example regions which are differentially methylated in cancer. For example, the genomic region may be known to have minimal or no methylation in a non-diseased cell, but is methylated (e.g. hypermethylated) in diseased cells, such as cancer cells. Such regions may have a high cancer-to-normal methylation ratio. A high cancer-to-normal methylation ratio can be defined as at least 5:1, at least 10:1, at least 20:1, at least 50:1, at least 100:1, or at least 1000:1. Such regions may comprise no detectable level of methylation in non-diseased tissue, but do exhibit methylation in cancer tissue. A high cancer-to-normal methylation ratio can be defined by ranking the cancer-to-normal methylation ratio of genomic regions and determining the genomic regions within the top 10%, the top 5%, the top 1%, the top 0.1%, the top 0.01%, the top 0.001% or the top 0.0001% of ranked genomic regions as having a high cancer-to-normal methylation ratio. The cancer-to-normal methylation ratio can be determined for a specific cancer type, or a specific cancer stage.

[0311] In some embodiments, the partition undergoes an error correction step to minimise the inclusion of erroneous mutations, such as amplification or sequencing errors or damaged bases, in the mutational profile. In some embodiments, the error correction step may comprise a sequencing-based method performed on the partition. In some embodiments, the error correction methods may comprise rolling circle amplification. In some embodiments, the error correction step may comprise duplex sequencing. In some embodiments, the error correction step may comprise the grouping of sequences subsequent to amplification according to their parent nucleic acid and determining whether mutations have double strand support, wherein double strand support is when the mutation is present in both strands of the same cell free nucleic acid. In some embodiments the error correction method is performed on the hypermethylated partitionAtty. Docket No. GH0264WOand identified mutations in the hypermethylated partition are used to determine the mutational profile. In some embodiments the error correction method is performed on the hypomethylated partition and identified mutations in the hypom ethylated partition are used to determine the mutational profile.

[0312] In some embodiments, genomic regions with mid cancer-to-normal methylation ratio are analyzed. Genomic regions with mid cancer-to-normal methylation ratio can be analyzed in combination with genomic regions high a cancer-to-normal methylation ratio. The additional analysis of genomic regions with mid cancer-to-normal methylation ratio increases the number of informative regions, but the genomic regions with mid cancer-to-normal methylation ratio are associated with higher biological noise. This increased noise can be mitigated, at least in part, by using error correction methods, as described elsewhere herein.

[0313] In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are those not classified as having a high cancer-to-normal methylation ratio but are within the top 10%, the top 5%, the top 1%, the top 0.1% or the top 0.01% of ranked genomic regions. In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are those not classified as having a high cancer-to-normal methylation ratio but have a cancer-to-normal methylation ratio of at least 2: 1, at least 5:1, at least 10:1, at least 20:1, at least 50:1, or at least 100:1. In some embodiments, the genomic regions with a mid cancer-to-normal methylation ratio are analyzed. In genomic regions with a mid cancer-to-normal methylation ratio, there will be an increase in biological noise compared to genomic regions with a high cancer-to-normal methylation ratio. Increased biological noise may be a result of increased background methylation in non-diseased cells. As such, error correction methods can be used to reduce the background noise in samples derived from workflows that analyze genomic regions with a mid cancer-to-normal methylation ratio.

[0314] Error correction methods can reduce the overall background noise by reducing technical noise in the method, such as by filtering for amplification or sequencing errors or damaged bases. In some embodiments, error correction can be performed subsequent to enrichment. In some embodiments, error correction can be performed subsequent to enrichment using capture probes. In some embodiments, the error correction method is performed in an analysis step. In some embodiments, this analysis step may compriseAtty. Docket No. GH0264WOcomputational error correction. In some embodiments, the analysis may be performed by a supervised machine learning model. In some embodiments, an error correction method is performed on the partition in addition to an error correction analysis step. In some embodiments, an error correction method is performed on the hypermethylated partition in addition to an error correction analysis step. In some embodiments, an error correction method is performed on the hypomethylated partition in addition to an error correction analysis step.

[0315] In some embodiments, the mutational profile comprises the frequency of mutations observed in the sequence reads relative to a reference sequence. In some embodiments, the mutational profile comprises the frequency of nucleotide transitions, including A— G, G— A, T— C, and / or C— T. In a further embodiment, the mutational profile comprises the frequency of nucleotide transversions, including A— T, G— C, T— A, and / or C— G. In some embodiments, the mutational profile comprises the frequency of both nucleotide transitions and nucleotide transversions. In some embodiments, the mutational profile may comprise a frequency of nucleotide transitions within a predefined length, for example nucleotide transitions per Mb. In some embodiments, the mutational profile may comprise a frequency of nucleotide transversions within a predefined length, for example nucleotide transversions per Mb. In some embodiments, the mutational profile may comprise a frequency of nucleotide transitions and transversions within a predefined length, for example nucleotide transitions and transversions per Mb. In some embodiments, the mutational profile may comprise a ratio of nucleotide transversions or transitions per Mb. In some embodiments, the mutation profile may comprise a ratio of transversion to transitions. In some embodiments, the frequency of transversions or transition may be normalised. In some embodiments, the frequency may be measured per Kb, per Mb, per Gb. In some embodiments, mutations can include single nucleotide variants (SNVs), insertions / deletions (indels); and / or structural rearrangements, such as gene fusions.

[0316] The mutational profile may comprise mutations at various genomic regions. In some embodiments, the mutational profile comprises the frequency of mutations within exons, introns, regulatory regions, or intergenic regions.

[0317] In some embodiments, the mutational profile may comprise mutations wherein the mutation is one of at least two mutations within a genomic region. In someAtty. Docket No. GH0264WOembodiments, the mutational profile may comprise the frequency of mutations wherein the mutation is one of at least two mutations within a genomic region. In some embodiments, the mutational profile may be determined using identified mutations which comprise at least two mutations within a genomic region. In some embodiments, the mutational profile may comprise mutations in a genomic region wherein there are at least two mutations within the genomic region. In some embodiments, a genomic region may be used for determining the mutational profile wherein there are at least two mutations within the genomic region.

[0318] In some embodiments, the genomic region may be between 50 and 400 nucleotides. In some embodiments, the genomic region that comprises at least two mutations may be between 100 and 300 nucleotides. In some embodiments, the genomic region that comprises at least two mutations may be at least 50, 75, 100, 125, 150, 175, 200, 225, or 250 nucleotides in length. In some embodiments, the genomic region that comprises at least two mutations may be no more than 400, 375, 350, 325, 300, 275, or 250 nucleotides in length.

[0319] In some embodiments, the at least two mutations may be identified on the same molecule. In some embodiments, the at least two mutations may be identified on the same cfDNA molecule. In some embodiments, the at least two mutations may be identified on different molecules. In some embodiments, the at least two mutations may be identified on different cfDNA molecules. In some embodiments, the at least two mutations may be SNVs.

[0320] In some embodiments, the mutational profile is computationally defined using a scoring system that assigns weights to mutations based on their frequency. In some embodiments, the mutational profile is computationally defined using a scoring system that assigns weights to mutations based on their type. For example, scoring systems can assign different weights mutations based on whether the mutation is an SNV, indel, or structural rearrangements. Wherein the mutational profile comprises SNVs, scoring systems can assign different mutations based on whether the mutations is a nucleotide transition or nucleotide transversion. The scoring system can assign different weights based on whether the mutation is A— G, G— A, T— C, and / or C— T. The scoring system can assign different weights based on whether the mutation is A— T, G— C, T— A, and / or C— G. In some embodiments, the mutational profile is computationallyAtty. Docket No. GH0264WOdefined using a scoring system that assigns weights to mutations based on their genomic context. For example, a scoring system can assign weights to mutation based on whether they are found in an intron, an exon, a regulatory region, or intergenic region. In some embodiments, a scoring system can assign weights to a mutation based on whether they are found within a genomic region known to be associated with cancer. In some embodiments, the mutational profile is computationally defined using a scoring system that assigns weights to mutations based on their frequency, type and genomic context.

[0321] In some embodiments, the mutational profile is computational defined using a scoring system that assigns weights to mutations found in particular genomic regions. These genomic regions can be regions known to be associated with diseases, such as cancer. In such embodiments, mutations found within genomic regions known to be associated with diseases, such as cancer, are assigned a higher weight in the scoring system, compared to mutations found in genomic regions not known to be associated with disease, such as cancer. In some embodiments, the mutational profile is computational defined using a scoring system that assigns weights to mutations found in particular genomic regions where the sequence reads utilised are derived from the hypermethylated partition. In some embodiments, the mutational profile is computationally defined using a scoring system that assigns weights to mutations found in particular genomic regions where the sequence reads utilised are derived from the hypomethylated partition. In the hypomethylated partition there is likely to be a greater degree of background noise because the partition is likely to contain a lower proportion of nucleic acids associated with cancer. Therefore, weighting the mutations of sequence reads that are derived from genomic regions known to be associated with cancer will decrease the background noise.Data analysis

[0322] In some embodiments, analysis of the mutational profile obtained from sequence reads comprises comparing the mutational profile to a reference dataset and optionally classifying the subject based on this comparison.

[0323] In some embodiments, analysis of the mutational profile comprises determining the somatic mutational profile by comparing the mutational profile to aAtty. Docket No. GH0264WOgermline sample from the same subject. Comparison of the mutational profile to a germline sample from the same subject enables the identification of the contribution of germline mutations to the mutational profile and, therefore, the contribution of somatic mutations to the mutational profile. In some embodiments, the somatic mutational profile can be identified by comparing the mutational profile determined from a biological sample (e.g. a sample comprising cfDNA) with a matched germline sample. This allows the determination of a somatic mutational profile, obtained by removing the germline mutations from the mutational profile. Germline samples can be derived from tissues or fluids that represent an individual’s inherited, non-cancerous genome. These samples provide a reference to distinguish germline mutations (inherited genetic variants) from somatic mutations (acquired alterations specific to disease, such as cancer). For example, germline samples may comprise buffy coat samples (leukocyte fraction) from blood, saliva or buccal swabs, skin biopsies, or plasma DNA from healthy pre-disease samples. In some embodiments, the somatic mutational profile can be used for further analysis, including classification processes using MRD models ensuring only the somatic mutations are analyzed. In some embodiments, the somatic mutational profile enables the analysis of the combination of the germline and somatic mutations in the mutational profile.

[0324] Reference data sets can contain mutational profiles of known cancer types. Comparison of the mutational profile of interest with such reference datasets can inform on classification of the subject. In some embodiments, this analysis may comprise classifying the subject as having cancer or a type of cancer through comparison of the mutational profile with the reference data sets. In some embodiments, the analysis may comprise classifying the subject as having cancer or a type of cancer by applying a supervised machine learning algorithm trained on a reference dataset containing mutational profiles of known cancer types. Supervised machine learning algorithms include, but are not limited to, classification algorithms or regression models.Supervised machine learning algorithms can be trained on reference datasets to predict whether the subject has MRD.

[0325] In some embodiments, the classification process may include the integration of clinical metadata with the mutational profile. Integration of clinical metadata of the subject with a mutational profile of interest can be use to compare with reference dataAtty. Docket No. GH0264WOwhich includes integrated metadata and mutational profiles. In some embodiments, the classification process involves the integration of clinical metadata with the mutational profile, and using a machine learning algorithm trained on combined datasets.

[0326] In some embodiments, the mutational profile is analyzed using a tumour-informed disease MRD model to classify whether the subject has MRD. Analysis comprises the comparison of the mutational profile with reference data sets. Reference datasets used in tumour-informed disease MRD models may comprise datasets originating from the tumour of the subject. Such datasets can be used to identify target regions for the analysis. For example, genomic regions containing mutations in the subject’s tumour, identified from tumour-derived data (e.g. from a tumor biopsy) obtained previously from the subject, can be enriched for (e.g. by target capture or selective amplification) in the provided methods. This facilitates the determination of mutational profiles for genomic regions known to contain mutations present in the subject’s tumour. In some embodiments, the mutational profile can be integrated with clinical metadata.

[0327] Tumour-informed analysis can further comprise the classification of whether the subject has MRD based on the comparison of the mutational profile and reference data sets. In some embodiments, supervised machine learning techniques can be used for the classification of the subject based on the comparison of mutational profiles with reference data sets. Supervised machine learning algorithms that are used to analyze a mutational profile using a tumour-informed disease MRD model can include classification algorithms or regression models.

[0328] In some embodiments, the mutational profile is analyzed for tumour-naive minimal residual disease (MRD) detection, to classify whether the subject has MRD. Analysis comprises the comparison of the mutational profile with reference data sets. Reference datasets used in tumour-naive disease MRD model comprise datasets that do not originate from the present subject. The mutational profile can be integrated with clinical metadata. The analysis can further comprise the classification of whether the subject has MRD based on the comparison of the mutational profile and reference data sets. In some embodiments, supervised machine learning techniques can be used for the classification of the subject based on the comparison of mutational profiles with reference data sets. Supervised machine learning algorithms that are used to analyze aAtty. Docket No. GH0264WOmutational profile using a tumour-naive disease MRD model can include classification algorithms or regression models.

[0329] In some embodiments, the provided methods may comprise combining data regarding the methylation and the mutation profiles of a sample. In some embodiments, the combined data can be used for the detection of diseases, such as cancer.Hypermethylated nucleic acids are known to be associated with cancer and cancer development. Therefore, the integration of methylation data and mutational data from genomic regions of interest can allow for more accurate detection of cancer. In some embodiments, a hypermethylated partition from a sample can undergo base conversion to detect methylation at single base resolution. This can be integrated with a mutational profile of the sample. In some embodiments, the data can be further integrated with clinical data. Integration of the mutational profile and methylation data can be used for the classification of subject based on the comparison of the resulting integrated data with reference data sets. Such data sets may be tumour-informed or tumour-naive. In some embodiments, the integrated data can be used for MRD detection, to classify whether or not a subject has MRD. In some embodiments, supervised machine learning techniques can be used for the classification the integrated data. Supervised machine learning algorithms that are used to analyze integrated mutational profile and methylation data can include classification algorithms and / or regression models.

[0330] In some embodiments, analysis of the mutational profile can be used for the diagnosis of disease. In some embodiments, analysis of the mutational profile can be used for the monitoring of disease progression, for example through the comparison of data obtained at different time points. In some embodiments, analysis of the mutational profile can be used for the prognosis of disease. In some embodiments, analysis of the mutational profile can be used for the prediction of disease outcome or trajectory. In some embodiments, analysis of the mutational profile can be used to determine the tumour mutational burden of the subject. Determining the tumour mutational burden using the mutational profile can be utilised in prognosis and predicting disease progression. In some embodiments, analysis of the mutational profile can be used to determine the tumour mutational burden of the subject which can be used to inform future treatment plans. In some embodiments, the analysis of the mutational profiles can be used to calculate a quantitative value. The quantitative value can be compared to aAtty. Docket No. GH0264WOthreshold value, the comparison of which can allow for the detection of disease, for example cancer.

[0331] In some embodiments, the disclosed methods comprise sequencing nucleic acids, wherein the sequencing comprises: (a) linking sample indexes to nucleic acid molecules in a plurality of samples, wherein nucleic acid molecules in the same sample receive the same sample index and nucleic acid molecules in different samples receive different sample indexes; (b) partitioning the nucleic acid molecules into a plurality of aliquots so that each aliquot receives nucleic acid molecules from two or more of the plurality of samples; (c) amplifying the nucleic acid molecules; (d) sequencing amplicons of the nucleic acid molecules to produce sequencing reads, wherein a sequencing read comprises a sequence of one of the nucleic acid molecules and a sample index; (e) grouping sequencing reads using at least the sample index, wherein sequencing reads within a group are from the same aliquot and are derived from the same starting molecule in the plurality of samples; and (f) calling sequences or variants present in the sample from the sequencing reads grouped into families

[0332] In some embodiments, the disclosed methods may comprise duplex sequencing techniques to minimise errors, for example, those caused by base damage. For example, the method may comprise sequencing nucleic acids by: (a) providing an amplified cluster of a plurality of nucleic acid molecules derived from a double-stranded nucleic acid molecule which comprises a first strand and a second strand, wherein a first subset of the plurality of nucleic acid molecules each comprises a first sequence that is a copy of at least a portion of the first strand sequence and wherein a second subset of the plurality of nucleic acid molecules each comprises a second sequence that is a reverse complement copy of at least a portion of the second strand sequence; (b) collecting sequencing signals from the amplified cluster to determine a disagreement between the first sequence and the second sequence at a locus; and (c) excluding the locus from single nucleotide variant (SNV) or single nucleotide polymorphism (SNP) calling based at least in part on the disagreement.

[0333] The disclosed methods may comprise rolling-circle amplification-based sequencing techniques. Such techniques can be used to minimise sequencing errors. For example, the disclosed methods may comprise identifying a sequence variant in a nucleic acid sample comprising a plurality of polynucleotides, each polynucleotide ofAtty. Docket No. GH0264WOthe plurality having a 5' end and a 3' end, the method comprising: (a) circularizing individual polynucleotides of said plurality to form a plurality of circular polynucleotides, each of which having a junction between the 5' end and 3' end; (b) amplifying the circular polynucleotides of (a); (c) sequencing the amplified polynucleotides to produce a plurality of sequencing reads; (d) identifying sequence differences between sequencing reads and a reference sequence; and (e) calling a sequence difference that occurs in at least two circular polynucleotides having different junctions as the sequence variant.Amplification

[0334] In methods of the present disclosure, parent nucleic acids within the nucleic acid sample or partitions are amplified to provide progeny nucleic acids. Sequence reads of the progeny nucleic acids are grouped according to their parent nucleic acids. In some embodiments, sequence reads of the partitions are grouped wherein a group contains sequence reads derived from the same nucleic acid in the original sample. In some embodiments, the grouping of sequencing reads can be achieved through the use of molecular barcodes, or such tags, which indicate the parent nucleic acid of the given sequence reads in the sample. In further embodiments, the sequence reads are grouped, wherein a group contains sequence reads derived from the same strand of a nucleic acid in the original sample. In some embodiments, groups can be paired such that the two groups in a pair represent opposing strands of the same nucleic acid from the original sample. Pairing of the group allows that identification of mutation with double strand support, wherein the mutation is classified as having double strand support when it is present in both strands of the same nucleic acids.

[0335] Amplification of the nucleic acids can maximise the likelihood that downstream analysis will detect target sequences or methylation patterns present within the nucleic acid sample or partitions. In some embodiments, the method includes partitioning steps wherein amplification of the nucleic acid sample can be performed before or after the partitioning steps.

[0336] Before amplification, adapters can be ligated to the sample nucleic acids, wherein the adapters comprise primer binding sites. The sample nucleic acids flanked by adapters can then be amplified by PCR and / or other amplification methods primed by primers binding to the primer binding sites in the adapters. Amplification methodsAtty. Docket No. GH0264WOcan involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. Other amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequencebased replication.

[0337] DNA ligase can be used to ligate DNA molecules (e.g. cfDNA) in the sample with an adapter on one or both ends, i.e. to form adapted DNA. As used herein, “adapter” refers to short nucleic acids (e.g., less than about 500, less than about 100 or less than about 50 nucleotides in length, or be 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases from end to end) that are typically at least partially double-stranded and can be ligated to the end of a given sample nucleic acid. In some instances, two adapters can be ligated to a single sample nucleic acid, with one adapter ligated to each end of the sample nucleic acid.

[0338] Ligation of adapters can comprise blunt end ligation or sticky-end ligation. In some embodiments, the present methods perform dsDNA ligations with T-tailed and C-tailed adapters when the sample nucleic acids have been subjected to A-tailing, e.g. using T4 polymerase or Klenow large fragment. This increases the efficiency of ligation and results in amplification of at least 50, 60, 70 or 80% of double stranded nucleic acids. Such methods can increase the amount or number of amplified molecules relative to control methods performed with T-tailed adapters alone by at least 10, 15 or 20%.

[0339] Adapters can include nucleic acid primer binding sites to permit amplification of a sample nucleic acid flanked by adapters at both ends, and / or a sequencing primer binding site, including primer binding sites for sequencing applications, such as various next generation sequencing (NGS) applications. Adapters can include a sequence for hybridizing to a solid support, e.g., a flow cell sequence. Adapters can also include binding sites for capture probes, such as an oligonucleotide attached to a flow cell support or the like. Adapters can also include sample indexes and / or molecular barcodes. These are typically positioned relative to amplification primer and sequencing primer binding sites, such that the sample index and / or molecular barcode is included in amplicons and sequencing reads of a given nucleic acid. Adapters of the same or different sequence can be linked to the respective ends of a sample nucleic acid. In some cases, adapters of the same or different sequence are linked to the respective ends of theAtty. Docket No. GH0264WOnucleic acid except that the sample index and / or molecular barcode differs in its sequence.

[0340] In some embodiments, primers relate to oligos which specifically target and enable amplification of amplicons within a set of amplicons. The primers may be of any suitable length depending on the particular needs and targeted sequences employed. In some embodiments, the primers may at least 10 nucleotides in length. Longer primers are also within the scope of the present disclosure as well known in the art. In some embodiments, primers may be more than 30, more than 40, more than 50 nucleotides in length.

[0341] In some embodiments, the primers used for amplification can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning — A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.). To enable hybridization to occur, primers may comprise an oligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to a portion of their target sequence. In some embodiments, primers may have complete sequence identity to their target sequences.

[0342] In some embodiments, primers may contain high affinity RNA analogs such as locked nucleic acids (LNAs). LNA oligos exhibit much better thermal stability when hybridised to complementary nucleic acids compared to typical oligos. For each incorporated LNA within a primer, the melting point of the duplex increases by 2-8 °C.Incorporation of LNAs into primers can be used in the disclosed methods to improve the specificity and sensitivity of the amplification reaction. In some embodiments, primers may contain molecular barcodes.

[0343] In some embodiments, amplification can comprise amplification-based enrichment. Such methods can be performed after the separating the nucleic acid population into at least two partitions according to nucleic acid length. Amplificationbased enrichment can comprise the use of target specific primers which specifically bind to a genomic region. The target specific primers may comprise molecular barcodes.Atty. Docket No. GH0264WOIn methods comprising amplification-based enrichment, methods may comprise: (i) amplification-based enrichment of parent nucleic acids to provide a first set of progeny nucleic acids; (ii) ligation of adapters to at least a subset of the first set of progeny nucleic acids to provide a first set of ligated progeny nucleic acids; and (iii) amplification of the first set of ligated progeny nucleic acids, using primers which bind to the adapters, to provide a second set of progeny nucleic acids. The second set of progeny nucleic acids, or derivatives thereof, may then be subjected to sequencing or further assays.

[0344] In some embodiments, amplification-based enrichment can be utilized to target genomic regions of interest in the sample. In some embodiments, the genomic region of interest may be known to have no or minimal methylation in non-diseased cells, but is methylated in diseased cells. In some embodiments, the diseased cells can be cancer cells. Where a subject is suspected of having a particular type of cancer, the genomic regions of interest may be differentially methylated in cancer cells of the particular cancer, e.g. relative to healthy cells. Further, the genomic regions of interest may be differentially methylated in cancer cells at a specific stage of cancer, e.g. relative to healthy cells. Such regions may have a high cancer-to-normal methylation ratio, as described elsewhere herein. In some embodiments, regions with mid cancer-to-normal methylation ratio are targeted. Regions with mid cancer-to-normal methylation ratio are as described elsewhere herein.

[0345] In some embodiments, DNA from one or more samples (e.g., the first, second, and / or third sample) or a subsample thereof (e.g., a hypermethylated or hypomethylated subsample thereof) is amplified. In some embodiments, the DNA from the sample or a subsample thereof (e.g., a hypermethylated or hypomethylated subsample thereof) can be subjected to a plurality of distinct amplification reactions. For example, DNA in a converted sample, as described herein, can be amplified.

[0346] In some embodiments, the amplification of the DNA in the converted sample comprises using a DNA polymerase. In some embodiments, the DNA polymerase is a uracil -tolerant DNA polymerase.

[0347] In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, OneTaq® DNA Polymerase, Taq DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo Klen ' / rit / , Epimark® Hot Start Taq DNAAtty. Docket No. GH0264WOPolymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, B st 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase I (E. coh)., DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3'— >5' exo-), or any combination thereof.

[0348] In some embodiments, DNA can be amplified by methylation-preserving amplification. In some embodiments, the methylation-preserving amplification can occur before the contacting the DNA in a sample with a CpG-binding protein.

[0349] Amplification, including methylation-preserving amplification, is typically primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. For example, DNA flanked by adapters added to the DNA as described herein can be amplified by PCR or other amplification methods.Amplification methods of use herein, including methylation-preserving amplification, can include any suitable methods, such as known to those of ordinary skill in the art. In some embodiments, amplification is primed by primers binding to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling, such as polymerase chain reaction (PCR), or can be isothermal, such as in linear amplification methods, transcription-mediated amplification, recombinase polymerase amplification (RPA), helices dependent amplification (HD A), loop-mediated isothermal amplification (LAMP) (Notomi et al., Nuc. Acids Res., 28, e63, 2000), rolling-circle amplification (RCA) (Blanco et al., J. Biol. Chem., 264, 8935-8940, 1989), or hyperbranched rolling circle amplification (Lizard et al., Nat. Genetics, 19, 225-232, 1998). Other amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and selfsustained sequence based replication. In some embodiments, the methylation-preserving amplification comprises linear amplification with thermocycling.

[0350] In some embodiments, methylation-preserving amplification comprises amplification performed in the presence of a methyl transferase. Methylating agents ofAtty. Docket No. GH0264WOuse in methylation-preserving amplification methods described herein are known to those of ordinary skill in the art, and can include, for example, any suitable methyl transferase. In some embodiments, the methylating agent is DNMT1. DNMT1 is the most abundant DNA methyl transferase in mammalian cells and predominantly methylates hemimethylated CpG di-nucleotides in the mammalian genome. For example, DNA molecules replicated using PCR amplification with DNMT1 incubation will maintain their methylation status post-amplification, for use in further analyses, such as those described herein (such as an epigenetic base conversion step and / or an enrichment step).

[0351] Additional methylating agents useful herein include the mammalian methyl transferases, DNMT3a and DNMT3b, the plant methyltransferases, MET1, and CMT3. In some embodiments, DNMT1 or another suitable methyltransferase is used with a methyl donor and may be used with or without cofactors known to those of ordinary skill in the art. DNMT1 works in vitro at 95% efficiency without a cofactor; however, DNMT1 may be used with a cofactor such as NP95(Uhrfl), such as described in Bashtrykov PI, et al. “The UHRF1 protein stimulates the activity and specificity of the maintenance DNA methyltransferase DNMT1 by an allosteric mechanism.” J Biol Chem. 2014. In some embodiments, DNMT1 is used at a concentration of about 50-10000 U / mL, such as about 50-2000, about 50-5000, about 2500-7500, or about 5000-10000 U / mL. In some embodiments, DNMT1 is used at a concentration of about 100-500, about 500-1000, about 100-1000, about 1000-1500, about 500-1500, about 600-1400, about 700-1300, about 800-1200, about 900-1100, or about 950-1050 U / mL. In some embodiments, DNMT1 is used at a concentration of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or about 2000 U / mL. In some embodiments, DNMT1 is used at a concentration of about 1,000 U / ml.

[0352] In some embodiments, enriching methylated DNA in a sample comprises amplification, such as embodiments comprising quantitative PCR (qPCR) or digital PCR. Some such embodiments comprising targeted detection of DNA sequences using qPCR or digital PCR do not comprise standard DNA library preparation steps, such as adapter ligation or tagging.Atty. Docket No. GH0264WO

[0353] In some embodiments, DNA in a converted sample, as described herein, can be amplified. In some embodiments, the amplification of the DNA in the converted sample comprises using a DNA polymerase. In some embodiments, the DNA polymerase is a uracil -tolerant DNA polymerase. In some embodiments, the uracil-tolerant polymerase may be Q5U® Hot Start High-Fidelity DNA Polymerase, OneTaq® DNA Polymerase, Taq DNA Polymerase, LongAmp® Taq DNA Polymerase, Hemo Klen ' / bt / , Epimark® Hot Start Taq DNA Polymerase, Bst DNA Polymerase, Full Length, Bst DNA Polymerase, Large Fragment, Bst 2.0 DNA Polymerase, Bst 3.0 DNA Polymerase, Bsu DNA Polymerase, Large Fragment, phi29 DNA Polymerase, phi29-XT DNA Polymerase, Therminator™ DNA Polymerase, DNA Polymerase I (E. coll), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”), Klenow Fragment (3 ' — >5' exo-), or any combination thereof.

[0354] In some embodiments, amplification occurs prior to a step of capturing the DNA. In some embodiments, amplification occurs prior to a step of sequencing the captured DNA.

[0355] In some embodiments, the present methods perform dsDNA ligations with T-tailed and C-tailed adapters. The addition of C-tailed adapters can increase ligation efficiency because the A-tailing reaction can also add G-tails to a small portion of the DNA molecules, when the A tailing is performed in the presence of dGTP, such as when the A-tailing is performed in the same reaction as the end repair. The use of T-tailed and C-tailed adapters can result in amplification of at least 50, 60, 70 or 80% of double stranded nucleic acids. The present methods can increase the amount or number of amplified molecules relative to control methods performed with T-tailed adapters alone by at least 10, 15, or 20%.

[0356] Rolling Circle Amplification (RCA)

[0357] In some embodiments, an amplification of the DNA in a sample, such as a converted sample, comprises rolling-circle amplification (RCA). In some embodiments, RCA comprises contacting the DNA in the sample with a ligase, thereby obtaining circularized DNA. In some embodiments RCA comprises contacting the circularized DNA with one or more primers and then amplifying the circularized DNA.Atty. Docket No. GH0264WO

[0358] In some embodiments, the circularized DNA is amplified using a DNA polymerase. In some embodiments, the DNA polymerase is a processive, strand displacing DNA polymerase. In some embodiments, the DNA polymerase is Phi29.

[0359] In some embodiments, the circularized DNA is contacted with a plurality of primers. In some embodiments, the one or more primers are random primers. In some embodiments, the one or more primers comprise one or more target-specific primers complementary to one or more target regions on the circularized DNA.

[0360] In some embodiments, the DNA is denatured to single stranded DNA (ssDNA) prior to the circularization. In some embodiments, the DNA is contacted with a mismatch endonuclease prior to the denaturation step. In some embodiments, the sample is contacted with a protease to inactivate the ligase. In some embodiments, contacting the sample with a protease occurs after the ligation but before the amplification step (e.g., before contacting the circularized DNA with one or more primers and then amplifying the circularized DNA). In some embodiments, the protease is inactivated following inactivation of the ligase. In some embodiments, the protease is thermolabile and the inactivating comprises heating the sample. In some embodiments, the sample is contacted with an exonuclease prior to contacting the circularized DNA with the one or more primers. In some embodiments, the exonuclease is used to digest non-circularized DNA.

[0361] In some embodiments, RCA is used to prepare a RCA sequencing library. In some embodiments, the RCA sequencing library comprises a plurality of concatemers. In some embodiments, the concatemers comprise double stranded DNA. In some embodiments, the methods provided herein further comprise shearing at least a portion of the concatemers into fragments comprising at least two copies of a sequence from a circularized DNA. In some embodiments, the shearing at least a portion of the concatemers comprises enzymatic shearing or mechanical shearing. In some embodiments, the fragments are less than 200 base pairs in length.

[0362] Exemplary methods of RCA are provided, e.g., in Lou et al., Proc. Natl. Acad. Sci. 110 (49) 19872-19877 (2013) and Li et al., EMBO Mol Med. 2024 Sep;16(9):2188-2209, as well as WO201508933, each of which is incorporated by reference herein. In some embodiments, the RCA occurs prior to a step of sequencingAtty. Docket No. GH0264WOthe DNA. In some embodiments, RCA occurs prior to a step of capturing the DNA. In some embodiments, RCA occurs prior to a step of sequencing the captured DNA.

[0363] In some embodiments, adapted DNA is amplified before sequencing. This may be an additional amplification step subsequent to an earlier amplification step. In some embodiments, amplification of adapted DNA comprises RCA, e.g., as described above. In some embodiments, RCA comprises copying the circularized DNA template using a rolling circle polymerase to generate a plurality of circularized DNA templates. In some embodiments, the rolling circle polymerase is a Phi29 DNA polymerase. Amplification may in some cases be before one or more capture steps. In some embodiments, the ligation step occurs after the conversion step. In some embodiments, the ligation occurs before or simultaneously with amplification.

[0364] In some embodiments, sequencing DNA that was amplified using RCA provides a plurality of sequence reads comprising multiple copies of the sequence of an original sample molecule or converted molecule and the copies are used to determine a consensus sequence of the original sample molecule or converted molecule. In some embodiments, the sequencing reads comprise a plurality of instances of an original sequence. In some embodiments, the plurality of sequencing reads comprise at least 2 reads obtained from circular DNA. In some embodiments, the plurality of sequencing reads comprise at least 3 reads obtained from circular DNA. In some embodiments, the plurality of sequencing reads comprise at least 4 reads obtained from circular DNA.

[0365] Emulsion PCR

[0366] In some embodiments, an amplification of the DNA in a sample comprises emulsion PCR. For emulsion PCR, template DNA molecules are compartmentalized into water-in-oil droplets, which allows for separation of individual template DNA molecules (e.g., dsDNA fragments) into individual droplets.

[0367] In some embodiments, emulsion PCR can be used to separately amplify a plurality of original DNA sequences (e.g., dsDNA fragments) to generate a family of sequencing reads that can be traced back to the original DNA sequence. In some such embodiments, the sequencing reads from the plus strand of the original dsDNA sequence can be paired with the minus strand of the original dsDNA sequence (e.g., through the use of barcodes or molecular tags). In some embodiments, emulsion PCR is used in a method of preparing an error-correcting sequencing library as describedAtty. Docket No. GH0264WOherein. Such methods are further described in Cheng et al., bioRxiv [Preprint] 2025 Aug 14:2025.08.11.669689 and WO2024159179, each of which is incorporated by reference herein. This method, called Paired Plus-Minus Sequencing (“PPM-Seq”) , can be used for performing error-correcting sequencing library preparation, as described herein.End repair and A-tailing

[0368] In some embodiments, the disclosed methods comprise subjecting DNA from one or more sample (e.g., the first, second, and / or third sample) or a subsample thereof (e.g., a hypermethylated and / or hypomethylated subsample thereof) to end repair to generate end-repaired DNA molecules. In some embodiments, end repair is performed prior to the sequencing. In some embodiments, end repair is performed prior to a capturing step. In some embodiments, end repair is performed after a capturing step, and prior to the sequencing. In some embodiments, the end repair is performed before separating at least a portion of probe-target complexes from other nucleic acids in the sample. In some embodiments, the end repair is performed after separating at least a portion of probe-target complexes from other nucleic acids in the sample. In some embodiments, the end repair is performed before two or more, or each, of the steps mentioned above. In some embodiments, the end repair is performed using deoxynucleotide triphosphates (dNTPs). In some embodiments, at least one type of dNTP comprises a modified base, and the at least one dNTP comprising a modified base is incorporated into repaired regions of the end-repaired DNA molecules at one or more locations.

[0369] End repair includes methods for repairing DNA by the conversion of non-blunt 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 DNA 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.Atty. Docket No. GH0264WO

[0370] 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. 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.

[0371] 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 concatenation 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.

[0372] 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 an 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. InAtty. Docket No. GH0264WOinstances 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.

[0373] 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.

[0374] 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.

[0375] 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 a 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 theAtty. Docket No. GH0264WOpolymerase 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.

[0376] 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).

[0377] 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 efficient nick translation cannot occur in the A tailing reaction because the three of the four nucleotide components are not present in the reaction mixture. In gapped DNA, 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. These filled gaps thereby generate synthesized regions.

[0378] In some embodiments, the end-repair is performed with a polymerase which lacks 5’to 3’ exonuclease activity and / or strand displacement activity. In some cases, the polymerase used in the end repair reaction may be Q5® High-Fidelity DNA Polymerase, Q5U® Hot Start High-Fidelity DNA Polymerase, Phusion® High-Fidelity DNA Polymerase, Hemo Klen ' / bt / , phi29 DNA Polymerase, T7 DNA Polymerase, DNA Polymerase I (E. coll), DNA Polymerase I, Large (Klenow) Fragment (“Klenow fragment”) or T4 DNA Polymerase. In some embodiments, the polymerase used in theAtty. Docket No. GH0264WOend repair is T4 DNA Polymerase or Klenow fragment. In some embodiments, the end repair is performed with a DNA polymerase which has 5 ’-3’ exonuclease activity and / or is a strand displacing DNA polymerase.

[0379] In some embodiments, the methods disclosed herein comprise an A tailing reaction after the end repair and before the ligation reaction, wherein the end repair and A tailing reactions are separated by a reaction cleanup. The A tailing reaction is typically performed in the presence of dATP, but in the absence of dCTP, dTTP and dGTP. In some embodiments, the A tailing reaction is performed using Klenow Fragment lacking 3'-5' exonuclease activity.

[0380] In some embodiments, a dNTP that comprises a modified base is used in end repair, which may be any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. In some embodiments, a dNTP comprising a modified base can be used in a combined end repair and A-tailing reaction. In some embodiments, the modified base is incorporated in the synthesized regions at both CpG sites and CpH sites (i.e. CpA, CpC and CpT sites). While methylation of cytosines in non-CpG contexts has been described, it is thought to comprise 0.02% of total methyl-cytosine in differentiated somatic cells (Jang et al. Genes (Basel). 2017 Jun; 8(6): 148). The presence of methylated cytosine in non-CpG contexts in the end-repaired DNA can therefore be interpreted as being introduced during the end repair and / or A-tailing reactions. Using a dNTP comprising a modified base can therefore be used to effectively label the synthesized regions of the end repaired DNA.

[0381] In some embodiments, a dNTP that comprises a modified base may comprise any modified base wherein the presence or the absence of the modification can be detected by a type of sequencing. The modified base may be 5-caryboxylcytosine (5-caC), 4-methylcytosine (4mC), 5-methylcytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), N6-methyladenosine (6mA), bromodeoxyuridine (BrdU), 5-fluorodeoxyuridine (FldU), 5-iododeoxyuridine (IdU), 5-ethynyldeoxyuridine (EdU) and / or 8-oxoguanine (8oxoG).

[0382] When a dNTP comprising a modified base is used, it may be used in place of the equivalent unmodified base in the end repair reaction. For instance, if a dCTP comprising 5mC is used in the end repair reaction, there may be no dCTP comprising anAtty. Docket No. GH0264WOunmodified cytosine. This would ensure that dCTPs incorporated into the DNA molecule during the end repair reaction contain 5mC. In some embodiments, multiple types of dNTP comprising a modified base are used in the end repair. For example, dATP comprising 6mA and dCTP comprising 5mC can be used in the end repair reaction in place of dATP comprising unmodified adenine and dCTP comprising unmodified cytosine. The use of multiple types of dNTP comprising a modified base is advantageous because it provides increased resolution in defining the regions of the end-repaired DNA molecule which have been synthesized during the end repair reaction. This is because, in this example, the end of a synthesized region can be defined as the first unmodified adenine or unmodified cytosine after a stretch of containing 6mAs and / or 5mCs, rather than relying on the detection of solely an unmodified adenine or solely an unmodified cytosine.

[0383] The sequencing method used will depend on the type of modified base used in the end-repair reaction such that the specific modification can be detected. Exemplary conversion-based methods are described above alongside the base modification which they can detect. Moreover, nanopore-based sequencing can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU, and EdU, and single-molecule real time (SMRT) sequencing from Pacific Biosciences can be used to detect 5-caC, 4mC, 5mC, 5hmC, 6mA, and 8oxoG.

[0384] In some embodiments, the disclosed methods use at least one type of dNTP which comprises a modified base (e.g. a methylated deoxy cytidine triphosphate, such as deoxy cytidine triphosphate comprising 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC)) in the end repair reaction. In particular embodiments, the dNTP is 5mC. During end repair reactions, the methylated deoxy cytidine triphosphate will be incorporated into the synthesized regions regardless of the sequence context. This will result in methylated cytosines in non-CpG positions (i.e., methylated cytosines in a CpH context), which are very rare in nature. These methylated non-CpG cytosines can therefore be used as labels for identifying synthesized regions in the end repaired DNA molecule. Similarly, other types of dNTP which comprise a modified base uncommon or absent in nature can be used. The identification of such modified bases can be performed using sequencing, and regions comprising these modifications can be interpreted as defining regions which were synthesized in the end repair reaction.Atty. Docket No. GH0264WO

[0385] In other embodiments of the disclosed methods, the modified base is a methylated cytosine, such as 5mC or 5hmC. In some embodiments, the methylated cytosine is 5mCpH (where H = A, T, or C). In other embodiments, the modified base is other than 5mC or 5hmC. In some embodiments wherein the modified base is other than 5mC or 5hmC, a repaired region is defined as (i) the sequence between two nonmodified bases spanning a modif...

Claims

1. Atty. Docket No. GH0264WOCLAIMS:

1. A method of analyzing cell free nucleic acids obtained from a biological sample of a subject, wherein the method comprises:a. partitioning the cell free nucleic acids based on the methylation status of the cell free nucleic acids, wherein the partitioning provides a hypermethylated partition and / or a hypomethylated partition;b. sequencing cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition, or derivatives thereof, to provide sequence reads;c. analyzing the sequence reads to obtain a mutational profile of the cell free nucleic acids from the hypermethylated partition and / or a hypomethylated partition.

2. The method of claim 1, wherein adaptors are ligated to the cell free nucleic acids either before or after partitioning.

3. The method of claim 2, wherein the adaptors comprise molecular barcodes.

4. The method of claim 1, wherein the sequence reads are grouped, wherein a group contains sequence reads derived from the same cell free nucleic acid in the biological sample.

5. The method of claim 1, wherein the sequence reads are grouped, wherein a group contains sequence reads derived from the same strand of a cell free nucleic acid in the biological sample.

6. The method of the preceding claim, wherein the groups are paired such that the two groups in a pair represent different strands of the same cell free nucleic acid.Atty. Docket No. GH0264WO7. The method of the preceding claim, wherein a mutation is classified as having double strand support when it is present in both strands of the same cell free nucleic acid.

8. The method of the preceding claim, wherein the mutational profile of the cell free nucleic acids is determined using only mutations with double strand support.

9. The method of claim 1, wherein the method comprises hybrid capture of nucleic acids derived from target genomic regions.

10. The method of the preceding claim, wherein the target genomic regions comprise differentially methylated regions.

11. The method of claim 1, wherein the method comprises sequencing cell free nucleic acids from the hypermethylated partition, or derivatives thereof.

12. The method of claim 1, wherein the method comprises sequencing cell free nucleic acids from the hypomethylated partition, or derivatives thereof.

13. The method of claim 1, wherein the mutational profile comprises the frequency of mutations observed in the sequence reads relative to a reference sequence.

14. The method of claim 1, wherein the mutational profile comprises the frequency of nucleotide transitions, including A— G, G— A, T— C, and / or C— >T.

15. The method of claim 1, wherein the mutational profile comprises the frequency of nucleotide transversions, including A— T, G— C, T— A, and / or C— >G.Atty. Docket No. GH0264WO16. The method of claim 1, wherein the mutational profile comprises the frequency of mutations within exons, introns, regulatory regions, and / or intergenic regions.

17. The method of claim 1, wherein the mutational profile is computationally defined using a scoring system that assigns weights to mutations based on their frequency, type, and / or genomic context.

18. The method of claim 1, wherein the hypermethylated partition is subjected to treatment with a methylation sensitive restriction enzyme (MSRE).

19. The method of claim 1, wherein the hypomethylated partition is subjected to treatment with a methylation dependent restriction enzyme (MDRE).

20. The method of claim 1, wherein the sequencing is whole genome or whole exome sequencing.

21. The method of claim 1, wherein analyzing the mutational profile comprises comparing the mutational profile to a reference dataset and classifying the subject based on the comparison.

22. The method of the preceding claim, comprising classifying the subject as having cancer or a type of cancer by applying a supervised machine learning algorithm trained on a reference dataset containing mutational profiles of known cancer types.

23. The method of claim 21, wherein the classification process involves the integration of clinical metadata with the mutational profile, and using a machine learning algorithm trained on combined datasets.

24. The method of claim 1, wherein the mutational profile is analyzed using a tumour-informed minimal residual disease (MRD) model to classify whether the subject has MRD.Atty. Docket No. GH0264WO25. The method of claim 1, wherein the mutational profile is analyzed for tumour-naive minimal residual disease (MRD) detection, to classify whether the subject has MRD.

26. The method of claim 1, wherein analyzing the mutational profile comprises determining the somatic mutational profile by comparing the mutational profile to a germline sample from the same subject.

27. The method of claim 1, wherein the method comprises analyzing sequence reads derived from target genomic regions with a high cancer-to-normal methylation ratio.

28. The method of claim 1, wherein the method comprises enrichment of nucleic acids derived from target genomic regions with a high cancer-to-normal methylation ratio.

29. The method of claim 1, wherein the method comprises analyzing sequence reads derived from target genomic regions with a mid cancer-to-normal methylation ratio and wherein the method comprises an additional step to reduce errors in the sequence reads.

30. The method of claim 1, wherein the method comprises enrichment of nucleic acids derived from target genomic regions with mid cancer-to-normal methylation ratio and wherein the method comprises an additional step to reduce errors in the sequence reads.

31. The method of claim 1, wherein the mutational profile comprises the frequency of copy number variations (CNVs), single nucleotide variations (SNVs), insertions / deletions (indels), fusions, and / or structural variants.

32. The method of claim 1, wherein the mutational profile comprises the frequency of SNVs.

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