Quality management method

Abstract

Provided herein are methods for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample using a base pair conversion procedure. The methods use nucleosides with known nucleoside identity and known modification state in an adaptor that is ligated to the DNA. In certain aspects, the present disclosure relates to methods for improving the quality control of such methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 274,868, filed November 2, 2021, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Single base resolution for the detection of epigenetic variants or nucleoside modifications in assays generally requires the conversion of modified nucleosides or corresponding unmodified nucleosides to change their base pair specificity. The conversion is then detected by sequencing. Examples of such methods include bisulfite and oxidized bisulfite and Tet-assisted bisulfite conversion, EM-seq, TAPS and TAPS β-conversion, and ACE-seq. See, e.g., Moss et al., Nat Commun. 2018;9:5068; Booth et al., Science 2012;336:934-937; Yu et al., Cell 2012;149:1368-80; Liu et al., Nature Biotechnology 2019;37:424-429; Schutsky, EK et al.; and Vaisvila et al. Genome Research 2021 31(7):1280-1289.

[0003] In bisulfite-based and EM-Seq methylation assays, unmethylated cytosines are converted to uracils, which are PCR amplified and NGS read as thymines. Incomplete (failed) conversion of an unmethylated base results in the base being incorrectly identified as methylated, i.e., a false positive signal. Conversely, incorrect conversion of a methylated base results in the base being incorrectly identified as unmethylated, i.e., a false negative signal. Because approximately 99% of non-CpG cytosines are unmethylated in the human genome, low conversion / non-conversion of CH cytosines (i.e., bases other than cytosine followed by guanine) in a given DNA molecule is sometimes used to filter out unconverted molecules before assessing (CpG) methylation levels.

[0004] In other methods, epigenetic conversion is reversed, i.e., modified nucleosides are converted rather than unmodified nucleosides. For example, in the TAPS method from Song's lab at the Ludwig Cancer Institute, methylated cytosines (5mC and 5hmC) are converted to DHU and PCR amplified and NGS read as thymine. Incomplete / failed conversion of a methylated residue in TAPS results in the base being inaccurately identified as unmethylated, i.e., a false negative signal. Conversely, erroneous conversion of an unmethylated base results in the base being inaccurately identified as methylated, i.e., a false positive signal. In these methods, the nucleosides being converted are generally much rarer in the sample, so ineffective conversions, and the resulting false negative signals, are more difficult to adequately detect. Given the challenges surrounding false positive and false negative signals in these assays, there is a need for quality control methods that allow estimation of these false signals and therefore interpretation of the data. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Moss et al.,Nat Commun.2018;9:5068 [Non-Patent Document 2] Booth et al.,Science 2012;336:934-937 [Non-Patent Document 3] Yu et al.,Cell 2012;149:1368-80 [Non-Patent Document 4] Liu et al.,Nature Biotechnology 2019;37:424-429 Summary of the Invention [Means for solving the problem]

[0006] Described herein are methods that provide improved quality control for the conversion step in methods for detecting and / or identifying modified nucleosides in DNA samples that rely on using a base pair specific conversion procedure that is sensitive to the modification state of the nucleoside. The present disclosure includes the following exemplary embodiments.

[0007] Embodiment 1 is a quality control method for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample, comprising: (a) ligating DNA to an oligonucleotide adaptor, the adaptor comprising a quality control nucleoside comprising a modified nucleoside, the quality control nucleoside having the same nucleoside identity and the same or a different modification state relative to a modified nucleoside to be detected in the DNA, the modification state of the quality control nucleoside being known; and (b) ligating adapted DNA to an oligonucleotide adaptor comprising a modified nucleoside. A conversion procedure for modifying the base pairing specificity of a quality control nucleoside in a sample of DNA (or a subsample thereof) depending on the modification state of the nucleoside, or not modifying the base pairing specificity of the quality control nucleoside, comprising: (i) modifying the base pairing specificity of an adapted DNA nucleoside having the same nucleoside identity and modification state as the quality control nucleoside in the adapter, and not modifying the base pairing specificity of an adapted DNA nucleoside having the same nucleoside identity as the quality control nucleoside in the adapter but a different modification state; and / or (ii) not modifying the base pairing specificity of an adapted DNA nucleoside having the same nucleoside identity and modification state as the quality control nucleoside in the adapter, and not modifying the base pairing specificity of an adapted DNA nucleoside having the same nucleoside identity ... but a different modification state. (c) sequencing the adapted DNA after conversion step (b); (d) using the sequence data obtained in step (c) to determine the base pair specificity conversion of quality control nucleosides in the adapter; and (e) using the base pair specificity conversion of quality control nucleosides in the adapter as a quality control measure for conversion step (b), wherein suboptimal conversion of adapter quality control nucleosides after the conversion procedure of step (b)(i) and / or erroneous conversion of adapter quality control nucleosides after the conversion procedure of step (b)(ii) predicts false negative and / or false positive detection of modified nucleosides in the DNA sample.

[0008] Embodiment 2 is the method of embodiment 1, wherein the conversion procedure is selected to alter the base pair specificity of modified quality control nucleosides in the adapter but not alter the base pair specificity of nucleosides of the DNA sample having the same nucleoside identity but different modification state and / or no modification; and wherein the suboptimal conversion of the modified quality control nucleoside predicts false negative detection of DNA sample nucleosides having the same nucleoside identity and modification state or different modification state and the same change in base pair specificity as the quality control nucleoside upon exposure to the conversion procedure.

[0009] Embodiment 3 is the method of embodiment 1 or embodiment 2, wherein the quality control nucleosides in the adapter include 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC).

[0010] Embodiment 4 is the method of embodiment 3, wherein the converting procedure comprises a 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.

[0011] Embodiment 5 is a quality control method for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample, comprising: (a) ligating DNA to an oligonucleotide adaptor, the adaptor comprising a quality control nucleoside comprising a modified nucleoside, the quality control nucleoside having the same nucleoside identity and the same or a different modification state relative to the modified nucleoside to be detected in the DNA, the modification state of the quality control nucleoside being known; (b) a conversion procedure for the adapted DNA, or a subsample thereof, which, depending on the modification state of the nucleoside, changes the base pair specificity of the quality control nucleoside or does not change the base pair specificity of the quality control nucleoside, (i) changes the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor, and does not change the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor; and / or (ii) subjecting the adaptor to a conversion procedure selected to not alter the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor, and to alter the base pair specificity of adapted DNA nucleosides having the same nucleoside identity but a different modification state as the quality control nucleoside in the adaptor; (c) sequencing the adapted DNA after the conversion step (b); (d) using the sequence data obtained in step (c) to determine the base pair specificity conversion of the quality control nucleoside in the adaptor; and (e) using the base pair specificity conversion of the quality control nucleoside in the adaptor as a quality control measure for the conversion step (b), wherein suboptimal conversion of the quality control nucleoside in the adaptor after the conversion procedure of step (b)(i) and / or erroneous conversion of the quality control nucleoside in the adaptor after the conversion procedure of step (b)(ii) predicts false negative and / or false positive detection of modified nucleosides in the DNA sample.

[0012] Example 6 is the method of Example 5, wherein the quality control nucleosides of the adapter comprise unmodified nucleosides.

[0013] Embodiment 7 is the method of embodiment 5 or embodiment 6, wherein the conversion procedure is selected to alter the base pair specificity of quality control nucleosides in the adapter but not alter the base pair specificity of DNA sample nucleosides having the same nucleoside identity and different modification state; and suboptimal conversion of the quality control nucleosides predicts false positive detection of DNA sample nucleosides having different modification states.

[0014] Embodiment 8 is the method of embodiment 7, wherein the quality control nucleoside in the adapter comprises a cytosine and / or the conversion procedure comprises a bisulfite conversion.

[0015] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the quality control nucleosides of the adapter include a first quality control nucleoside having a first modification state and a second quality control nucleoside having a second modification state different from the first modification state.

[0016] Embodiment 10 is the method of embodiment 9, wherein the first quality control nucleoside is modified and the second quality control nucleoside is unmodified.

[0017] Embodiment 11 is the method of embodiment 9, wherein the first quality control nucleoside is a modified cytosine and the second quality control nucleoside is an unmodified cytosine.

[0018] Embodiment 12 is the method of embodiment 11, wherein the first quality control nucleoside is 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).

[0019] Embodiment 13 is the method of any one of embodiments 9 to 11, wherein the conversion procedure is selected to alter the base pair specificity of the first quality control nucleoside but not the second quality control nucleoside, or the conversion procedure is selected to alter the base pair specificity of the second quality control nucleoside but not the first quality control nucleoside.

[0020] Embodiment 14 is the method of any one of embodiments 9 to 11, wherein the conversion procedure is selected to alter the base pair specificity of modified quality control nucleosides in the adapter but not alter the base pair specificity of DNA sample nucleosides having the same nucleoside identity but a different modification state and / or no modification; and wherein suboptimal conversion of the modified quality control nucleoside predicts false negative detection of DNA sample nucleosides having the same nucleoside identity and modification state as the quality control nucleoside, or a different modification state and the same change in base pair specificity, upon exposure to the conversion procedure.

[0021] Embodiment 15 is the method of any one of embodiments 1 to 14, further comprising using the sequence data obtained in step (c) to: (i) identify adapted DNA molecules with suboptimal or erroneous conversion of quality control nucleosides in the adapter sequence; and (ii) infer suboptimal or erroneous conversion of nucleosides having the same nucleoside identity and modification state in the full-length molecule identified in step (i).

[0022] Embodiment 16 includes determining a conversion rate of a quality control nucleoside in the adapted DNA or in an individual adapted DNA molecule, (i) applying a weighting that depends on the conversion rate to an analysis of the detection of modified nucleosides in (A) the DNA sample; or (B) the individual adapted DNA molecule; (ii) detecting (A) suboptimal conversion of an adaptor quality control nucleoside or a conversion rate of an adaptor quality control nucleoside below a predefined quality control threshold; and / or (B) erroneous conversion of an adaptor quality control nucleoside or a conversion rate of an adaptor quality control nucleoside below a predefined quality control threshold from further analysis to detect modified nucleosides. and / or (iii) removing adapted DNA molecules from further analysis to detect modified nucleosides, the adapted DNA molecules having (A) suboptimal conversion of adapter quality control nucleosides or a conversion rate of adapter quality control nucleoside below a predetermined quality control threshold; and / or (B) erroneous conversion of adapter quality control nucleosides or a conversion rate of adapter quality control nucleoside above a predetermined quality control threshold.

[0023] Embodiment 17 is the method of any one of embodiments 1 to 16, further comprising enriching DNA by capturing the set of target regions from the sample, the capture step occurring before, after or during the ligation step (a) and the conversion step (b).

[0024] Embodiment 18 is the method of any one of embodiments 1 to 17, further comprising: (i) comparing the sequence data obtained in step (c) with (A) a predetermined reference sequence; and / or (B) sequence data obtained by sequencing a subsample of DNA that has not been subjected to the conversion procedure; and (ii) identifying point differences between the converted DNA sequence and the reference sequence (A) or the unconverted DNA sequence data (B) as nucleosides having a modification state that allows for a change in base pair specificity upon exposure to the conversion procedure.

[0025] Embodiment 19 is the method of any one of embodiments 1-18, wherein the DNA comprises cell-free DNA (cfDNA), optionally cfDNA obtained from a test subject, and optionally, the test subject is a patient having or suspected of having cancer.

[0026] Embodiment 20 is the method of any one of embodiments 1-19, further comprising using detection of modified nucleosides in the DNA sample to determine or predict the presence of DNA produced by a cancer cell or tumor, to determine the probability that a test subject has a tumor or cancer, or to characterize a cancer or tumor in a subject.

[0027] Embodiment 21 is the method of any one of embodiments 1 to 20, wherein the subsamples of DNA do not undergo a conversion step prior to sequencing, the converted and unconverted subsamples have different adapter sequences, and the converted and unconverted subsamples are recombined for sequencing step (c).

[0028] Embodiment 22 is the method of any one of embodiments 1 to 21, further comprising analyzing the DNA to detect copy number variations, single base mutations, insertions, deletions, methylations, and / or fusions.

[0029] Embodiment 23 is the method of any one of embodiments 1 to 22, further comprising capturing the epigenetic target region from the adaptor-ligated DNA and amplifying and sequencing the epigenetic target region.

[0030] Embodiment 24 is the method of embodiment 23, wherein the captured epigenetic target regions form a set of epigenetic target regions.

[0031] Embodiment 25 is the method of embodiment 24, wherein the set of epigenetic target regions comprises a plurality of type-specific epigenetic target regions, and the type-specific epigenetic target regions are type-specific differentially methylated regions and / or type-specific fragments.

[0032] Embodiment 26 is the method of embodiment 25, wherein the plurality of type-specific epigenetic target regions comprises type-specific hypomethylated regions.

[0033] Embodiment 27 is the method of any one of embodiments 1 to 26, wherein the sample is a blood sample.

[0034]

[0031] Embodiment 28 is the method of any one of embodiments 25-27, wherein the plurality of type-specific epigenetic target regions comprises a target region that is hypermethylated in immune cells compared to non-immune cell types present in the blood sample; differentially methylated in colon compared to other tissue types; differentially methylated in breast compared to other tissue types; differentially methylated in liver compared to other tissue types; differentially methylated in kidney compared to other tissue types; differentially methylated in pancreas compared to other tissue types; differentially methylated in prostate compared to other tissue types; differentially methylated in skin compared to other tissue types; or differentially methylated in bladder compared to other tissue types.

[0035] Embodiment 29 is the method of any one of embodiments 25-28, wherein the multiple type-specific epigenetic target regions include target regions that are hypomethylated in non-immune blood cells compared to methylation levels of the target regions in different cell or tissue types in the sample; in immune cell-specific fragments compared to non-immune cell types present in the blood sample; or in colon-, lung-, breast-, liver-, kidney-, pancreas-, prostate-, skin-, or bladder-specific fragments compared to other tissue types.

[0036] Embodiment 30 is the method of any one of embodiments 23 to 29, further comprising identifying at least one cell type or tissue type that is the source of the type-specific epigenetic target region.

[0037] Embodiment 31 is the method of any one of embodiments 23 to 30, wherein the level of a type-specific epigenetic target region originating from a cell type or tissue type is determined.

[0038] Embodiment 32 is the method of embodiment 31, wherein the level of a type-specific epigenetic target region originating from an immune cell, a non-immune blood cell, colon, lung, breast, liver, kidney, prostate, skin, bladder, or pancreas is determined.

[0039] Embodiment 33 is the method of embodiment 32, wherein the type-specific epigenetic target region comprises a cell type-specific, tissue type-specific, and / or cancer type-specific epigenetic target region.

[0040] Embodiment 34 is the method of any one of embodiments 27 to 33, wherein the blood sample is fractionated prior to capturing at least the set of epigenetic target regions of DNA.

[0041] Embodiment 35 includes dividing a sample or an aliquot thereof into a plurality of divided subsamples, including a first divided subsample and a second divided subsample; contacting the second divided subsample with a methylation-dependent nuclease, thereby degrading non-specifically divided DNA in the second subsample to produce a treated second subsample, and optionally contacting the first divided subsample with a methylation-sensitive endonuclease, thereby degrading non-specifically divided DNA in the first divided subsample to produce a treated first subsample. 35. The method of any one of embodiments 1-34, further comprising generating a subsample of a first subsample of DNA having a greater proportion of cytosine modifications than the second subsample, wherein the epigenetic target region is captured from at least a portion of the first subsample or the processed first subsample, and optionally, the DNA from the subject that has been contacted with the one or more capture probes comprises DNA from the first divided subsample, the processed first subsample, the second divided subsample, and / or the processed second subsample.

[0042] Embodiment 36 is the method of embodiment 35, wherein the cytosine modification is methylation, optionally wherein the cytosine modification is methylation at the 5 position of cytosine.

[0043] Embodiment 37 is the method of embodiment 35 or embodiment 36, wherein the first subsample is contacted with a methylation-sensitive endonuclease.

[0044] Embodiment 38 is the method of embodiment 37, wherein the methylation-sensitive endonuclease cleaves unmethylated CpG sequences.

[0045] Embodiment 39 is the method of any one of embodiments 35 to 38, wherein the methylation-sensitive endonuclease is one or more of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr1OI, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI.

[0046] Embodiment 40 is the method of any one of embodiments 1 to 39, wherein the conversion procedure comprises bisulfite conversion; protection of 5hmC; Tet-assisted bisulfite conversion; optionally, Tet-assisted conversion with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; protection of hmC followed by Tet-assisted conversion, optionally, with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; protection of hmC followed by deamination of mC and / or C; optionally, chemical-assisted conversion with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; or enzymatic protection of a modified cytosine followed by deamination of the unprotected cytosine to uracil.

[0047] Embodiment 41 is the method of embodiment 40, wherein the substituted borane reducing agent is 2-picoline borane or borane pyridine.

[0048] Embodiment 42 is the method of embodiment 40, wherein deamination of mC and / or C comprises treatment with an AID / APOBEC family DNA deaminase enzyme.

[0049] Embodiment 43 is the method of embodiment 40, wherein the protection of hmC comprises glycosylation of hmC.

[0050] Embodiment 44 is the method of any one of embodiments 1 to 43, further comprising detecting the presence or absence of sequence variation and / or determining the fragmentation pattern, wherein adapted DNA comprising quality control nucleosides representing suboptimal or erroneous conversion of the quality control nucleosides is included in detecting the presence or absence of sequence variation and / or determining the fragmentation pattern.

[0051]

[0023] Embodiment 45 is a kit comprising an adaptor comprising a quality control nucleoside, a modified nucleoside, and one or more of a conversion reagent and a ligase.

[0052] Embodiment 46 is the kit of embodiment 45, comprising a conversion reagent capable of altering the base pair specificity of the quality control nucleosides.

[0053] Embodiment 47 is the kit of embodiment 45, comprising a conversion reagent that is not capable of substantially altering the base pair specificity of the quality control nucleosides.

[0054] Embodiment 48 is the kit of any one of embodiments 45 to 47, wherein the conversion reagent is a conversion reagent used in bisulfite conversion, oxidized bisulfite conversion, Tet-assisted (TAB) conversion, Tet-assisted conversion with a substituted borane reducing agent, protection of hmC combined with Tet-assisted conversion with a substituted borane reducing agent, APOBEC-linked epigenetic (ACE) conversion, or enzymatic conversion of a nucleobase.

[0055] Embodiment 49 is the method of any one of embodiments 1 to 44 or the kit of any one of embodiments 45 to 48, wherein the oligonucleotide adaptor comprises a sequencing primer binding site and the quality control nucleoside is located downstream of the sequencing primer binding site. "Downstream of" the primer binding site for an adaptor at the 5' end of a strand means located 3' of the primer binding site, and for an adaptor at the 3' end of a strand means located 5' of the primer binding site.

[0056] Embodiment 50 is the method of any one of embodiments 1 to 44 or 49, further comprising amplifying the DNA using a primer that targets the adapter, wherein the amplification step occurs between the conversion step (b) and the sequencing step (c).

[0057] In an aspect, the disclosure provides a quality control method for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample, comprising: (a) ligating DNA to an oligonucleotide adaptor, the adaptor comprising a quality control nucleoside comprising a modified nucleoside, the quality control nucleoside having the same nucleoside identity and the same or a different modification state relative to a modified nucleoside to be detected in the DNA, the modification state of the quality control nucleoside being known; (b) applying to the adapted DNA, or a subsample thereof, a conversion procedure that alters the base pair specificity of the quality control nucleoside or does not alter the base pair specificity of the quality control nucleoside depending on the modification state of the nucleoside, (i) altering the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor, and not altering the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor, and not altering the base pair specificity of adapted DNA nucleosides having the same nucleoside identity but a different modification state as the quality control nucleoside in the adaptor. and / or (ii) subjecting the adaptor to a conversion procedure selected to not alter the base pair specificity of adapted DNA nucleosides having the same nucleoside identity and modification state as the quality control nucleoside in the adaptor, and to alter the base pair specificity of adapted DNA nucleosides having the same pair identity as the quality control nucleoside in the adaptor but a different modification state; (c) sequencing the adapted DNA after the conversion step (b); (d) using the sequence data obtained in step (c) to determine the base pair specificity conversion of the quality control nucleoside in the adaptor; and (e) using the base pair specificity conversion of the quality control nucleoside in the adaptor as a quality control measure for the conversion step (b), wherein suboptimal conversion of the quality control nucleoside in the adaptor after the conversion procedure of step (b)(i) and / or erroneous conversion of the quality control nucleoside in the adaptor after the conversion procedure of step (b)(ii) predicts false negative and / or false positive detection of modified nucleosides in the DNA sample.

[0058] In some embodiments, the method further comprises using the sequence data obtained in step (c) to (i) identify adapted DNA molecules with suboptimal or erroneous conversions of quality control nucleosides in the adapter sequence; and (ii) infer (additional) suboptimal or erroneous conversions of nucleosides with the same nucleoside identity and modification state in the full-length molecules identified in step (i).

[0059] In some embodiments, the method includes determining a conversion rate of a quality control nucleoside in the adapted DNA or individual adapted DNA molecules, and (i) applying a weighting that depends on the conversion rate to an analysis of detection of modified nucleosides in (A) the DNA sample; or (B) the individual adapted DNA molecule; and (ii) detecting from further analysis to detect modified nucleosides: (A) suboptimal conversion of an adaptor quality control nucleoside, or a conversion rate of an adaptor quality control nucleoside below a quality control threshold or a predefined quality control threshold; and / or (B) erroneous conversion of an adaptor quality control nucleoside, or a quality control rate of an adaptor quality control nucleoside below a quality control threshold or a predefined quality control threshold; and / or (iii) removing from further analysis to detect modified nucleosides adapted DNA molecules having (A) suboptimal conversion of adapter quality control nucleosides or a conversion rate of adapter quality control nucleosides below the quality control threshold or a pre-defined quality control threshold; and / or (B) erroneous conversion of adapter quality control nucleosides or a conversion rate of adapter quality control nucleosides above the quality control threshold or a pre-defined quality control threshold.

[0060] In some embodiments, the adapter quality control nucleosides comprise modified nucleosides.

[0061] When using a conversion procedure that uses quality control nucleosides of modified adapters to convert the base pair specificity of the modified nucleosides, false negatives (i.e., nucleosides incorrectly identified as unmodified) can be detected. Thus, in some embodiments, the conversion procedure is selected to change the base pair specificity of the modified quality control nucleosides in the adapter, but not the base pair specificity of nucleosides of DNA samples that have the same nucleoside identity but different modification state and / or no modification compared to the quality control nucleoside; suboptimal conversion of the modified quality control nucleoside predicts false negative detection of nucleosides of DNA samples that have the same identity and modification state as the quality control nucleoside, or the same change in base pair specificity when exposed to the conversion procedure. That is, conversion of the base pair specificity of an unmodified quality control nucleoside would predict that other unmodified nucleosides in the DNA sample having the same nucleoside identity (or modified nucleosides in the DNA sample having the same nucleoside identity as the quality control nucleoside but a different modification state that protects the nucleoside from a change in base pair specificity upon exposure to the conversion procedure) would be erroneously identified as having the modification state of the quality control nucleoside (or, if applicable, a different modification that still allows the same change in base pair specificity upon exposure to the conversion procedure).

[0062] In some embodiments, the quality control nucleosides in the adaptor include 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC). In some embodiments, the conversion procedure includes Tet-assisted conversion with a substituted borane reducing agent, optionally the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0063] Also, false negatives (i.e., nucleosides incorrectly identified as unmodified) can be detected when using a conversion procedure that converts unmodified nucleosides using quality control nucleosides of modified adapters. Thus, in some embodiments, the conversion procedure is selected not to change the base pair specificity of the quality control nucleosides in the adapter, but to change the base pair specificity of nucleosides of a DNA sample that have the same nucleoside identity but a different modification state and / or no modification compared to the quality control nucleoside, where erroneous conversion of the quality control nucleoside predicts a false negative detection / identification of a nucleoside of a DNA sample that has the same nucleoside identity and modification state or the same base pair specificity change as the quality control nucleoside upon exposure to the conversion procedure. That is, conversion of the base pair specificity of the modified quality control nucleoside predicts that nucleosides of other DNA samples having the same nucleoside identity and the same modification (or a different modification state, but the same expected change in base pair specificity upon exposure to the conversion procedure) will be erroneously identified as having an unmodified (or, if applicable, a different modification that protects the nucleoside from a change in base pair specificity upon exposure to the conversion procedure). In some embodiments, the quality control nucleoside in the adapter comprises 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC). In some embodiments, the conversion procedure comprises bisulfite conversion.

[0064] In some embodiments, the adapter quality control nucleosides comprise unmodified nucleosides.

[0065] In other cases, when using a conversion procedure that converts unmodified nucleosides using quality control nucleosides of unmodified adapters, false positives (i.e., nucleosides incorrectly identified as modified) can be detected. Thus, in some embodiments, the conversion procedure is selected to change the base pair specificity of the quality control nucleosides in the adapters, but not the base pair specificity of nucleosides of DNA samples that have the same nucleoside identity but different modification states compared to the quality control nucleosides; here suboptimal conversion of the quality control nucleosides predicts false positive detection of nucleosides of DNA samples with different modification states. That is, non-conversion of the base pair specificity of the unmodified quality control nucleoside predicts that unmodified nucleosides of other DNA samples having the same nucleoside identity (and / or, if applicable, the same nucleoside identity and a different modification state, specifically, if the nucleoside does not protect against a change in base pair specificity upon exposure to the conversion procedure) as the quality control nucleoside will be erroneously identified as modified (or having a modification, if applicable, that does not protect the nucleoside against a change in base pair specificity upon exposure to the conversion procedure). In some embodiments, the quality control nucleoside in the adapter comprises a cytosine. In some embodiments, the conversion procedure comprises a bisulfite conversion.

[0066] Also, false positives (i.e., nucleosides incorrectly identified as modified) can be detected when a conversion procedure is used that converts modified nucleosides using quality control nucleosides of unmodified adapters. Thus, in some embodiments, the conversion procedure is selected to not change the base pair specificity of the quality control nucleosides in the adapter, but to change the base pair specificity of nucleosides of a DNA sample that has the same nucleoside identity but a different modification state compared to the quality control nucleoside; here, incorrect conversion of the quality control nucleoside predicts a false positive detection of the nucleoside of the DNA sample with a different modification state. That is, conversion of the base pair specificity of an unmodified quality control nucleoside predicts that other unmodified nucleosides in the DNA sample having the same nucleoside identity (or modified nucleosides in the DNA sample having the same nucleoside identity as the quality control nucleoside but a different modification state that protects the nucleoside from changes in base pair specificity upon exposure to the conversion procedure) will be erroneously identified as having the modification state of the quality control nucleoside (or, if applicable, a different modification that still allows the same change in base pair specificity upon exposure to the conversion procedure). In some embodiments, the quality control nucleoside in the adapter comprises a cytosine. In some embodiments, the conversion procedure comprises Tet-assisted conversion with a substituted borane reducing agent, optionally the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane.

[0067] The method may further comprise enriching DNA by capturing the set of target regions from the sample. The capture step may occur before, after or during the ligation step (a) and the conversion step (b).

[0068] The sequencing step (c) allows the identification of modified nucleosides in the initial sample as those that have been converted. For example, the method may comprise: (i) comparing the sequence data obtained in step (c) with (A) a (predetermined) reference sequence; and / or (B) sequence data obtained by sequencing a subsample of DNA that has not been subjected to the conversion procedure; and (ii) identifying point differences between the converted DNA sequence and the reference sequence (A) or the unconverted DNA sequence data (B) as nucleotides having a modification state that allows a change in base pair specificity upon exposure to the conversion procedure.

[0069] In some embodiments, the DNA comprises cell-free DNA (cfDNA). The cfDNA may be obtained, for example, from a test subject. In some cases, the test subject is a patient who has or is suspected of having cancer.

[0070] In some cases, the methods may further include using detection of modified nucleosides in the DNA sample to determine or predict the presence of DNA produced by a cancer cell or tumor, to determine the probability that a test subject has a tumor or cancer, or to characterize a cancer or tumor in a subject.

[0071] In some embodiments, the subsamples of DNA are not subjected to a conversion procedure prior to sequencing. In some cases, the converted and unconverted subsamples may have different adapter sequences. The converted and unconverted subsamples may be recombined for sequencing step (c). The different adapter sequences may be used to distinguish sequences or molecules from exposed and unexposed subsamples in a later step or analysis.

[0072] In a further aspect, the disclosure provides a method for analyzing a modified nucleoside profile of DNA in a sample, the method comprising: (a) ligating the DNA to an oligonucleotide adaptor comprising one or more known modified nucleosides; (b) subjecting the adapted DNA, or a subsample thereof, to a conversion procedure that alters the base pair specificity of the known modified nucleosides in the adaptor; (c) sequencing the adapted DNA of step (b); (d) using the sequence data to determine the conversion rate of the known modified nucleosides in the adaptor; and (e) using the conversion rate determined in step (d) to estimate the conversion rate of the modified nucleoside in the DNA sample. In other embodiments, step (d) comprises using the sequence data obtained in step (c) to determine the base pair specificity conversion of a quality control nucleoside in the adaptor; and step (e) comprises using the base pair specificity conversion of the quality control nucleoside in the adaptor as a quality control measure in the conversion step (b). The sequencing step (c) allows the identification of modified nucleosides in the initial sample as having been converted. For example, the method may comprise: (f) comparing the sequence data obtained in step (c) with (A) a predefined reference sequence; and / or (B) sequence data obtained by sequencing a subsample of DNA that has not been subjected to the conversion procedure; and (g) identifying point differences between the converted DNA sequence and the reference or unconverted DNA sequence as modified nucleotides in the DNA sample.

[0073] The method of the present disclosure can be used to estimate the conversion rate on either the sample level or the molecular level. Thus, in a further aspect, the present disclosure provides a method for analyzing the modified nucleoside profile of DNA in a sample, comprising: (a) ligating the DNA to an oligonucleotide adaptor containing one or more known modified nucleosides; (b) subjecting the adapted DNA, or a subsample thereof, to a conversion procedure that changes the base pair specificity of the known modified nucleosides in the adaptor; (c) sequencing the adapted DNA of step (b); (d) using the sequence data to identify adapted DNA molecules with suboptimal conversion of the known modified nucleosides in the adaptor; and (e) inferring / predicting suboptimal conversion of the modified nucleotides in the full-length adapted DNA molecules identified in step (d). The method may further comprise: using the sequence data to determine the conversion rate of the known modified nucleosides in the adaptor of one or more molecules identified in step (d); and using the determined conversion rate to estimate the conversion rate of the full-length adapted DNA molecule.

[0074] In a further aspect, the present disclosure provides a method for detecting modified nucleosides in a DNA sample. The method may include any set of steps (a) to (e) described above. Other features described above are also applicable to such a method, as appropriate. For example, the method may further include: (f) comparing the sequence data obtained in step (c) with (A) a predefined reference sequence; and / or (B) sequence data obtained by sequencing a subsample of DNA that was not subjected to the conversion procedure; and (g) identifying point differences between the converted DNA sequence and the reference or unconverted DNA sequence as modified nucleotides in the DNA sample. Step (e) provides a quality control measure for the method.

[0075] The conversion rate determined in step (d) provides a quality control measure for the conversion procedure and can be used to estimate the proportion of false negatives, i.e. modified residues in the initial sample that are not effectively converted by the conversion procedure and therefore erroneously identified.

[0076] Thus, in a further aspect, the present disclosure provides a quality control method for analyzing the modified nucleoside profile of DNA in a sample or for detecting modified nucleosides in a DNA sample. The method may comprise any set of steps (a) to (e) described above. Other features described above are also applicable to such a method, as appropriate.

[0077] In some embodiments, the method further comprises applying analytical weights to the DNA samples or individual adapted DNA molecules in the samples, where the weights depend on the conversion rates determined in step (d). Typically, a higher weight is given to samples or molecules with higher determined conversion rates, and a lower weight is given to samples or molecules with lower determined conversion rates. The weights reflect the level of confidence that can be assigned to the modified nucleoside profile determined by sequencing the DNA molecules of the sample after conversion. In some cases, samples or molecules with suboptimal conversion or conversion rates below a (predetermined) quality control threshold may be excluded from further analysis.

[0078] In some embodiments, the results of the methods disclosed herein are used as input to generate a report. The report may be in paper or electronic format. For example, the detection of false positives and / or false negatives as obtained by the methods disclosed herein or information obtained therefrom can be presented directly in such a report. Alternatively, or in addition, diagnostic information or treatment recommendations based at least in part on the methods disclosed herein can be included in the report.

[0079] Various steps of the methods disclosed herein may be performed at the same or different times, in the same or different geographic locations, eg, countries, and / or by the same or different people.

[0080] 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]

[0081] [Figure 1] FIG. 1 illustrates the differences between three single-base-resolution methylation assays: bisulfite sequencing, NEB EM-seq, and TAPS. All three methods distinguish methylcytosine and hydroxymethyl-cytosine from unmethylated cytosine. However, TAPS has an inverse conversion logic to the other two methods, changing the base pair of methylated cytosine (sequences decoded as "T") and leaving unmethylated cytosine (further decoded as "C") unchanged, whereas bisulfite sequencing and EM-seq change the base pair of unmethylated cytosine (sequences decoded as "T") and leaving methylated cytosine (further decoded as "C"). [Diagram 2] FIG. 2 is a schematic diagram of an example system suitable for use in some embodiments of the present disclosure. [Diagram 3] 3 illustrates an embodiment of a quality control method for monitoring false negative and / or false positive detection of DNA undergoing a TAPS base conversion procedure. Adapters containing 5mC (e.g., in molecular barcodes) are ligated to DNA and then undergo the TAPS conversion procedure, altering the base pair specificity of methylated cytosines (sequences decoded as "T"), but not unmethylated cytosines (further decoded as "C"). DNA molecules containing unconverted 5mC in the adapter can be used to infer suboptimal conversion of 5mC in the full-length molecule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] 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 encompass all alternatives, modifications, and equivalents that may be included within the invention as defined by the appended claims.

[0083] Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, as they may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, "nucleic acid" includes a plurality of nucleic acids.

[0084] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​are understood to be approximations that take into account significant figures and errors associated with the measurements.

[0085] Unless specifically stated in the specification above, embodiments herein that recite various components as "comprising" are also intended to "consist of" or "consisting essentially of" the recited components.

[0086] The section headings used herein are for organizational purposes only and are not to be construed as limiting the disclosed subject matter in any way.

[0087] All patents, patent applications, websites, other publications or documents, etc. 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 was specifically and individually indicated to be incorporated by reference. Where different versions of publications, websites, etc. are published at different times, the most recent version published as of the effective filing date of this application is meant, unless otherwise indicated.

[0088] definition As used herein, "base pair specificity" refers to the standard DNA base (A, C, G, or T) with which a given base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pair specificity (i.e., specificity for G), while uracil and cytosine have different base pair specificities, since uracil has base pair specificity for A, while cytosine has base pair specificity for G. For example, the ability of uracil to wobble pair with G is irrelevant, since uracil also most preferentially pairs with A of the four standard DNA bases.

[0089] Nucleosides of the "same identity" or "same nucleoside identity" refer to nucleosides having the same base, regardless of the modification state of that base. For example, cytosine is considered to be of the "same identity" as 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), regardless of their different modification states.

[0090] "Conversion reagent" refers to a reagent that can be used to change the base pair specificity of at least one modified or unmodified nucleoside in a nucleic acid. For example, bisulfite is a conversion reagent that can be used to change an unmodified cytosine (having base pair specificity for G) to uracil (having base pair specificity for A), and Tet enzyme and pyridine borane are conversion reagents that can be used together to change a methylated or hydroxymethylated cytosine (having base pair specificity for G) to dihydrouracil (having base pair specificity for A). A conversion procedure is a procedure that uses one or more conversion reagents to change the base pair specificity of at least one modified or unmodified nucleoside in a nucleic acid.

[0091] A conversion reagent or procedure "cannot substantially alter the base pairing specificity" of a nucleoside having a first modification state (which may be modified or unmodified) if the conversion reagent or procedure preferentially alters the base pairing specificity of that nucleoside having a second, different modification state to the point where an alteration to the base pairing specificity of a nucleoside having a first modification state would be properly considered erroneous. For example, bisulfite conversion cannot substantially alter the base pairing specificity of 5mC (a nucleoside having a first modification state), but preferentially alters the base pairing specificity of unmodified cytosine (a nucleoside having a second modification state). In contrast, TAPS preferentially alters the base pairing specificity of 5mC (and 5hmC) (either of which may be considered to have a second modification state), but TAPS cannot substantially alter the base pairing specificity of unmodified C (a nucleoside having a first modification state).

[0092] "Capturing" one or more target nucleic acids refers to preferentially isolating or separating one or more target nucleic acids from non-target nucleic acids.

[0093] A "captured set" of nucleic acids refers to the nucleic acids that are undergoing capture.

[0094] A "target region set" or "set of target regions" refers to multiple genomic loci that are targeted for capture and / or targeted by a set of probes (eg, through sequence complementarity).

[0095] "Corresponding to a set of target regions" means that a nucleic acid, e.g., cfDNA, originates from a locus in the set of target regions or specifically binds to one or more probes for the set of target regions.

[0096] "Sequence variable target region" refers to a target region that may exhibit sequence changes, such as nucleotide substitutions (i.e., single nucleotide variations), insertions, deletions, or gene fusions or rearrangements, in neoplastic cells (e.g., tumor and cancer cells) relative to normal cells. A set of sequence variable target regions is a set of sequence variable target regions. In some embodiments, a sequence variable target region is a target region that may exhibit changes affecting 50 or fewer contiguous nucleotides, e.g., 40, 30, 20, 10, 5, 4, 3, or 2 or fewer nucleotides, or affecting one nucleotide.

[0097] "Epigenetic target region" refers to a target region that may exhibit sequence-independent variation across tissue types (e.g., a target region that has a different degree of methylation in solid tissue types than in hematopoietic cells) or that may exhibit differences in neoplastic cells, e.g., tumor cells or cancer cells, from normal cells. In some embodiments, an epigenetic target region exhibits sequence-independent differences in cfDNA originating from tissue types that do not normally contribute substantially to cfDNA, e.g., lung, colon, etc., relative to background cfDNA, e.g., cfDNA originating from hematopoietic cells. In some embodiments, an epigenetic target region exhibits sequence-independent differences in cfDNA from subjects with cancer relative to cfDNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, methylation changes (increases or decreases), nucleosome distribution, cfDNA fragmentation patterns, CCCTC-binding factor ("CTCF") binding, transcription start sites, and regulatory protein binding regions. An epigenetic target region set is a set of epigenetic target regions. Thus, epigenetic target region sets include, but are not limited to, hypermethylated variable target region sets, hypomethylated variable target region sets, and fragmented variable target region sets, such as CTCF binding sites and transcription start sites. For present purposes, loci that are subject to neoplasia, tumor, or cancer-associated focal amplifications and / or gene fusions can also be included in the epigenetic target region set, for example, because detection of copy number changes or fused sequences that map to two or more loci in a reference genome by sequencing tends to be more similar to detection of the representative epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, in that focal amplifications and / or gene fusions are detectable at relatively shallow sequencing depths because their detection does not depend on the accuracy of base requirements at one or a few individual locations.

[0098] As used herein, "epigenetic features" refers to any feature of DNA or chromatin other than the primary sequence (i.e., the sequence of A, C, G, and T bases). Epigenetic features include covalent modifications of bases, such as methylation, and the modification and positioning of histones and other stable DNA-associated proteins.

[0099] As used herein, a "differentially methylated region" refers to a region that has a detectably different degree of methylation in at least one type of tissue relative to the degree of methylation in cell-free DNA from a healthy subject. In some embodiments, a differentially methylated region of DNA has a detectably different degree of methylation in at least one type of tissue relative to the degree of methylation in another type of tissue; or in a sample from a healthy subject relative to the degree of methylation in a subject with a precancer, cancer, or neoplasm. In some embodiments, a differentially methylated region has a detectably higher degree of methylation in at least one type of tissue relative to the degree of methylation in cell-free DNA from a healthy subject. In some embodiments, a differentially methylated region has a detectably lower degree of methylation in at least one type of tissue relative to the degree of methylation in cell-free DNA from a healthy subject. In some embodiments, the differentially methylated region is hypomethylated in the erythroid lineage or in immature erythrocytes (e.g., reticulocytes) and hypermethylated in at least one non-erythroid cell or tissue type (e.g., leukocytes or solid tissue cell types, e.g., epithelial cells, muscle cells, etc.).

[0100] As used herein, "type-specific" in the context of epigenetic mutations refers to epigenetic mutations that are present in one cell or tissue type, or in a number of related cell or tissue types, to a detectably different degree relative to other cell or tissue types. Similarly, a "type-specific epigenetic target region" is an epigenetic target region that has a detectably different epigenetic feature in one cell or tissue type, or in a number of related cell or tissue types, to other cell or tissue types. Exemplary epigenetic features are discussed in the definition of epigenetic target region above. For example, a "type-specific differentially methylated region" is a region of DNA that has a detectably different degree of methylation in one cell or tissue type, or in a number of related cell or tissue types, to other cell or tissue types. Examples of type-specific differentially methylated regions include tissue-specific differentially methylated regions, such as those with copy number gain in early cancers. In some embodiments, the capture, identification, and / or detection of type-specific differentially methylated regions facilitates the identification of the cell type or tissue type from which the DNA originates. The cell or tissue from which the type-specific differentially methylated regions originate may be a wild-type cell or tissue or a neoplastic cell or tissue. In another example, a "type-specific fragment" of DNA is a DNA fragment resulting from a type-specific fragmentation pattern that is detectably present in one cell or tissue type, or in multiple related cell or tissue types, relative to other cell or tissue types. In some embodiments, the type-specific fragment is present only in a particular cell or tissue type. In some embodiments, the type-specific fragment is detectably present to a greater extent in a particular cell or tissue type.

[0101] As used herein, a "blood sample" refers to a sample containing whole blood or components thereof (eg, plasma, serum, buffy coat, plasma pellet).

[0102] "Buffy coat" refers to a portion of a blood (such as whole blood) or bone marrow sample that contains all or most of the sample's white blood cells and platelets. A buffy coat fraction of a sample can be prepared from the sample using centrifugation to separate sample components by density. For example, after centrifugation of a whole blood sample, the buffy coat fraction is located between the plasma layer and the erythrocyte (red blood cell) layer. Buffy coats 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.

[0103] As used herein, "DNA annealed to a primer" means a DNA to which at least one primer is annealed.

[0104] An "intron region" of DNA herein encodes an intron or a portion thereof. Intron regions include "J intron regions," which are sequences that intervene between germline J gene segments (e.g., in immunoglobulin or T cell receptor loci) and are removed during somatic V(D)J recombination, as well as regions that encode introns that are removed during post-transcriptional splicing of the pre-mRNA to generate the mRNA. An "exon region" of DNA herein encodes at least one exon or a portion thereof in a pre-mRNA or mRNA. In some embodiments, the exon region is a VDJ exon region, meaning that it encodes one or more V, D, or J exons in the pre-mRNA or mRNA. An "exon-exon junction region" of DNA herein encodes at least one exon-exon junction in a pre-mRNA (e.g., an immunoglobulin or T cell receptor pre-mRNA) before any introns are removed by splicing. Exon-exon junction regions can be formed, for example, by V(D)J recombination, when a J segment is joined to a D or V segment, or a V segment is joined to a D or J segment.

[0105] As used herein, a DNA "structural variation" or "structural variant" is a mutation that comprises a DNA sequence not present in the wild-type genome other than a point mutation (e.g., where at least 5, 10, 20, or 50 contiguous nucleotides differ from the wild-type sequence at the corresponding locus). Examples of DNA structural variations include rearrangements, such as transitions, insertions, deletions, duplications, copy number variations, and inversions. As used herein, a "structural variation sequence" or "structural variant sequence" is a DNA sequence that comprises or consists of a portion or the entirety of a structural variation.

[0106] As used herein, "partitioning" of nucleic acids, such as DNA molecules, refers to the separation, fractionation, selection, or enrichment of a sample or population of nucleic acids into multiple subsamples or subpopulations of nucleic acids based on one or more modifications or characteristics that are in different proportions in each of the multiple subsamples or subpopulations. Partitioning may include physically dividing the nucleic acid molecules based on the presence or absence of one or more methylated nucleic acid bases. A sample or population may be divided into one or more divided subsamples or subpopulations based on characteristics that signify genetic or epigenetic changes or disease states.

[0107] As used herein, the form of a sample "as originally isolated" refers to the composition or chemical structure of the sample at the time it is isolated and before it is subjected to any procedure that alters the chemical structure of the isolated sample. Similarly, a feature "as originally present" in a molecule refers to the feature present in the "original molecule" or in the molecule "originally comprising" the feature before that molecule is subjected to any procedure that alters the chemical structure of the molecule.

[0108] As used herein, "without substantial alteration of base pair specificity" of a given nucleobase means that the majority of molecules that contain that nucleobase that are sequenceable have no alteration of the base pair specificity of the given nucleobase relative to the base pair specificity that was present in the sample that was originally isolated. In some embodiments, 75%, 90%, 95%, or 99% of the molecules that contain that nucleobase that are sequenceable have no alteration of the base pair specificity relative to the base pair specificity that was present in the sample that was originally isolated. As used herein, "altered base pair specificity" of a given nucleobase means that the majority of molecules that contain that nucleobase that are sequenceable have an alteration of the base pair specificity at that nucleobase relative to the base pair specificity in the sample that was originally isolated.

[0109] As used herein, a "combination" containing multiple members refers to either a single composition or a set of contiguous compositions containing the members, e.g., in separate containers or compartments that are contained within a larger container, e.g., a multi-well plate, tube rack, refrigerator, freezer, incubator, aquarium, ice bucket, appliance, or other form of storage.

[0110] As used herein, a "label" is a capture moiety, fluorophore, oligonucleotide, or other moiety that facilitates detection, separation, or isolation of the one to which it is attached.

[0111] As used herein, a "capture moiety" is a molecule that allows for affinity separation of molecules linked to the capture moiety from molecules lacking the capture moiety. Exemplary capture moieties include biotin, which allows for affinity separation by binding to streptavidin that is linked or linkable to a solid phase, or oligonucleotides that allow for affinity separation through binding to complementary oligonucleotides that are linked or linkable to a solid phase.

[0112] As used herein, "capture probe" refers to a probe that includes a capture moiety and is generated by amplification, and thus includes an amplicon of a template DNA. In some embodiments, the amplification includes polymerase chain reaction (PCR).

[0113] As used herein, "antiparallel orientation" of two primers means that the primers anneal to a nucleic acid in opposite orientations relative to each other (e.g., to opposite strands of the nucleic acid) and / or in an orientation compatible with exponential PCR amplification. For example, primers that anneal to a rearrangement in an antiparallel orientation can facilitate amplification of an amplicon that includes a rearrangement breakpoint.

[0114] As used herein, a "tag" is a molecule, such as a nucleic acid, label, fluorophore, or peptide, that contains information that indicates a characteristic of the molecule with which the tag is associated. For example, a molecule may have a sample tag (that distinguishes a molecule in one sample from a molecule in a different sample), a molecular tag / molecular barcode / barcode (that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios)), a purification tag, and / or a detectable tag or label.

[0115] "Specifically binds" in the context of a probe or other oligonucleotide and a target sequence means that, under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or a copy thereof, to form a stable probe:target hybrid, while at the same time minimizing the formation of stable probe:non-target hybrids. Thus, a probe hybridizes to a target sequence, or a copy thereof, to a sufficiently greater extent than non-target sequences to allow capture or detection of the target sequence. Suitable hybridization conditions, which are well known in the art, can be predicted based on sequence composition or can be determined by using routine testing methods (see, e.g., §§ 1.90-1.91, 7.37-7.57, 9.47-9.51, and 11.47-11.57 of Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), which is incorporated herein by reference, especially §§ 9.50-9.51, 11.12-11.13, 11.45-11.47, and 11.55-11.57).

[0116] A nucleic acid is "produced by a tumor" if it originates from a tumor cell, or is ctDNA or circulating tumor DNA. Tumor cells are neoplastic cells that originate from a tumor, whether they remain within the tumor or become diverged from the tumor (such as in the case of metastatic cancer cells and circulating tumor cells).

[0117] A "target region" in the context of a nucleic acid refers to a locus that is targeted for identification and / or capture, e.g., by using a probe (e.g., through sequence complementarity). A "target region set" or "set of target regions" refers to a plurality of genomic loci that are targeted for identification and / or capture, e.g., by using a set of probes (e.g., through sequence complementarity).

[0118] The "capture yield" of a collection of probes for a given set of target regions refers to the amount of nucleic acid corresponding to the set of target regions that the collection of probes captures under typical conditions (e.g., the amount relative to another set of target regions or the absolute amount). Exemplary typical capture conditions are incubation of sample nucleic acid and probes in a small reaction volume (about 20 μL) containing a stringent hybridization buffer at 65° C. for 10-18 hours. Capture yields can be expressed in absolute terms, or in the case of a collection of multiple probes, in relative terms. When capture yields for a set of multiple target regions are compared, they are normalized to the footprint size of the set of target regions (e.g., on a per kilobase basis). Thus, for example, if the footprint sizes of a first and second target region are 50 kb and 500 kb, respectively (representing a normalization factor of 0.1), then: When the mass per volume concentration of the captured DNA corresponding to the first target region set is greater than 0.1 times the mass per volume concentration of the captured DNA corresponding to the second target region set, the DNA corresponding to the first target region set is captured with a higher yield than the DNA corresponding to the second target region set. As a further example, using the same footprint size, when the captured DNA corresponding to the first target region set has a mass per volume concentration that is 0.2 times the mass per volume concentration of the captured DNA corresponding to the second target region set, the DNA corresponding to the first target region set is captured with a capture yield that is 2 times greater than the DNA corresponding to the second target region set.

[0119] The term "methylation" or "DNA methylation" refers to the addition of a methyl group to a nucleotide base in a nucleic acid molecule. In some embodiments, methylation refers to the addition of a methyl group to a cytosine at a CpG site (a cytosine-phosphate-guanine site (i.e., a cytosine followed by a guanine in the 5'→3' direction of a nucleic acid sequence)). In some embodiments, DNA methylation refers to the addition of a methyl group to an adenine, such as in the case of N6-methyladenine. In some embodiments, DNA methylation is 5-methylation (modification of the carbon at the 5 position of the cytosine ring). In some embodiments, 5-methylation refers to the addition of a methyl group to the 5C position of cytosine to generate 5-methylcytosine (5mC). In some embodiments, methylation includes derivatives of 5mC. Derivatives of 5mC include, but are not limited to, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-caryboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon at the 3 position of the cytosine ring). In some embodiments, 3C methylation includes the addition of a methyl group to the 3C position of cytosine to generate 3-methylcytosine (3mC). Methylation can also occur at non-CpG sites, for example, methylation can occur at CpA, CpT, or CpC sites. DNA methylation can alter the activity of methylated DNA regions. For example, if DNA within a promoter region is methylated, transcription of a gene can be repressed. DNA methylation is critical for normal expression, and abnormalities in methylation can disrupt epigenetic regulation. Disruptions in epigenetic regulation, for example repression, can cause diseases such as cancer. Promoter methylation in DNA can indicate cancer.

[0120] "Modified nucleoside profile of DNA" refers to the location and identity of nucleosides within a DNA sequence as well as the modification state of the nucleoside, e.g., methylation. As described above, different methods of conversion and subsequent sequencing detect one or more different types of modified or unmodified nucleosides. For example, the TAPS method detects 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosine (5hmC) without distinguishing between them. Therefore, a method for analyzing the modified nucleoside profile of DNA in a sample typically means identifying a specific modification or group of modifications, e.g., 5mC and / or 5hmC. Modified nucleosides are identified according to the specific method / conversion procedure used as described above. This generally involves comparing sequence data obtained from DNA undergoing a conversion procedure with a reference sequence. Typically, the method includes (i) comparing the sequence data to (A) one or more predefined reference sequences, typically corresponding to one or more epigenetic target regions where the modified nucleoside profile is of particular significance, e.g., in cancer diagnosis, prognosis or characterization; or (B) sequence data obtained by sequencing subsamples of DNA that have not been subjected to the conversion procedure, e.g., subsamples separated before subjecting the separate subsamples to the conversion procedure, e.g., as described herein; and (ii) identifying point differences between the converted DNA sequence and the reference sequence (A) or the unconverted DNA sequence (B) as nucleosides (in the initial sample) having a modification state that allows for a change in base pair specificity upon exposure to the conversion procedure.

[0121] It is understood that the modified nucleoside profile being determined by standard conversion and sequencing methods may contain errors due to incomplete or erroneous conversion of modified or unmodified nucleosides in the sample. The methods of the present disclosure provide a means to assess the conversion rate on either a sample or molecular basis.

[0122] The term "hypermethylation" refers to an increase in the level or extent of methylation of a nucleic acid molecule relative to other nucleic acid molecules in a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypermethylated DNA may include DNA molecules that contain at least one methylated residue, at least two methylated residues, at least three methylated residues, at least five methylated residues, or at least ten methylated residues. As used herein, "type-specific hypermethylation" refers to an increase in the level or extent of methylation of DNA in one cell or tissue type, or in multiple related cell or tissue types, relative to other cell or tissue types. In some embodiments, the capture, identification, and / or detection of type-specific hypermethylated regions facilitates the identification of the cell or tissue type from which the DNA originated. The cells or tissues from which the type-specific hypermethylated regions originated may be wild-type cells or tissues or neoplastic cells or tissues.

[0123] The term "hypomethylation" refers to a reduction in the level or degree of methylation of a nucleic acid molecule relative to other nucleic acid molecules in a population (e.g., a sample) of nucleic acid molecules. In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules that include zero methylated residues, up to one methylated residue, up to two methylated residues, up to three methylated residues, up to four methylated residues, or up to five methylated residues. As used herein, "type-specific hypomethylation" refers to a reduction in the level or degree of methylation of DNA in one cell or tissue type, or in multiple related cell or tissue types, relative to other cell or tissue types. In some embodiments, the capture, identification, and / or detection of type-specific hypomethylated regions facilitates the identification of the cell or tissue type from which the DNA originates. The cells or tissues from which the type-specific hypomethylated regions originate can be wild-type cells or tissues or neoplastic cells or tissues.

[0124] The term "agent that recognizes modified nucleobases in DNA", e.g., "agent that recognizes modified cytosines in DNA", refers to a molecule or reagent that binds to or detects one or more modified nucleobases, e.g., methylcytosines, in DNA.

[0125] A "modified nucleoside" is a nucleoside that contains a difference in chemical structure from an unmodified nucleoside. In the case of DNA, unmodified nucleosides include deoxyribosyl and one of adenine, cytosine, guanine, or thymine. In some embodiments, the modified nucleoside includes a modified cytosine. In some embodiments, the modified nucleoside includes a methylated nucleobase. In some embodiments, the modified cytosine is a methylcytosine, e.g., 5-methylcytosine. In such embodiments, the cytosine modification is methyl. Agents that recognize methylcytosine in DNA include, but are not limited to, "methyl-binding reagents," which herein refer to reagents that bind to methylcytosine. Methyl-binding reagents include, but are not limited to, methyl-binding domains (MBDs) and methyl-binding proteins (MBPs), as well as antibodies specific for methylcytosine. In some embodiments, such antibodies bind to 5-methylcytosine in DNA. In some such embodiments, the DNA may be single-stranded or double-stranded.

[0126] "Or a combination thereof" and "or combinations thereof," as used herein, refer to any and all permutations and combinations of the listed terms preceding the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in a particular situation, also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing this example, expressly included are combinations that include one or more repeats of an item or term, such as, for example, BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of skill in the art will understand that there is typically no limit to the number of items or terms in any combination unless otherwise clear from the context.

[0127] "Or" is used in its inclusive sense, ie, equivalent to "and / or", unless the context requires otherwise.

[0128] Samples and Subjects The present disclosure relates to a method for analyzing modified nucleoside profiles of nucleic acids or quality control methods for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample, e.g., DNA in a sample. In some cases, the nucleic acid is obtained or obtained from a subject. In some embodiments, the nucleic acid sample may comprise or consist of nucleic acid, e.g., DNA, from a biological sample obtained from a subject. The subject may be a human, mammal, animal, primate, rodent (including mouse and rat), or other common laboratory, domestic, companion, service, or agricultural animal, e.g., rabbit, dog, cat, horse, cow, sheep, goat, or pig. The subject may have or be suspected of having cancer, tumor, or neoplasm in some cases. In other cases, the subject may not have cancer or detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, e.g., any one or more of chemotherapy, antibodies, vaccines, or biologies. The subject may, for example, be in remission from a tumor, cancer, or neoplasm (e.g., after treatment such as chemotherapy, surgical resection, radiation, or a combination thereof). The subject may or may not have been diagnosed as predisposed to cancer or any cancer-related genetic mutation / disorder. In some embodiments, the sample is a polynucleotide sample obtained from a tumor tissue biopsy. The cancer, tumor, or neoplasm may generally be of any type, for example, a cancer, tumor, or neoplasm of the lung, colon, rectum (or colorectal), kidney, breast, prostate, or liver, or other type of cancer described herein. In some embodiments, the sample is obtained from a subject in remission from a tumor, cancer, or neoplasm (e.g., after chemotherapy, surgical resection, radiation, or a combination thereof). In any of the above embodiments, the pre-cancer, cancer, tumor, or neoplasm or suspected pre-cancer, cancer, tumor, or neoplasm may belong to the bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin, or liver. In some embodiments, the precancer, cancer, tumor, or neoplasm or suspected precancer, cancer, tumor, or neoplasm is in the lung.In some embodiments, the precancer, cancer, tumor, or neoplasm or suspected precancer, cancer, tumor, or neoplasm is of the colon or rectum. In some embodiments, the precancer, cancer, tumor, or neoplasm or suspected precancer, cancer, tumor, or neoplasm is of the breast. In some embodiments, the precancer, cancer, tumor, or neoplasm or suspected precancer, cancer, tumor, or neoplasm is of the prostate. In any of the above embodiments, the subject may be a human subject. In some embodiments, the sample is obtained from a subject with stage I cancer, stage II cancer, stage III cancer, or stage IV cancer.

[0129] The sample may be any biological sample that has been isolated from a subject. The sample may be a body sample. The sample may include body tissues, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsies, cerebrospinal fluid, synovial fluid, lymphatic fluid, peritoneal fluid, interstitial or extracellular fluid, bodily fluids in the spaces between cells, such as gingival crevicular fluid, bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine. The sample is preferably a bodily fluid, in particular blood and its fractions, and urine. The sample may be in the form in which it is first isolated from the subject, or may have undergone further processing to remove or add components, such as cells, or to enrich one component for another.

[0130] In some embodiments, the population of nucleic acids is obtained from serum, plasma or blood samples from subjects suspected of having or previously diagnosed with a neoplasm, tumor, precancer or cancer. The population includes nucleic acids with different levels of sequence diversity, epigenetic mutations, and / or post-replicative or transcriptional modifications. Post-replicative modifications include modifications of cytosine, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, particularly at the 5-position of the nucleobase.

[0131] The sample may be isolated or obtained from the subject and transported to a location for sample analysis. The sample may be stored and transported at a desired temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. The sample may be isolated or obtained from the subject at the location for sample analysis. The subject may be a human, mammal, animal, companion animal, service animal, or pet. The subject may have cancer, pre-cancer, infection, transplant rejection, or other disease or disorder associated with alterations in the immune system. The subject may not have cancer or detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, e.g., any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may or may not have been diagnosed with cancer or susceptible to any cancer-related genetic mutation / disorder.

[0132] The volume of plasma may depend on the desired read depth of the sequenced region. Exemplary volumes are 0.4-40 mL, 5-20 mL, and 10-20 mL. For example, the volume may be 0.5 mL, 1 mL, 5 mL, 10 mL, 20 mL, 30 mL, or 40 mL. The volume of plasma collected may be 5-20 mL.

[0133] A sample may contain nucleic acids containing various amounts of genome equivalents. For example, a sample of about 30 ng of DNA may contain about 10,000 (10 4 ) haploid human genome equivalents, and approximately 200 billion (2 × l0 11 Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, about 600 billion individual molecules.

[0134] The sample may include nucleic acids from different sources, e.g., from cells and acellular of the same subject, from cells and acellular of different subjects. The sample may include nucleic acids having mutations. For example, the sample may include DNA having germline mutations and / or somatic mutations. A germline mutation refers to a mutation present in the germline DNA of a subject. A somatic mutation refers to a mutation that originates within the somatic cells of a subject, e.g., within a cancer cell. The sample may include DNA having a cancer-associated mutation (e.g., a cancer-associated somatic mutation). The sample may include epigenetic variants (i.e., chemical or protein modifications), where the epigenetic variants are associated with the presence of a genetic variant, such as a cancer-associated mutation. In some embodiments, the sample includes epigenetic variants associated with the presence of a genetic variant, where the sample does not include a genetic variant.

[0135] The sample may be or include cell-free nucleic acid or cfDNA. cfDNA may be obtained from a test subject, for example, as described above. For example, the sample for analysis may be plasma or serum containing cell-free nucleic acid. "Cell-free DNA," "cfDNA molecule," or "cfDNA" includes, for example, DNA molecules naturally present in a subject in an extracellular form (e.g., blood, serum, plasma, or other bodily fluids, such as lymph, cerebrospinal fluid, urine, or sputum). While cfDNA was originally present within one or more cells in a large complex organism, such as a mammal, it has undergone release from the cells into bodily fluids found in the organism in vivo, and can be obtained by obtaining a sample of the bodily fluid without the need to perform an in vitro cell lysis step. In other words, cell-free nucleic acid or DNA is nucleic acid or DNA that is not contained within or otherwise bound to a cell, or that remains in a sample after removal of intact cells. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, such as genomic DNA, mitochondrial DNA, siRNA, miRNA, circular RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids can be released into bodily fluids through secretion or cell death processes, such as cell necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluids from cancer cells, including, for example, circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, the cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, the cell-free nucleic acids are produced by tumor cells. In some embodiments, the cell-free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.

[0136] Exemplary amounts of cell-free nucleic acid in a sample prior to amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can include obtaining between 1 femtogram (fg) and 200 ng of cell-free nucleic acid molecules from a sample.

[0137] Cell-free DNA refers to DNA that is not contained within cells at the time of its isolation from a subject. For example, cfDNA can be isolated from a sample as DNA remaining in the sample after removing intact cells without lysing the cells or otherwise extracting intracellular DNA. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, such as genomic DNA, mitochondrial DNA, siRNA, miRNA, circular RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-translated RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids can be released into bodily fluids through secretion or cell death processes, such as cell necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluids from cancer cells, including, for example, circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell-free nucleic acids are produced by tumor cells. In some embodiments, the cell-free nucleic acid is produced by a mixture of tumor cells and non-tumor cells.

[0138] Cell-free nucleic acids have a typical size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of the molecules, with a mode at about 168 nucleotides and a second minor peak in the range of 240-440 nucleotides.

[0139] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step, where the cell-free nucleic acids found in solution are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, the cells in the bodily fluid can be lysed and the cell-free and cellular nucleic acids can be processed together. Generally, after addition of buffer and washing steps, the nucleic acids can be precipitated with alcohol. Additionally, clean-up steps, such as silica-based columns, can be used to remove contaminants or salts. Non-specific bulk carrier nucleic acids, such as C1 DNA, DNA or proteins for bisulfite sequencing, hybridization, and / or ligation, can be added throughout the reaction to optimize certain aspects of the procedure, such as yield.

[0140] After such processing, the sample may contain various forms of nucleic acid, including double-stranded DNA, single-stranded DNA, and single-stranded RNA, hi some embodiments, single-stranded DNA and RNA may be converted to double-stranded form for inclusion in subsequent processing and analysis steps.

[0141] The double-stranded DNA molecules in the sample and the single-stranded nucleic acid molecules that have been converted to double-stranded DNA molecules can be ligated to adapters at either one or both ends. Typically, the double-stranded molecules are blunt-ended by treatment with a polymerase that has a 5'-3' polymerase and a 3'-5' exonuclease (or proofreading function) in the presence of all four standard nucleotides. Klenow large fragment and T4 polymerase are examples of suitable polymerases. The blunt-ended DNA molecules can be ligated, at least in part, with double-stranded adapters (e.g., Y-shaped or bell-shaped adapters). Alternatively, complementary nucleotides can be added to the blunt ends of the sample nucleic acid and the adapter to facilitate ligation. Contemplated herein are both blunt-end ligation and sticky-end ligation. In blunt-end ligation, both the nucleic acid molecule and the adapter tag have blunt ends. In sticky end ligation, typically the nucleic acid molecule has an "A" overhang and the adaptor has a "T" overhang.

[0142] Ligation to adapters Double-stranded nucleic acids, such as DNA molecules in a sample, and single-stranded nucleic acid molecules that have been converted to double-stranded molecules, can be ligated to adapters at either one or both ends. In some cases, DNA can be ligated, for example, by extending the terminal overhangs of the DNA molecules, adding adenosine residues to the 3' ends of the fragments, and phosphorylating the 5' ends of each DNA fragment. Typically, double-stranded molecules are blunt-ended by treatment with a polymerase that has a 5'-3' polymerase and a 3'-5' exonuclease (or proofreading function) in the presence of all four standard nucleotides. Klenow large fragment and T4 polymerase are examples of suitable polymerases.

[0143] The blunt-ended DNA molecule can be ligated at least partially with a double-stranded adaptor (e.g., a Y-shaped or bell-shaped adaptor). Alternatively, complementary nucleotides can be added to the blunt ends of the sample nucleic acid and the adaptor to facilitate ligation. Contemplated herein are both blunt-end ligation and sticky-end ligation. In blunt-end ligation, both the nucleic acid molecule and the adaptor tag have blunt ends. In sticky-end ligation, typically the nucleic acid molecule has an "A" overhang and the adaptor has a "T" overhang.

[0144] A DNA ligase and an adaptor are added to ligate the DNA molecules in the sample to the adaptors at one or both ends, i.e., to form adapted DNA. As used herein, an "adaptor" typically refers to a short nucleic acid (e.g., less than about 500, less than about 100, or less than about 50 nucleotides in length, or 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases end-to-end) that is at least partially double-stranded and can be ligated to the ends of a given sample nucleic acid molecule. In some cases, two adaptors can be ligated to a single sample nucleic acid molecule, one adaptor ligated to each end of the sample nucleic acid molecule.

[0145] The adaptor may include a nucleic acid primer binding site that allows amplification of the sample nucleic acid molecule flanked by the adaptor at both ends, and / or a sequencing primer binding site, including primer binding sites in sequencing applications, such as various next generation sequencing (NGS) applications. The adaptor may include a sequence for hybridizing to a solid support, such as a flow cell sequence. The adaptor may also include a binding site for a capture probe, such as an oligonucleotide attached to a flow cell support. The adaptor may also include a sample index and / or a molecular barcode. These are typically positioned relative to the amplification primer and sequencing primer binding sites such that the sample index and / or molecular barcode are included in the amplicon and sequencing reads of a given nucleic acid molecule. Adapters of the same or different sequences may be ligated to each end of the sample nucleic acid molecule. In some cases, adapters of the same or different sequences are ligated to each end of the nucleic acid molecule, except that the sample index and / or molecular barcode differ in their sequences. In some embodiments, the adaptor is a Y-shaped adaptor, where one end is blunt-ended or tailed as described herein, when ligated to a nucleic acid molecule that is further blunt-ended or tailed with one or more complementary nucleotides within the tail of the adaptor. In another exemplary embodiment, the adaptor is a bell-shaped adaptor that includes a blunt or tailed end for ligating to a nucleic acid molecule to be analyzed. Other exemplary adaptors include T-tail, C-tail or hairpin-shaped adaptors. For example, a hairpin-shaped adaptor can include a complementary double-stranded portion and a loop portion, where the double-stranded portion can be attached (e.g., ligated) to a double-stranded polynucleotide. A hairpin-shaped sequencing adaptor can be attached to both ends of a polynucleotide fragment to generate a circular molecule that can be sequenced multiple times. The adaptors used in the methods of the present disclosure include one or more known modified nucleosides, e.g., methylated nucleosides. When two adaptors are ligated to a sample nucleic acid (one at each end), one or both of the adaptors may include one or more known modified nucleosides.Typically, the primer binding site, the sequencing primer binding site, the sample index and / or the molecular barcode, if present, do not contain known modified nucleosides that alter base pair specificity as a result of the conversion procedure.

[0146] In some embodiments, the sample nucleic acids flanked by the adaptors can be amplified by PCR and other amplification methods. Such amplification can be used to increase the amount of DNA available for subsequent steps such as sequencing, and can be performed in addition to selective amplification of DNA containing rearranged sequences. For example, this type of amplification can be performed as part of library preparation (e.g., before selective amplification of DNA containing rearranged sequences) and / or after preparation of a targeted library (which would be after selective amplification of DNA containing rearranged sequences). Amplification can be stimulated by primers that bind to primer binding sites in the adaptors adjacent to the DNA molecules to be amplified. Amplification methods can include cycles of denaturation, annealing and extension resulting from thermocycling, or can be isothermal, such as in the case of transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustaining sequence-based replication.

[0147] In some embodiments, the method performs dsDNA ligation with T-tail and C-tail adapters resulting in at least 50, 60, 70, or 80% amplification of double stranded nucleic acid prior to ligation to the adapters. Preferably, the method increases the amount or number of amplified molecules by at least 10, 15, or 20% compared to a control method performed with T-tail adapters alone.

[0148] In some embodiments, an adaptor can be added to the DNA or a subsample thereof. The adaptor can be ligated to the DNA at any point in the methods herein. In some embodiments, the adaptor is ligated to the DNA of the sample or subsample before annealing of a primer to the DNA to generate a capture probe. In some such embodiments, the adaptor-ligated DNA is amplified before annealing of a primer to the DNA to generate a capture probe. In some embodiments, the adaptor is ligated to the DNA of the sample or subsample before the DNA is contacted with the capture probe. In some embodiments, the DNA to which the adaptor is ligated is present in the same sample or subsample as the DNA used as a template to generate the capture probe. In some embodiments, the DNA to which the adaptor is ligated is present in a different sample or subsample than the DNA used as a template to generate the capture probe, for example, a second sample or a second subsample of the first sample. In some embodiments, the adaptor ligated to the DNA is captured by the capture probe.

[0149] In some embodiments, the primers used to generate the capture probe are not complementary to the adapter, and thus the resulting capture probe does not contain an adapter.Therefore, the adapter-ligated DNA can be selectively amplified in the presence of the capture probe that does not contain an adapter.Similarly, the adapter-ligated DNA can be separated from the DNA that does not contain an adapter.

[0150] In some embodiments, the disclosed methods include analyzing DNA in a sample. In such methods, adapters can be added to the DNA. This can be done simultaneously with the amplification procedure (if PCR is used, this can be referred to as library prep-PCR or LP-PCR), before the amplification step, or after, for example, by providing an adapter at the 5' portion of the primer. In some embodiments, the adapter is added by other techniques, for example, ligation. In some such methods, a first adapter is added to the 3' end of the nucleic acid by ligation, which can include ligation to single-stranded DNA. In some embodiments, prior to any splitting or capture step, a first adapter is added to the nucleic acid by ligation, which can include ligation to (e.g., to its 3' end) single-stranded DNA. In some embodiments, capture probes can be isolated after splitting and ligation. For example, the hypomethylated fraction can be ligated with an adapter, and then a portion of the ligated hypomethylated fraction can be used to generate a capture probe for rearrangement. The adaptor can be used as a priming site for double stranded synthesis, for example, using a universal primer and a DNA polymerase. A second adaptor can then be ligated to at least the 3' end of the second strand of the double stranded molecule. In some embodiments, the first adaptor includes an affinity tag, such as biotin, and the nucleic acid ligated to the first adaptor is attached to a solid support (e.g., a bead) that may include a binding partner for the affinity tag, such as streptavidin. For further discussion of related procedures, see Gansauge et al., Nature Protocols 8:737-748 (2013). Commercial kits for sequencing library preparation that are compatible with single stranded nucleic acids are available, for example, the Accel-NGS® Methyl-Seq DNA Library Kit from Swift Biosciences. In some embodiments, after adaptor ligation, the nucleic acid is amplified,

[0151] In some embodiments, the adaptors contain a sufficient number of different tags such that the number of tag combinations results in a low probability, e.g., 95, 99 or 99.9%, that two nucleic acids with the same start and stop points will receive the same combination of tags. Adaptors, whether they have the same or different tags, can contain the same or different primer binding sites, although preferably the adaptors contain the same primer binding sites.

[0152] In some embodiments, after attachment of the adaptors, the nucleic acid undergoes amplification, which can, for example, use a universal primer that recognizes a primer binding site in the adaptor.

[0153] In some embodiments, after attachment of the adaptors, the DNA or a subsample or portion of the DNA is split, including contacting the DNA with an agent that preferentially binds to nucleic acids with epigenetic modifications. The nucleic acid is split into at least two split subsamples that differ in the extent to which the nucleic acid has the modification from binding to the agent. For example, if the agent has an affinity for nucleic acids with the modification, nucleic acids that are over-represented in the modification (compared to the median representation in the population) will preferentially bind to the agent, while nucleic acids that are under-represented in the modification will not bind to the agent or will be more easily eluted from the agent. The nucleic acid can then be amplified from primers that are bound to the primer binding sites in the adaptors. Splitting can alternatively be performed before adapter attachment, and in either case, the adaptors may include differential tags that include moieties that identify which split has occurred in the molecule.

[0154] In some embodiments, the nucleic acid is ligated at both ends of a Y-shaped adaptor that contains a primer binding site and a tag, and the molecule is amplified.

[0155] Molecular tagging In some embodiments, the nucleic acid molecules of a sample may be tagged with a sample index and / or a molecular barcode (commonly referred to as a "tag").

[0156] A tag or index can be a molecule, e.g., a nucleic acid, that contains information that indicates the characteristics of the molecule with which it is associated. For example, a molecule can have a sample tag or sample index (that distinguishes a molecule in one sample from a molecule in a different sample), a split tag (that distinguishes a molecule in one split from a molecule in a different split), and / or a molecular tag / molecular barcode / barcode that distinguishes different molecules from each other (in both unique and non-unique tagging scenarios).

[0157] Tagging strategies can be divided into unique tagging strategies and non-unique tagging strategies. In unique tagging, all or substantially all of the molecules in a sample have different tags, such that the reads can be assigned to the original molecules based on the tag information alone. The tags used in such methods are sometimes referred to as "unique tags". In non-unique tagging, different molecules in the same sample may have the same tag, such that the sequence reads are assigned to the original molecules using other information in addition to the tag information. Such information may include start and stop coordinates, coordinates that position the molecule, start or stop coordinates alone, etc. The tags used in such methods are sometimes referred to as "non-unique tags". Thus, it is not necessary to uniquely tag all molecules in a sample. It is sufficient to uniquely tag molecules that fall within an identifiable class in a sample. Thus, molecules in different identifiable families may have the same tag, which does not lack information about the identity of the tagged molecule.

[0158] In certain embodiments, the tag may comprise one barcode or a combination of barcodes. As used herein, the term "barcode" refers to a nucleic acid molecule having a particular nucleotide sequence, or, depending on the context, the nucleotide sequence itself. A barcode may have, for example, 10-100 nucleotides. A collection of barcodes may have degenerate sequences or may have sequences with a particular Hamming distance, as desired for a particular purpose. Thus, for example, a molecular barcode may consist of one barcode or a combination of two barcodes, each attached to a different end of the molecule. Additionally or alternatively, for different fractions and / or samples, different sets of molecular barcodes, molecular tags, or molecular indexes may be used, such that the barcodes not only serve as molecular tags through their individual sequences, but also serve to identify the fractions and / or samples to which they correspond based on the sets they are members of.

[0159] In some embodiments, two or more fractions, e.g., each fraction, are differentially tagged. Tags can be used to label fractions of distinct polynucleotide populations, allowing the tag (or tags) to be correlated with a particular fraction. Alternatively, tags can be used in embodiments of the disclosure that do not use a splitting step. In some embodiments, a single tag can be used to label a particular fraction. In some embodiments, multiple different tags can be used to label a particular fraction. In embodiments where multiple different tags are used to label a particular fraction, the set of tags used to label one fraction can be easily distinguished from the set of tags used to label another fraction. In some embodiments, the tag may have additional functionality, e.g., the tag may be used to indicate the source of a sample or to serve as a unique molecular identifier (which may improve the quality of sequencing data by distinguishing sequencing errors from mutations, e.g., Kinde et al., Proc Nat'l Acad Sci USA 108:9530-9535 (2011), Kou et al., PLoS 0NE, 11:e0146638 (2016)), or as a non-unique molecular identifier, e.g., as described in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, the tag may have additional functionality, e.g., the tag may be used to indicate the source of a sample or to serve as a non-unique molecular identifier (which may improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0160] The tags may be incorporated into or otherwise linked 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 others. Such adapters are eventually linked to the target nucleic acid molecules. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) may be applied to introduce the sample index into the nucleic acid molecule using conventional nucleic acid amplification methods. Amplification may be performed in one or more reaction mixtures (e.g., multiple microwells in an array). The molecular barcodes and / or sample indexes may be introduced simultaneously or in any sequential order. In some embodiments, the molecular barcodes and / or sample indexes are introduced before and / or after the conversion procedure. In some embodiments, the molecular barcodes and / or sample indexes are introduced before and / or after the sequence capture step (if present) is performed. In some embodiments, only the molecular barcodes are introduced before probe capture and the sample index is introduced after the sequence capture step is performed. In some embodiments, both the molecular barcode and the sample index are introduced before performing a probe-based capture step (if present). In some embodiments, the sample index is introduced after performing a sequence capture step (if present). In some embodiments, the sample index is incorporated through overlap extension polymerase chain reaction (PCR).

[0161] In some embodiments, the tags may be located at one or both ends of the sample nucleic acid molecule. In some embodiments, the tags are predetermined or random or quasi-random oligonucleotide sequences. In some embodiments, the tags may be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides in length together. Typically, the tags are about 5-20 or 6-15 nucleotides in length. The tags may be randomly or non-randomly linked to the sample nucleic acid.

[0162] In some embodiments, each sample or fraction (discussed below) is uniquely tagged with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or subsample is uniquely tagged with a molecular barcode or a combination of molecular barcodes. In other embodiments, multiple molecular barcodes may be used such that the molecular barcodes are not necessarily unique to each other in the plurality (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are typically attached (e.g., by ligation) to individual molecules such that the combination of the molecular barcode and the sequence to which it may be attached creates a unique sequence that can be individually tracked. Detection of the non-unique molecular barcode in combination with endogenous sequence information (e.g., initial (start) and / or end (stop) genomic positions / positions corresponding to the sequence of the original nucleic acid molecule in the sample, start and stop genomic positions corresponding to the sequence of the original nucleic acid molecule in the sample, initial (start) and / or end (stop) genomic positions / positions of the sequence read mapped to the reference sequence, start and stop genomic positions of the sequence read mapped to the reference sequence, subsequence of the sequence read at one or both ends, length of the sequence read, and / or length of the original nucleic acid molecule in the sample) typically allows for assignment of a unique identity to a particular molecule. In some embodiments, the initial region includes the first 1, first 2, first 5, first 10, first 15, first 20, first 25, first 30, or at least the first 30 base positions at the 5' end of the sequencing read that align to the reference sequence. In some embodiments, the terminal regions include the last 1, the last 2, the last 5, the last 10, the last 15, the last 20, the last 25, the last 30, or at least the last 30 base positions at the 3' end of the sequencing read that aligns to the reference sequence. The length of the individual sequence reads, or the number of base pairs, is also optionally used to assign a unique identity to a given molecule. Thus, as described herein, fragments from a single strand of nucleic acid that have been assigned a unique identity may allow for subsequent identification of fragments from the parental and / or complementary strands.

[0163] In certain embodiments of non-unique tagging, the number of different tags used may be sufficient such that the probability that all DNA molecules of a particular group have different tags is very high (e.g., at least 99%, at least 99.9%, at least 99.99% or at least 99.999%. It should be noted that when barcodes are used as tags, and when barcodes are attached, for example randomly, to both ends of the molecules, a combination of barcodes together may constitute a tag. This number is a function of the number of molecules that fall under the requirements in the terms. For example, a class may be all molecules that are located at the same start-stop position on a reference genome. A class may be all molecules that are located at a particular locus, for example, a particular base or throughout a particular region (e.g., up to 100 bases or genes or exons of a gene). In certain embodiments, the number of different tags used to uniquely identify the number of molecules z in a class is 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 * Either z (for example, the lower limit) and 100,000 * z, 10,000 * z, 1000 * z or 100 *z can be between 0 and 1 (e.g., an upper limit). In some embodiments, molecular barcodes are introduced in an expected ratio of a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) to molecules in a sample. In one example format, about 2 to about 1,000,000 different molecular barcode sequences, or about 5 to about 150 different molecular barcode sequences, or about 20 to about 50 different molecular barcode sequences, ligated to both ends of a target molecule are used. Alternatively, 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 20-50 different molecular barcode sequences may be attached to each end of a target molecule) can be used. Such a number of identifiers is typically sufficient for different molecules to have the same start and stop points to have a high probability (e.g., at least 94%, 99.5%, 99.99%, or 99.999%) of receiving different combinations of identifiers. In some embodiments, about 80%, about 90%, about 95%, or about 99% of the molecules have the same combination of molecular barcodes.

[0164] For example, in a sample of about 5 ng to 30 ng of cell-free DNA, approximately 3000 molecules are expected to map to a particular nucleotide coordinate, and about 3 to 10 molecules with any start coordinate to share the same stop coordinate. Thus, about 50 to about 50,000 different tags (e.g., about 6 to 220 barcode combinations) may be sufficient to uniquely tag all such molecules. To uniquely tag a total of 3000 molecules that map through nucleotide coordinates, about 1 million to about 20 million different tags would be required.

[0165] In some embodiments, assignment of unique or non-unique molecular barcodes in reactions is performed using methods and systems described, for example, in US Patent Publication Nos. 20010053519, 20030152490, and 20110160078, and in U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is incorporated herein by reference in its entirety. Alternatively, in some embodiments, different nucleic acid molecules of a sample may be identified using only intrinsic sequence information (e.g., start and / or stop positions, subsequences at one or both ends of the sequence, and / or length).

[0166] In some embodiments, the tagging nucleic acid is sequenced after loading into a microwell plate. The microwell plate may have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, unique tags may be loaded such that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genome sample. In some cases, unique tags may be loaded such that less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genomic sample. In some cases, the average number of unique tags loaded per genomic sample is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags per genomic sample.

[0167] In a preferred format, 20-50 different tags (e.g., barcodes) ligated to both ends of a target nucleic acid are used. For example, 35 different tags (e.g., barcodes) ligated to both ends of a target molecule would result in 35x35 permutations, which is equivalent to 1225 for 35 tags. Such a membership of tags is sufficient that different molecules with the same start and stop points have a high probability (e.g., at least 94%, 99.5%, 99.99%, 99.999%) of receiving different combinations of tags. Other barcode combinations include any number between 10 and 500, such as about 15x15, about 35x35, about 75x75, about 100x100, about 250x250, about 500x500.

[0168] In some cases, the unique tag may be a predetermined or random or quasi-random oligonucleotide sequence. In other cases, multiple barcodes may be used such that the barcodes are not necessarily unique to each other in the plurality. In this example, the barcode may be ligated to an individual molecule, such that the combination of the barcode and the sequence to which it may be ligated creates a unique sequence that can be tracked individually. As described herein, detection of a non-unique barcode in combination with sequence data of the initial (start) and final (stop) portions of a sequence read may allow for the assignment of a unique identity to a particular molecule. Also, the length or number of base pairs of the individual sequence reads may be used to assign a unique identity to such a molecule. Thus, as described herein, fragments from a single strand of nucleic acid that have been assigned a unique identity may allow subsequent identification of the fragment from the parent strand.

[0169] In some embodiments, the method includes adding one or more internal control DNAs and forward and reverse primers to amplify the internal control DNA. The internal control DNA may be added prior to amplification using primers that anneal upstream and downstream of the rearrangement breakpoint. The forward and reverse primers for amplifying the internal control DNA may be included with or added simultaneously with the primers that anneal upstream and downstream of the rearrangement breakpoint. The internal control DNA may comprise or consist of sequences that are not present in the genome of the subject or that are not present in the genome of the species of which the subject is a member (e.g., the human genome). The forward and / or reverse primers for amplifying the internal control DNA may comprise sequences that are not complementary to any sequence in the genome of the subject, e.g., the human genome. The use of the internal control DNA can ensure that the amplification process proceeded as designed. As such, the method may include detecting (e.g., sequencing) molecules that have been amplified from and / or captured by the one or more internal control DNAs. The method may include comparing the amount of internal control DNA (e.g., the number of molecules or reads detected that correspond to the internal control DNA sequence) to a predetermined threshold and rejecting the sequencing result if the predetermined threshold is not met or accepting the sequencing result if the predetermined threshold is met. The predetermined threshold can be established, for example, based on historical data or by testing the method on samples of DNA from test subjects, such as healthy volunteers. For example, amplification and detection of one or more internal control DNAs provides confirmation that the amplification process is proceeding properly, thus reducing the chance of false negatives.

[0170] Conversion Steps As described in the methods disclosed herein, the use of quality control nucleosides in an adaptor can be advantageously employed with a conversion procedure that converts the base pair specificity of a modified nucleoside (e.g., Tet-assisted conversion with a substituted borane reducing agent) or an unmodified nucleoside (e.g., bisulfite conversion). For example, in some embodiments, when a molecule that includes an adaptor containing two or more quality control nucleosides is exposed to a conversion procedure selected to alter the base pair specificity of the quality control nucleosides, the base pair specificity of a first portion (e.g., at least one) of the quality control nucleosides is altered, but the base pair specificity of a second portion (e.g., at least one) of the quality control nucleosides in the adaptor is unaffected, which may indicate suboptimal conversion. As described in the methods disclosed herein, the use of quality control nucleosides in adapters can be advantageously used to predict / infer / indicate false negative detection and / or identification of modified nucleosides in a DNA sample (i.e., incorrectly identifying a base as unmodified) and / or false positive detection and / or identification of modified nucleosides in a DNA sample (i.e., incorrectly identifying a base as modified). Quality control nucleosides described herein intended for use to detect the occurrence of false positive detection of modified nucleosides may be referred to as "false positive quality control nucleosides". Quality control nucleosides described herein intended for use to detect the occurrence of false negative detection of modified nucleosides may be referred to as "false negative quality control nucleosides". Nucleosides having a modification state, meaning that their base pair specificity is not altered when exposed to a particular conversion procedure, may in some cases be referred to as having a "protected" nucleoside or a "protected modification state" or the like.

[0171] When false negatives are detected using a conversion procedure that changes the base pair specificity of modified nucleosides, the quality control nucleosides in the adapter may include modified nucleosides such that the conversion efficiency of the conversion procedure / suboptimal conversion can be measured and therefore the frequency of false negatives can be predicted. Suboptimal conversion refers to the conversion of less than all nucleosides of the type that normally converts, such as the reagents used in the suboptimal conversion with Tet enzyme and pyridine borane (e.g., in TAPS) result in the conversion of some, but not all, 5mC and 5hmC to dihydrouracil. The terms suboptimal and suboptimal have equivalent meanings. Suboptimal conversion may also be referred to as incomplete conversion, in the sense that some nucleosides (modified or unmodified) that should have been converted by the conversion procedure in a complete reaction were not actually converted.

[0172] When a conversion procedure that converts the base pair specificity of unmodified nucleosides is used to detect false positives, the quality control nucleosides in the adapter may include modified nucleosides such that the frequency of misconversion of the modified nucleoside can be measured and therefore the frequency of false positives can be predicted. Misconversion typically refers to the conversion of a nucleoside other than the nucleoside that is converted by the conversion procedure. Conversion of a methylated cytosine by bisulfite conversion (which typically converts only unmodified cytosines) is an example of a misconversion.

[0173] When false positives are detected using a conversion procedure that converts the base pair specificity of modified nucleosides, quality control nucleosides in the adapter may include unmodified nucleosides such that the frequency of incorrect conversion of unmodified nucleosides can be measured and therefore the frequency of false positives can be predicted.

[0174] When false positives are detected using a conversion procedure that converts the base pair specificity of unmodified nucleosides, quality control nucleosides in the adapter may include unmodified nucleosides such that the conversion efficiency of the conversion procedure / suboptimal conversion can be measured and therefore the frequency of false positives can be predicted.

[0175] There are various methods to detect and / or identify modified nucleosides, depending on the conversion procedure that changes the base pair specificity of the nucleoside based on the modification state of the nucleoside. These changes in base pair specificity can then be detected by sequencing, and therefore the modification state of the nucleoside can be inferred.

[0176] In some cases, the conversion procedure used in the disclosed method changes the base pair specificity of modified nucleosides (e.g., methylated cytosine) but does not change the base pair specificity of the corresponding unmodified nucleoside (e.g., cytosine) or does not change the base pair specificity of any unmodified nucleoside (e.g., cytosine, adenosine, guanosine, and thymidine (or uracil)). The advantages of a method that does not change the base pair specificity of unmodified nucleosides include reduced loss of sequence complexity, higher sequencing efficiency, and reduced alignment defects. In addition, methods such as TAPS may be preferred over methods such as bisulfite sequencing and EM-seq in some cases because they are less destructive (particularly important for low-yield samples such as cfDNA) and do not require denaturation, meaning that non-conversion errors are more likely to be theoretically random (see FIG. 1). Methods that require denaturation for conversion do not denature the DNA molecule, resulting in non-conversion of all bases in the DNA molecule. These non-random (localized) conversions can be seen as false negatives (non-methylated regions) because biological changes in methylation are mainly localized to the localized region of interest. Random non-conversion methods may maximally affect a low percentage of bases in a region, and therefore the specificity of methylation change detection may be maximized (reducing false positives) by thresholding the % of bases in a region that are methylated / non-methylated. Therefore, in some cases, conversion procedures that do not involve denaturation are preferred.

[0177] In some embodiments, the adaptor comprises a first quality control nucleoside having a first modification state (e.g., modified, e.g., methylated) and a second quality control nucleoside having a second modification state (e.g., unmodified). Such adaptors can be used to detect both suboptimal and erroneous conversions.

[0178] FIG. 3 shows an embodiment of a quality control method for monitoring false negative and / or false positive detection of DNA undergoing a TAPS base conversion procedure. Adapters containing 5mC (e.g., in molecular barcodes) are ligated to DNA and then undergo a TAPS conversion procedure to change the base pairing of methylated cytosines (sequences read as "T") and not for unmethylated cytosines (also read as "C"). Each strand is sequenced. Molecules that have undergone suboptimal conversion can be identified and filtered out, at least for purposes of determining methylation. In some embodiments, molecules that have undergone suboptimal conversion include ssDNA molecules where 0 / 2 barcode 5mCs are converted to T. The sample conversion rate can be calculated by dividing all converted barcode 5mCs by the sum of barcode 5mCs (in this case 31 / 36=86% TAPS conversion rate).

[0179] In other cases, the conversion procedure used in the disclosed methods alters the base pairing specificity of an unmodified nucleoside (e.g., cytosine) but does not alter the base pairing specificity of the corresponding modified nucleoside (e.g., methylated cytosine). Such methods include, for example, bisulfite sequencing.

[0180] Those skilled in the art can select a suitable method depending on their needs, including which nucleoside modifications are to be detected and / or identified.

[0181] In some embodiments, the conversion procedure converts a modified nucleoside. In some embodiments, the conversion procedure for converting a modified nucleoside includes Tet-assisted conversion with a substituted borane reducing agent, optionally the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, ammonia borane, or pyridine borane. In the Tet-assisted pic-borane conversion with substituted borane reducing agent conversion, mC and hmC are converted to caC using a TET protein without affecting unmodified C. Then, caC and fC, if present, are 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, for example, Liu et al., Nature Biotechnology 2019;37:424-429 (e.g., Appendix Figure 1 and Appendix Note 7). DHU is decoded as 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 an unmodified cytosine. Sequencing of the converted DNA identifies positions that are decoded as cytosine, which is an unmodified C position. Meanwhile, positions that are decoded as T are identified as being T, mC, fC, caC, or hmC. Thus, performing TAP conversion facilitates the identification of positions containing unmodified C using the resulting sequence reads. Thus, in these embodiments, the quality control nucleosides in the adapters used in the method comprise methyl-cytosine (5mC) and / or hydroxymethyl-cytosine (hmC). This procedure involves Tet-assisted pyridine borane sequencing (TAPS), described in further detail in Liu et al. 2019, supra, in which the Tet enzyme is used to progressively oxidize 5mC and 5hmC to 5fC or 5CaC, followed by pyridine borane deamination of 5fC and 5CaC to DHU (amplified as T).This procedure involves Tet-assisted pyridine borane sequencing (TAPS), which is described in further detail in Liu et al. 2019, supra.

[0182] Alternatively, protection of hmC (e.g., with βGT) can be combined with Tet-assisted conversion with a substituted borane reducing agent, e.g., as described above. In this method (TAPS-β), hmC can be protected from conversion, e.g., through glucosylation with β-glucosyltransferase (βGT) to form ghmC (forming 5-glucosylhydroxymethylcytosine). This is described in Yu et al., Cell 2012;149:1368-80. Treatment with a TET protein, such as mTet1, then converts mC to caC, but not C or ghmC. caC is then converted to DHU by treatment with pic-borane or another substituted borane reducing agent, such as borane pyridine, tert-butylamine borane, or ammonia borane, again without affecting unmodified C or ghmC. Sequencing of the converted DNA identifies positions that are decoded as cytosine, either at hmC or at unmodified C. On the other hand, positions that are decoded as T are identified as being T, fC, caC, or mC. Thus, performing TAPSβ conversion on samples as described herein facilitates distinguishing positions containing unmodified C or hmC from positions containing mC on the other hand using the sequence reads obtained. Thus, in these embodiments, the quality control nucleosides in the adapter used in the method may typically include both methyl-cytosine (5mC) and hydroxymethyl-cytosine (hmC). This allows the efficiency of each of the two steps to be determined separately. For example, if sequencing of the adapter shows that both 5mC and hmC nucleosides have altered base pair specificity, this indicates that the protection step of hmC was ineffective. If neither 5mC nor hmC nucleosides have altered base pair specificity, this indicates that (at least) Tet-assisted conversion was ineffective. Both steps were effective when base pairing of mC nucleosides in the adapter, but not of hmC nucleosides, converted base pairing specificity.Alternatively, the efficiency of just one step or the other could be determined by just including mC or hmC as a quality control nucleoside in the adaptor. The efficiency of conversion and / or a determination of whether a protection step was ineffective can be used to determine whether suboptimal conversion is present, as defined in the methods disclosed herein. For an exemplary description of this type of conversion, see, for example, Liu et al., Nature Biotechnology 2019;37:424-429.

[0183] In addition to controlling for suboptimal conversion of modified nucleosides, quality control nucleosides in the adapter can also be used to predict false positives (i.e., nucleosides that are misclassified as modified). In this case, the quality control nucleoside in the adapter contains an unmodified C, given the proper conversion procedure. If sequencing of the adapter shows that the quality control nucleoside has converted base pair specificity, this indicates that an unmodified base (e.g., unmodified C) has been erroneously converted. This information can then be used to predict false positive detection of modified nucleosides (e.g., modified C) in a DNA sample.

[0184] In some embodiments, the conversion procedure for converting the modified nucleoside includes chemically assisted conversion with a substituted borane reducing agent, optionally the substituted borane reducing agent is 2-picoline borane, borane pyridine, tert-butylamine borane, borane pyridine, or ammonia borane. In the chemically assisted conversion with a substituted borane reducing agent, hmC is specifically oxidized to fC using an oxidizing agent such as potassium perruthenate (KRuO4) (also suitable for use in ox-BS conversion). Treatment with pic-borane or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane, or ammonia borane converts fC and caC to DHU, but does not affect mC or unmodified C. Sequencing of the converted DNA identifies positions that are decoded as cytosine, which are either mC or unmodified C positions. Meanwhile, positions that are decoded as T are identified as being T, fC, caC, or hmC. Therefore, by implementing this type of conversion described herein, it becomes easier to distinguish positions containing unmodified C or mC from positions containing hmC using the sequence reads obtained. Therefore, in these embodiments, and when controlling for false negatives (i.e., nucleosides that are misclassified as unmodified), the adaptors used in the methods include hydroxymethyl-cytosine (hmC). For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019;37:424-429.

[0185] Exemplary conversion procedures that change the base pair specificity of modified cytosines are described. However, in principle, any modified nucleoside and suitable conversion procedure that changes the base pair specificity of modified nucleosides (i.e., single-base epigenetic conversion assays) could be used in the methods described herein, thereby allowing the modified base to be distinguished from the corresponding unmodified nucleosides and / or other types of modifications during sequencing. For example, any conversion procedure could be used to change the base pair specificity of modified nucleosides to N 6 -Methyladenine (mA), N 6-Hydroxymethyladenine (hmA), or N 6 -formyl adenine (fA) 6 -Methyladenine (mA), N 6 -Hydroxymethyladenine (hmA), or N 6 -formyl adenine (fA) from any other one or more and / or the corresponding unmodified nucleoside, i.e. unmodified adenosine.

[0186] In some embodiments, the conversion procedure converts unmodified nucleosides. In some embodiments, the conversion procedure for converting unmodified nucleosides includes bisulfite conversion. Treatment with bisulfite converts unmodified cytosine and certain modified cytosine nucleotides (e.g., 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) to uracil, while other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted. Thus, when bisulfite conversion is used, the converted nucleobase is presumed to include one or more of unmodified cytosine, 5-formylcytosine, 5-carboxylcytosine, or other cytosine forms affected by bisulfite. The unconverted nucleobase is presumed to include one or more of mC and hmC. Sequencing of bisulfite-treated DNA identifies positions that are decoded as cytosine, which are mC or hmC positions. Meanwhile, positions that are decoded as T are identified as bisulfite-sensitive forms of T or C, such as unmodified cytosine, 5-formylcytosine, or 5-carboxylcytosine. Thus, performing the bisulfite conversion described herein facilitates identifying positions that contain mC or hmC. For an exemplary description of bisulfite conversion, see, for example, Moss et al., Nat Commun. 2018;9:5068.

[0187] In some embodiments, the procedure for converting unmodified nucleosides includes oxidative bisulfite (Ox-BS) conversion. In this procedure, hmC is first converted to fC (which is bisulfite-sensitive), followed by bisulfite conversion. Thus, when oxidative bisulfite conversion is used, the converted nucleobase is presumed to include one or more of unmodified cytosine, fC, caC, hmC, or other cytosine forms that are affected by bisulfite. The unconverted nucleobase is presumed to include mC. Sequencing of Ox-BS converted DNA identifies positions that are decoded as cytosine, which is an mC position. Meanwhile, positions that are decoded as T are identified as being bisulfite-sensitive forms of T, hmC, or C, e.g., unmodified cytosine, fC, or hmC. Thus, performing Ox-BS conversion makes it easier to identify positions that contain mC. For an exemplary description of oxidative bisulfite conversion, see, e.g., Booth et al., Science 2012;336:934-937.

[0188] In some embodiments, the procedure for converting unmodified nucleosides comprises Tet-assisted bisulfite (TAB) conversion. In TAB conversion, hmC is protected from conversion and mC is oxidized prior to bisulfite treatment, such that the position originally occupied by mC is converted to U while the position originally occupied by hmC remains as a protected form of cytosine. For example, as described in Yu et al., Cell 2012;149:1368-80, β-glucosyltransferase can be used to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), then a TET protein such as mTet1 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. Thus, when TAB conversion is used, the converted nucleobases are predicted to include one or more of unmodified cytosine, fC, caC, mC, or other cytosine forms that are affected by bisulfite. The unconverted nucleobases are predicted to include hmC. Sequencing of TAB-converted DNA identifies positions that are decoded as cytosine, which are hmC positions. Meanwhile, positions that are decoded as T are identified as being T, mC, or bisulfite-sensitive forms of C, such as unmodified cytosine, fC, or caC. Thus, performing TAB conversion on the first subsample as described herein facilitates identifying positions that contain hmC.

[0189] In some embodiments, the conversion procedure for converting unmodified nucleosides comprises APOBEC-linked epigenetic (ACE) conversion. In ACE conversion, unmodified cytosine and mC are deaminated using an AID / APOBEC family DNA deaminase enzyme, such as APOBEC3A (A3A), but not hmC, fC, or caC. Sequencing of ACE-converted DNA identifies positions that are decoded as cytosine, which are hmC, fC, or caC positions. Meanwhile, positions that are decoded as T are identified as being T, unmodified C, or mC. Thus, performing ACE conversion as described herein facilitates distinguishing positions that contain hmC from positions that contain mC or unmodified C using sequence reads obtained from the first subsample. For an exemplary description of ACE conversion, see, for example, Schutsky et al., Nature Biotechnology 2018;36:1083-1090.

[0190] In some embodiments, the conversion procedure involves enzymatic protection of modified cytosines followed by deamination of unprotected cytosines to uracil (which is then decoded as T). Prior to enzymatic protection and deamination of modified cytosines, a hairpin is ligated to the DNA, where the hairpin joins to a complementary DNA strand. The DNA strands are then separated and the missing strand is synthesized. An adaptor is ligated to the DNA, the hairpin is opened, and PCR amplification is performed. Each side of the hairpin is decoded and represents the same stretch of DNA, and a set of resolution rules resolves the reads into one of A, unmodified C, modified C, G, or T. For an exemplary description of this conversion procedure, see, for example, Fullgrabe et al., bioRxiv 2022;07.08.499285.

[0191] In certain embodiments, the disclosed methods have utility in providing quality control methods for identifying methylated cytosines (i.e., CpG and CpH cytosines) that are not present in any sequence context. Methylated CpH or non-CpG cytosines are rare and therefore require a high level of sensitivity to reliably detect. In addition, methylated CpGs co-located with methylated non-CpGs cannot be detected by methods that use the methylation state of non-CpG cytosines as an indicator of suboptimal molecular conversion. The disclosed methods accomplish this by providing quality control nucleosides known to have specific modification states, thus providing a reliable measure of the frequency of erroneous and / or suboptimal conversions.

[0192] In some embodiments, the method of the present disclosure includes an analysis of sequence diversity and / or fragmentation patterns, and does not exclude adapted DNA with suboptimal or erroneous conversion of quality control nucleosides from the analysis of sequence diversity and / or fragmentation patterns. For example, the method may include detecting the presence or absence of sequence diversity and / or determining fragmentation patterns, where adapted DNA with quality control nucleosides, meaning suboptimal or erroneous conversion of quality control nucleosides, is included in detecting the presence or absence of sequence diversity and / or determining fragmentation patterns. In this way, the method may reduce the possibility of false negatives and / or false positives in the detection of modified nucleosides (e.g., mC or hmC) by removing adapted DNA that is not suitable for the purpose due to suboptimal or erroneous conversion, while retaining such adapted DNA for the analysis of sequence diversity and / or fragmentation patterns (not affected by suboptimal or erroneous conversion), thus avoiding the impact on sensitivity.

[0193] Analysis and / or partitioning of DNA In some cases, a heterogeneous nucleic acid sample is divided into two or more fractions (subsamples). In some embodiments, each fraction is differentially tagged. The tagged fractions can then be pooled together for population sample prep and / or sequencing. The splitting-tagging-pooling step can be performed two or more times, with each round of splitting being based on a different feature, and tagging using differential tags that are distinct from other fractions and splitting means.

[0194] Examples of features that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins binding to DNA. The resulting fractions may 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 cytosine modification (e.g., cytosine methylation) or methylation in general is performed, optionally combined with at least one additional partitioning step that may be based on any of the aforementioned features or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids in the presence of one or more epigenetic modifications and in the absence of one or more epigenetic modifications. Examples of epigenetic modifications include the presence or absence of methylation; the level of methylation; the type of methylation (e.g., 5-methylcytosine and other types of methylation, e.g., adenine methylation and / or cytosine hydroxymethylation); and the association and level of association with one or more proteins, e.g., histones. Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into nucleic acid molecules associated with nucleosomes and nucleic acid molecules lacking nucleosomes.Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).Alternatively or additionally, the heterogeneous population of nucleic acids can be divided based on the length of the nucleic acid (e.g., molecules up to 160 bp and molecules having a length greater than 160 bp).

[0195] In some cases, different procedures are applied to different fractions to determine different characteristics of the initial sample. At least one fraction of nucleic acid, such as DNA, undergoes a conversion procedure according to the disclosed method described herein. In some embodiments, at least one fraction does not undergo a conversion procedure. The corresponding sequences from the converted and unconverted fractions can be compared to identify the single base that has undergone conversion, and therefore identify the corresponding modified nucleoside in the initial sample.

[0196] For methods that include a partitioning step, partition tags (that distinguish molecules in one fraction from molecules in a different fraction) may be included within the adaptors or may be added to the sample molecules.

[0197] In some embodiments, two or more fractions, e.g., each fraction, are differentially tagged. Tags are used to label fractions of distinct polynucleotide populations, allowing a tag (or tags) to be correlated with a particular fraction. In some embodiments, a single tag can be used to label a particular fraction. In some embodiments, multiple different tags can be used to label a particular fraction. In embodiments where multiple different tags are used to label a particular fraction, the set of tags used to label one fraction can be easily distinguished from the set of tags used to label another fraction. In some embodiments, the tag may have additional functionality, e.g., the tag may be used to indicate the source of a sample or to serve as a unique molecular identifier (which may improve the quality of sequencing data by distinguishing sequencing errors from mutations, e.g., Kinde et al., Proc Nat'l Acad Sci USA 108:9530-9535 (2011), Kou et al., PLoS 0NE, 11:e0146638 (2016)), or as a non-unique molecular identifier, e.g., as described in U.S. Pat. No. 9,598,731. Similarly, in some embodiments, the tag may have additional functionality, e.g., the tag may be used to indicate the source of a sample or to serve as a non-unique molecular identifier (which may improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0198] In some embodiments, split tagging involves tagging molecules in each fraction with split tags. After recombining the fractions (e.g., to reduce the number of required sequencing runs and avoid unnecessary costs) and sequencing the molecules, the split tags identify the fraction of the source. In another embodiment, different fractions are tagged with different sets of molecular tags, e.g., composed of pairs of barcodes. In this way, each molecular barcode is useful for indicating the fraction of the source and identifying the molecules in the fraction. For example, a first set of 35 barcodes can be used to tag molecules in the first fraction, while a second set of 35 barcodes can be used to tag molecules in the second fraction.

[0199] In some embodiments, after splitting and tagging with split tags, the molecules may be pooled for sequencing in a single run. In some embodiments, for example, in a step subsequent to the addition and pooling of split tags, a sample tag is added to the molecules. The sample tag may facilitate pooling of material generated from multiple samples for sequencing in a single sequencing run.

[0200] Alternatively, in some embodiments, the split tags can be correlated with samples and fractions. As a simple example, a first tag can indicate a first fraction of a first sample; a second tag can indicate a second fraction of the first sample; a third tag can indicate a first fraction of a second sample; and a fourth tag can indicate a second fraction of the second sample.

[0201] While tags may be attached to molecules that have already been split based on one or more features, the final tagged molecules in the library may no longer have those features. For example, single-stranded DNA molecules may be split and tagged, while the final tagged molecules in the library are likely to be double-stranded. Similarly, DNA may undergo splitting based on different levels of methylation in the final library, while the tagged molecules derived from these molecules are likely to be unmethylated. Thus, the tags attached to molecules in the library typically represent the characteristics of the "parent molecule" from which the final tagged molecule is derived, and not necessarily the characteristics of the tagged molecule itself.

[0202] For example, molecules in a first fraction are tagged and labeled using barcodes 1, 2, 3, 4, etc.; molecules in a second fraction are tagged and labeled using barcodes A, B, C, D, etc.; molecules in a third fraction are tagged and labeled using barcodes a, b, c, d, etc. Differentially tagged fractions can be pooled before sequencing. Differentially tagged fractions can be sequenced separately or simultaneously sequenced together, for example, in the same flow cell of an Illumina sequencer.

[0203] After sequencing, analysis of the reads can be performed at the level of each fraction and at the level of the total DNA population. Tags are used to separate the reads from different fractions. Analysis can include in silico analysis to determine genes and epigenetic variations (one or more of methylation, chromatin structure, etc.) using sequence information, genome conformation length, coverage, and / or copy number. In some embodiments, higher coverage can be correlated with higher nucleosome occupancy in a genomic region, while lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0204] The methods disclosed herein include analyzing DNA in a sample. In some embodiments described herein, the disclosed methods include dividing the DNA. In such methods, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically divided based on one or more characteristics of the DNA. This approach can be used to determine, for example, whether a particular sequence is hypermethylated or hypomethylated. In some embodiments, a first subsample or aliquot of the sample is subjected to a step for generating capture probes as described elsewhere herein, and a second subsample or aliquot of the sample is subjected to division. In some embodiments, the sample or a subsample or aliquot thereof is subjected to division and differential tagging followed by a capture step using capture probes for the rearranged sequences and optionally additional capture probes, for example, for sequence variables and / or epigenetic target regions.

[0205] Methylation profiling can include determining the methylation pattern through different regions of a genome. For example, after dividing and sequencing molecules based on the degree of methylation (e.g., the relative number of methylated nucleobases per molecule), the sequences of molecules in different fractions can be mapped to a reference genome. This can indicate regions of the genome that are more highly methylated or less highly methylated compared to other regions. In this way, genomic regions can differ in their degree of methylation for individual molecules.

[0206] Partitioning of nucleic acid molecules in a sample can increase rare signals, for example, by enriching rare nucleic acid molecules that are more prevalent in one fraction of the sample. For example, genetic variations that are present in hypermethylated DNA but less present (or absent) in hypomethylated DNA can be more easily detected by dividing the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing multiple fractions of a sample, multidimensional analysis of single molecules can be performed, and therefore greater sensitivity can be achieved. Partitioning can include physically dividing nucleic acid molecules into fractions or subsamples based on the presence or absence of one or more methylated nucleic acid bases. Samples can be divided into fractions or subsamples based on features that signify differential gene expression or pathology. Samples can be divided based on features that result in signal differences between normal and pathological conditions, or combinations thereof, during the analysis of nucleic acids, for example, cell-free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell-free nucleic acid (cfNA).

[0207] In some embodiments, hypermethylated and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they exhibit differential methylation characteristics of tumor cells or cell types that do not normally contribute to the DNA sample being analyzed (such as cfDNA), and / or specific immune cell types.

[0208] In some cases, the heterogeneous DNA in the sample is divided into two or more fractions (e.g., at least 3, 4, 5, 6, or 7 fractions). In some embodiments, each fraction is differentially tagged. The tagged fractions can then be pooled together for population sample prep and / or sequencing. The split-tagging-pooling step can be performed two or more times, with each round of splitting being based on a different feature (examples provided herein), and tagging using differential tags that are distinct from other fractions and splitting means. In other cases, the differentially tagged fractions are sequenced separately.

[0209] In some embodiments, sequence reads from the differentially tagged and pooled DNA are obtained and analyzed in silico. The tags are used to sort the reads from different fractions. Analysis to detect genetic variants can be performed at the fraction-by-fraction level and at the total nucleic acid population level. For example, the analysis can include in silico analysis to determine genetic variants such as CNVs, SNVs, indels, and nucleic acid fusions in each fraction. In some cases, the in silico analysis can include determining chromatin structure. For example, the coverage of sequence reads can be used to determine nucleosome positioning in chromatin. Higher coverage can be correlated with higher nucleosome occupancy in a genomic region, while lower coverage can be correlated with lower nucleosome occupancy or nucleosome depleted regions (NDRs).

[0210] Examples of features that can be used for partitioning include sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation, and / or proteins binding to DNA. The resulting fractions may 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 is generally performed based on cytosine modification (e.g., cytosine methylation) or methylation, and is optionally combined with at least one additional partitioning step that may be based on any of the aforementioned features or forms of DNA. In some embodiments, a heterogeneous population of nucleic acids is partitioned into nucleic acids in the presence of one or more epigenetic modifications and in the absence of one or more epigenetic modifications. Examples of epigenetic modifications include the presence or absence of methylation; the level of methylation; the type of methylation (e.g., 5-methylcytosine and other types of methylation, e.g., adenine methylation and / or cytosine hydroxymethylation); and the association and level of association with one or more proteins, e.g., histones. Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into nucleic acid molecules associated with nucleosomes and nucleic acid molecules lacking nucleosomes.Alternatively or additionally, the heterogeneous population of nucleic acids can be divided into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).Alternatively or additionally, the heterogeneous population of nucleic acids can be divided based on the length of the nucleic acid (e.g., molecules up to 160 bp and molecules having a length greater than 160 bp).

[0211] The agent used to split the population of nucleic acids in the sample can be an affinity agent, e.g., an antibody with the desired specificity, a natural binding partner or variant thereof (Bock et al., Nat Biotech 28:1106-1114 (2010); Song et al., Nat Biotech 29:68-72 (2011)), or an artificial peptide selected, e.g., by phage display, to have specificity for a given target. In some embodiments, the agent used in the splitting is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, e.g., methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is the product of a procedure that affects a first nucleobase in DNA differently than a second nucleobase in the DNA of the sample. In some embodiments, the modified nucleobase can be a "converted nucleobase," meaning that its base pair specificity has been altered by the 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 splitting agents include antibodies, such as antibodies that recognize modified nucleobases, which may be modified cytosines, such as methylcytosines (e.g., 5-methylcytosine). In some embodiments, the splitting agent is an antibody that recognizes modified cytosines other than 5-methylcytosine, such as 5-carboxylcytosine (5caC). Alternative splitting agents include methyl-binding domains (MBDs) and methyl-binding proteins (MBPs) as described herein, including proteins such as MeCP2.

[0212] Additional non-limiting examples of partitioning agents are histone binding proteins that 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.

[0213] In some embodiments, partitioning may include both binary partitioning and partitioning based on the degree / level of modification. For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments can be partitioned from non-methylated fragments using a methyl-binding domain protein (e.g., MethylMinder Methylated DNA Enrichment Kit (ThermoFisher Scientific). Additional partitioning may then involve eluting fragments with different levels of methylation by adjusting the salt concentration in the solution with the methyl-binding domain and bound fragments. As the salt concentration increases, fragments with greater methylation levels are eluted.

[0214] Analysis of the DNA may include detecting or quantifying the DNA of interest. Analysis of the DNA may include detecting genetic variants and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation).

[0215] In some embodiments, the methylation level can be determined using partitioning, modification-sensitive conversion such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction enzyme digestion, methylation-dependent restriction enzyme digestion, or any other suitable technique. 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., MBD such as MBD2, MBD4, or MeCP2) or an antibody specific for 5-methylcytosine (such as in the case of MeDIP) can be used to partition the DNA. This technique can be used to determine, for example, whether a particular sequence is hypermethylated or hypomethylated. In some embodiments, the DNA fragmentation pattern can be determined based on the end points and / or midpoints of the DNA molecule, e.g., cfDNA molecule.

[0216] In some cases, the final fraction is enriched in nucleic acids with different degrees of modification (over- or under-representation of modification). Over- and under-representation can be defined by the number of modifications carried by a nucleic acid relative to the median number of modifications per strand in the population. For example, if the median number of 5-methylcytosine residues in the nucleic acids in a sample is 2, then nucleic acids containing 3 or more 5-methylcytosine residues will be over-represented in this modification, and nucleic acids with 1 or 0 5-methylcytosine residues will be under-represented. The effect of affinity separation is to enrich for nucleic acids that are over-represented in the modification in the binding phase and under-represented in the modification in the non-binding phase (i.e., in solution). Nucleic acids in the binding phase can be eluted before further processing.

[0217] When using MeDIP or MethylMiner® Methylated DNA Enrichment Kit (ThermoFisher Scientific), sequential elution can be used to resolve different levels of methylation. For example, the hypomethylated fraction (unmethylated) can be separated from the methylated fraction by contacting the nucleic acid population with MBD from the kit, which is attached to magnetic beads. Using the beads, the methylated nucleic acids are separated and removed from the unmethylated nucleic acids. Nucleic acids with different levels of methylation are then eluted by performing one or more sequential elution steps. For example, a first set of methylated nucleic acids can be eluted at a salt concentration of 160 mM or more, 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 these methylated nucleic acids are eluted, magnetic separation is again used to separate the more highly methylated nucleic acids from the less methylated nucleic acids. The elution and magnetic separation steps can be repeated to create various fractions, such as a hypomethylated fraction (enriched in nucleic acids that contain unmethylation), a methylated fraction (enriched in nucleic acids that contain low levels of methylation), and a hypermethylated fraction (enriched in nucleic acids that contain high levels of methylation).

[0218] In some methods, the nucleic acids bound to the agents used for affinity separation-based partitioning are subjected to a washing step, in which nucleic acids that are weakly bound to the affinity agents are washed away. These nucleic acids can be enriched in nucleic acids with modifications to a degree close to the mean or median (i.e., halfway between the nucleic acids that remain bound to the solid phase and those that are not bound to the solid phase upon initial contact of the sample with the agent).

[0219] Affinity separation results in at least two, and sometimes three or more, fractions of nucleic acids with different degrees of modification. While the fractions are still separated, the nucleic acids of at least one fraction, and usually two or three (or more) fractions, are linked to nucleic acid tags, usually provided as components of an adaptor, so that the nucleic acids in the different fractions receive different tags that distinguish the members of one fraction from the other. The tags linked to the nucleic acid molecules of the same fraction can be identical or different from each other. However, when different from each other, the tags share part of their code, allowing them to identify the molecules to which they are attached as being of a particular fraction.

[0220] For further details regarding partitioning nucleic acid samples based on features such as methylation, see WO 2018 / 119452, which is incorporated by reference herein.

[0221] In some embodiments, the partitioning comprises contacting the DNA with a methylation-sensitive restriction enzyme (MSRE) and / or a methylation-dependent restriction enzyme (MDRE). After treatment of the DNA with an MSRE or MDRE, the DNA can be partitioned based on size to generate highly methylated (longest DNA molecules after MSRE treatment and shortest DNA fragments after MDRE treatment), intermediate (intermediate length DNA molecules after MSRE or MDRE treatment), and hypomethylated (shortest DNA molecules after MSRE treatment and longest DNA fragments after MDRE treatment) subsamples.

[0222] In some embodiments, the partitioning is performed by contacting the nucleic acid with a methyl-binding domain ("MBD") of a methyl-binding protein ("MBP"). In some such embodiments, the nucleic acid is contacted with the entire MBP. In some embodiments, the MBD binds 5-methylcytosine (5mC), and the MBP comprises the MBD, herein interchangeably referred to as a methyl-binding protein or a methyl-binding domain protein. In some embodiments, the MBD is coupled to paramagnetic beads, e.g., Dynabeads® M-280 streptavidin, via a biotin linker. Partitioning into fractions with different degrees of methylation can be performed by eluting the fractions by increasing NaCl concentrations.

[0223] In some embodiments, the bound DNA is eluted by contacting the antibody or MBD with a protease, such as proteinase K. This can be performed instead of or in addition to the elution step using NaCl, as discussed above.

[0224] Examples of agents that recognize modified nucleobases contemplated herein include, but are not limited to, the following: (a) MeCP2 is a protein that preferentially binds 5-methyl-cytosine over unmodified cytosine. (b) RPL26, PRP8 and the DNA mismatch repair protein MHS6 bind preferentially to 5-hydroxymethyl-cytosine over unmodified cytosine. (c) FOXK1, FOXK2, FOXP1, FOXP4 and FOXI3 preferably bind 5-formyl-cytosine over unmodified cytosine (Iurlaro et al., Genome Biol. 14:R119 (2013)). (d) an antibody specific for one or more methylated or modified nucleobases or their conversion products, e.g., 5mC, 5caC, or DHU.

[0225] In general, elution is a function of the number of modifications, e.g., the number of methylation sites per molecule, with molecules with more methylation eluting under increasing salt concentrations. A series of elution buffers with increasing NaCl concentrations can be used to elute DNA into different populations based on the degree of methylation. Salt concentrations can range from about 100 nM to about 2500 mM NaCl. In one embodiment, the process results in three fractions. Molecules are contacted with a solution at a first salt concentration, including molecules that include an agent that recognizes modified nucleobases, and the molecules can be attached to a capture moiety, e.g., streptavidin. At the first salt concentration, a population of molecules will bind to the agent and a population will remain unbound. The unbound population can be separated as a "hypomethylated" population. For example, the first fraction, enriched in hypomethylated forms of DNA, is the one that remains unbound at a low salt concentration, e.g., 100 mM or 160 mM. A second fraction enriched in moderately methylated DNA is eluted using a moderate salt concentration, for example, 100 mM to 2000 mM, which is also separated from the sample. A third fraction enriched in highly methylated forms of DNA is eluted using a high salt concentration, for example, at least about 2000 mM.

[0226] In some embodiments, methylated DNA is purified using a monoclonal antibody raised against 5-methylcytidine (5mC). The DNA is denatured, for example at 95° C., to obtain single-stranded DNA fragments. Antibody-bound DNA is immunoprecipitated using standard or protein G coupled to magnetic beads and washing followed by incubation with anti-5mC antibody. Such DNA can then be eluted. Fractions may include unprecipitated DNA and one or more fractions eluted from the beads.

[0227] In some embodiments, a fraction of the DNA is desalted and enriched in preparation for the enzymatic steps of library preparation.

[0228] Sequences that contain abnormally high copy numbers may tend to be hypermethylated. Thus, in some embodiments, the DNA that is contacted with the capture probe specific to the members of the epigenetic target region set that includes multiple target regions that are both type-specific differentially methylated regions and copy number polymorphisms comprises at least a portion of a hypermethylated fraction. The DNA that is derived from or comprises at least a portion of a hypermethylated fraction may or may not be combined with the DNA that is derived from or comprises at least a portion of one or more other fractions, such as at least a portion of an intermediate fraction or a hypomethylated fraction.

[0229] In some embodiments, methylation is detected using a conversion procedure. The conversion procedure includes any technique that differentially modifies a first nucleobase but not a second nucleobase in a modification-dependent manner, e.g., methylated versus unmodified (or hydroxymethylated, or formylated, or carboxylated, etc.) and / or modified in one manner versus another (e.g., methylated versus hydroxymethylated). An example of such a conversion procedure includes bisulfite conversion, which converts unmodified cytosine and certain modified cytosines (e.g., 5-formylcytosine (fC) or 5-carboxylcytosine (caC)) to uracil, while other modified cytosines (e.g., 5-methylcytosine, 5-hydroxylmethylcystosine) are not converted. Performing bisulfite conversion can facilitate identifying positions containing mC or hmC using sequence reads. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018;9:5068.

[0230] Examples of such conversion procedures include oxidized bisulfite (Ox-BS) conversion.Performing Ox-BS conversion can facilitate identifying the position containing mC using sequence reads.For an exemplary description of oxidized bisulfite conversion, see, for example, Booth et al., Science 2012;336:934-937.

[0231] Examples of such conversion procedures include Tet-assisted bisulfite (TAB) conversion. For example, as described in Yu et al., Cell 2012;149:1368-80, β-glucosyltransferase can be used to protect hmC (forming 5-glucosylhydroxymethylcytosine (ghmC)), then a TET protein such as mTet1 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. 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. Performing TAB conversion can facilitate identifying positions containing hmC using sequence reads.

[0232] Examples of such conversion procedures also include Tet-assisted conversion with a substituted borane reducing agent, optionally 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane. See, e.g., Liu et al., Nature Biotechnology 2019;37:424-429 (e.g., Appendix Figure 1 and Appendix Note 7). Performing a TAP conversion can facilitate identifying positions containing unmodified C using sequence reads. This procedure encompasses Tet-assisted pyridine borane sequencing (TAPS), described in more detail in Liu et al. 2019, supra.

[0233] Alternatively, protection of hmC (e.g., using βGT) can be combined with Tet-assisted conversion with a substituted borane reducing agent. Performing such a TAPSβ conversion can facilitate using sequence reads to distinguish positions containing unmodified C or hmC from positions containing mC on the other hand. For an exemplary description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019;37:424-429.

[0234] Examples of such conversion procedures include APOBEC-linked epigenetic (ACE) conversion. Performing ACE conversion can facilitate using sequence reads to distinguish positions containing hmC from positions containing mC or unmodified C. For an exemplary description of ACE conversion, see, for example, Schutsky et al., Nature Biotechnology 2018;36:1083-1090.

[0235] Examples of such conversion procedures include enzymatic conversion of nucleobases, such as EM-Seq. See, for example, Vaisvila R, et al. (2019) EM-seq: Detection of DNA methylation at single base resolution from picograms of DNA. bioRxiv; DOI: 10.1101 / 2019.12.20.884692vl, available at: www.biorxiv.org / content / 10.1101 / 2019.12.20.884692vl.

[0236] In some embodiments, methylation is detected using methylation-sensitive restriction enzymes (MSREs). For example, a sample, a subsample, or a portion of a sample may be subjected to digestion with one or more MSREs to cleave unmethylated sequences. Exemplary MSREs include AatII, AccII, AciI, Aorl3HI, Aorl5HI, BspT104I, BssHII, BstUI, Cfr1OI, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, 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 methylation sensitive nucleases include BstUI and HpaII. In some embodiments, the two methylation sensitive nucleases include HhaI and AccII. In some embodiments, the methylation sensitive nucleases include BstUI, HpaII and Hin6I. In some embodiments, the portion of the sample that is contacted with one or more MSREs comprises hypermethylated DNA or is or comprises a hypermethylated DNA fraction, which may be obtained as described elsewhere herein.

[0237] In some embodiments, DNA fragmentation is detected by determining the end and / or midpoint of sequenced fragments of DNA (e.g., cfDNA).For example, differences in fragmentation patterns may occur depending on whether the fragments originate from tumor or from healthy cells.To detect tumor cell-derived DNA of cfDNA based on fragmentation, the presence or absence of increased levels of abnormal fragments can be determined in regions with copy number amplification (e.g., proportional to the degree of amplification), for example, when the increase and abnormality are relative to control or healthy samples.

[0238] In some embodiments, a sample or subsample (e.g., a first, second, or third subsample that has been prepared by dividing a sample described herein based on the level of cytosine modification, such as methylation, e.g., 5-methylation of cytosine, etc.) is contacted with a methylation-dependent nuclease or a methylation-sensitive nuclease. Unless otherwise indicated, when division is performed based on cytosine modification, the first subsample is the subsample having a higher level of modification; the second subsample is the subsample having a lower level of modification; and, if present, the third subsample has an intermediate level of modification between the first and second subsamples.

[0239] As discussed above, the division procedure may result in incomplete sorting of DNA molecules in the subsample. Selection of methylation-dependent nucleases or methylation-sensitive nucleases may be performed to degrade non-specifically divided DNA. For example, the second subsample may be contacted with a methylation-dependent nuclease, such as a methylation-dependent restriction enzyme, which may degrade non-specifically divided DNA (e.g., methylated DNA) in the second subsample to generate a processed second subsample. Alternatively or additionally, the first subsample may be contacted with a methylation-sensitive endonuclease, such as a methylation-sensitive restriction enzyme, which may degrade non-specifically divided DNA in the first subsample to generate a processed first subsample. Degradation of non-specifically split DNA in either or both of the first or second subsamples is proposed as an improvement to the performance of methods that rely on accurate division of DNA based on cytosine modifications, for example, to detect the presence of abnormally modified DNA in a sample, to determine the tissue of origin of the DNA, and / or to determine whether a subject has cancer. For example, such degradation may provide improved sensitivity and / or simplify downstream analysis. In general, if the non-specifically split DNA is highly methylated, for example in a low methylation fraction, a methylation-dependent nuclease, for example a methylation-dependent restriction enzyme, should be used. Conversely, if the non-specifically split DNA is low methylated, for example in a high methylation fraction, a methylation-sensitive nuclease, for example a methylation-sensitive restriction enzyme, should be used. A methylation-dependent nuclease, for example a methylation-dependent restriction enzyme, preferentially cuts methylated DNA relative to unmethylated DNA, while a methylation-sensitive nuclease, for example a methylation-sensitive restriction enzyme, preferentially cuts unmethylated DNA relative to methylated DNA.

[0240] When contacting the subsample with a nuclease, one or more nucleases can be used. In some embodiments, the subsample is contacted with multiple nucleases. The subsample may be contacted with the nucleases sequentially or simultaneously. The simultaneous use of nucleases can be advantageous if the nucleases are active under similar conditions (e.g., buffer composition) to avoid unnecessary sample manipulation. By contacting the second subsample with two or more methylation-dependent restriction enzymes, non-specifically cleaved hypermethylated DNA can be more completely degraded. Similarly, by contacting the first subsample with two or more methylation-sensitive restriction enzymes, non-specifically cleaved hypomethylated and / or unmethylated DNA can be more completely degraded.

[0241] In some embodiments, the 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 methylation dependent nuclease comprises FspEI. In some embodiments, the methylation dependent nuclease comprises FspEI and MspJI, e.g., used sequentially.

[0242] In some embodiments, the methylation sensitive nuclease comprises one or more of AatII, AccII, AciI, Aorl3HI, Aorl5HI, BspT104I, BssHII, BstUI, Cfr1OI, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, MspI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, 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 methylation sensitive nuclease comprises BstUI and HpaII. In some embodiments, the two methylation sensitive nucleases comprise HhaI and AccII. In some embodiments, the methylation sensitive nucleases include BstUI, HpaII, and Hin6I.

[0243] In some embodiments, FspEI is used to digest nucleic acid molecules in at least one subsample (e.g., the low methylation fraction). In some embodiments, BstUI, HpaII and Hin6I are used to digest nucleic acid molecules in at least one subsample (e.g., the high methylation fraction) and FspEI is used to digest nucleic acid molecules in at least one other subsample (e.g., the low methylation fraction). In embodiments that include a moderately methylated fraction, 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 the moderately methylated fraction are digested with the same nuclease as the high methylation fraction. For example, the moderately methylated fraction can be pooled with the high methylation fraction, and then the pooled fractions may be subjected to digestion. In some embodiments, the nucleic acid molecules in the moderately methylated fraction are digested with the same nuclease as the low methylation fraction. For example, the moderately methylated fraction can be pooled with the low methylated fraction and the pooled fractions can then be subjected to digestion.

[0244] In some embodiments, the subsample is contacted with the nuclease described above after the step of tagging or attaching adapters to both ends of the DNA. The tag or adapter may be resistant to cleavage by the nuclease using any of the techniques described above. In this approach, cleavage may prevent analysis from being performed on non-specifically cleaved molecules, since the cleavage products lack the tag or adapter at both ends.

[0245] Alternatively, the step of tagging or attaching the adapters can be performed after cleavage by the nuclease described above. The cleaved molecules can then be identified in the sequence reads based on having an end (attachment point to the tag or adapter) that corresponds to the nuclease recognition site. Treating the molecules in this way may also allow for obtaining information from the cleaved molecules, such as observing somatic mutations. It may be desirable to tag or attach the adapters after contacting the subsample with the nuclease, and when low molecular weight DNA such as cfDNA is being analyzed, 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 a nuclease that can be heat inactivated at a relatively low temperature (e.g., 65°C or less, or 60°C or less) to avoid denaturing the DNA, as denaturation may interfere with the subsequent ligation step.

[0246] If the sample is divided into three subsamples, including a third subsample containing moderately 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 in relation to the contacting step, and may be performed before or after the step of tagging or attaching an adaptor as discussed above. In some embodiments, the first and third subsamples are combined before contacting with the methylation-sensitive nuclease. Such a step may have any of the features described elsewhere herein in relation to the contacting step, and may be performed before or after the step of tagging or attaching an adaptor as discussed above. In some embodiments, the first and third subsamples are differentially tagged before being combined.

[0247] Alternatively, if the sample is divided into three subsamples, including a third subsample containing moderately methylated molecules, the third subsample is contacted in some embodiments with a methylation-dependent nuclease. Such a step may have any of the features described elsewhere herein in relation to the contacting step, and may be performed before or after the step of tagging or attaching an adaptor as discussed above. In some embodiments, the second and third subsamples are combined before contacting with the methylation-dependent nuclease. Such a step may have any of the features described elsewhere herein in relation to the contacting step, and may be performed before or after the step of tagging or attaching an adaptor as discussed above. In some embodiments, the second and third subsamples are differentially tagged before being combined.

[0248] In some embodiments, the DNA is purified after contacting with a nuclease, for example using SPRI beads. Such purification may be performed after heat inactivation of the nuclease. Alternatively, purification can be omitted; thus, subsequent steps, such as, for example, amplification, can be performed on a subsample containing the heat-inactivated nuclease. In another embodiment, the contacting step can be performed in the presence of a purification reagent, such as SPRI beads, for example, to minimize losses associated with tube exchange. After cleavage and heat inactivation, the SPRI beads can be reused for purification by adding a molecular crowding reagent (e.g., PEG) and salt.

[0249] In some embodiments, when a conversion procedure is performed on a sample or subsample, subsequent capture from that sample or subsample of one or more sets of target regions (e.g., at least a set of epigenetic target regions) uses capture probes that are specific for the modification state (e.g., of at least one base in the sequence to which the probe hybridizes), e.g., complementary to a target sequence that has undergone a conversion (e.g., conversion of a modified or unmodified cytosine to uracil or its analog, e.g., DHU, which preferentially pairs with adenine) or has not undergone a conversion, as desired. As such, the probes can be specific for sequences in which a modification of interest, e.g., methylation, was present or absent. In some embodiments, when modification sensitive conversion is performed on a sample or subsample, subsequent capture from that sample or subsample of one or more sets of target regions (e.g., at least a set of epigenetic target regions) uses capture probes that include probes that can hybridize to target sequences regardless of modification state (e.g., including nucleobases that randomly pair with modified or unmodified cytosine, that preferentially pair with adenine (e.g., inosine may pair with C or U), and that may or may not have been converted to uracil or an analog thereof, such as DHU).

[0250] In some embodiments, the method includes preparing a pool comprising at least a portion of the DNA of the second subsample (also referred to as the hypomethylated fraction) and at least a portion of the DNA of the first subsample (also referred to as the hypermethylated fraction). For example, target regions comprising epigenetic target regions and / or sequence variable target regions may be captured from the pool. Capturing a set of target regions from at least a portion of the subsamples as described elsewhere herein includes a capture step performed on a pool comprising DNA from the first and second subsamples. Amplifying the DNA in the pool may be performed prior to capturing the target regions from the pool. The capture step may have any of the features as described elsewhere herein.

[0251] Epigenetic target regions may exhibit differences in methylation levels and / or fragmentation patterns depending on whether they originate from a tumor or a healthy cell, or what type of tissue they originate from, as discussed elsewhere herein. Sequence variable target regions may exhibit differences in sequence depending on whether they originate from a tumor or a healthy cell.

[0252] Analysis of epigenetic target regions from low methylation fractions may be less useful in some applications than analysis of sequence variable target regions from high methylation fractions and low methylation fractions and epigenetic target regions from high methylation fractions. As such, in methods where sequence variable target regions and epigenetic target regions are being captured, the latter may be captured to a lesser extent than one or more of sequence variable target regions from high methylation fractions and low methylation fractions and epigenetic target regions from high methylation fractions. For example, sequence variable target regions can be captured from a portion of low methylation fractions that is not pooled with high methylation fractions, and pools can be prepared with a portion (e.g., most, substantially all, or all) of DNA from high methylation fractions and none or some (e.g., a minority) of DNA from low methylation fractions. Such approaches can reduce or eliminate sequencing of epigenetic target regions from low methylation fractions, thereby reducing the amount of sequencing data that is sufficient for further analysis.

[0253] In some embodiments, the inclusion of a minority of DNA from the hypomethylated fraction in the pool facilitates quantification, e.g., on a relative basis, of one or more epigenetic features (e.g., methylation or other epigenetic features, as discussed in detail elsewhere herein).

[0254] In some embodiments, the pool comprises a small number of DNA from the hypomethylated fraction, e.g., less than about 50% of the DNA from the hypomethylated fraction, e.g., about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the DNA from the hypomethylated fraction. In some embodiments, the pool comprises about 5%-25% of the DNA from the hypomethylated fraction. In some embodiments, the pool comprises about 10%-20% of the DNA from the hypomethylated fraction. In some embodiments, the pool comprises about 10% of the DNA from the hypomethylated fraction. In some embodiments, the pool comprises about 15% of the DNA from the hypomethylated fraction. In some embodiments, the pool comprises about 20% of the DNA from the hypomethylated fraction.

[0255] In some embodiments, the pool comprises a portion of the hypermethylated fraction, which may be at least about 50% of the DNA of the hypermethylated fraction. For example, the pool may comprise at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the DNA of the hypermethylated fraction. In some embodiments, the pool comprises 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% of the DNA of the hypermethylated fraction. In some embodiments, the second pool comprises all or substantially all of the hypermethylated fraction.

[0256] In some embodiments, the method includes preparing a first pool that includes at least a portion of the DNA of the low methylated fraction. In some embodiments, the method includes preparing a second pool that includes at least a portion of the DNA of the high methylated fraction. In some embodiments, the first pool further includes a portion of the DNA of the high methylated fraction. In some embodiments, the second pool further includes a portion of the DNA of the low methylated fraction. In some embodiments, the first pool includes a majority of the DNA of the low methylated fraction, and optionally a minority of the DNA of the high methylated fraction. In some embodiments, the second pool includes a majority of the DNA of the high methylated fraction and a minority of the DNA of the low methylated fraction. In some embodiments that include a moderately methylated fraction, the second pool includes at least a portion of the DNA of the moderately methylated fraction, e.g., a majority of the DNA of the moderately methylated fraction. In some embodiments, the first pool includes a majority of the DNA of the low methylated fraction and the second pool includes a majority of the DNA of the high methylated fraction and a majority of the DNA of the moderately methylated fraction.

[0257] In some embodiments, the method comprises capturing at least a first set of target regions from a first pool, e.g., the first pool is as described in any of the above embodiments. In some embodiments, the first set comprises sequence variable target regions. In some embodiments, the first set comprises hypomethylated variable target regions and / or fragmented variable target regions. In some embodiments, the first set comprises sequence variable target regions and fragmented variable target regions. In some embodiments, the first set comprises sequence variable target regions, hypomethylated variable target regions and fragmented variable target regions. A step of amplifying the DNA in the first pool may be performed prior to this capturing step. In some embodiments, capturing the first set of target regions from the first pool comprises contacting the DNA of the first pool with a first set of capture probes. In some embodiments, the first set of capture probes comprises target binding probes specific for sequence variable target regions. In some embodiments, the first set of capture probes comprises target binding probes specific for sequence variable target regions, hypomethylated variable target regions and / or fragmented variable target regions.

[0258] In some embodiments, the method includes capturing a second set of target regions or a plurality of sets of target regions from a second pool, e.g., the first pool is as described in any of the above embodiments. In some embodiments, the second plurality includes epigenetic target regions, e.g., hypermethylated variable target regions and / or fragmented variable target regions. In some embodiments, the second plurality includes sequence variable target regions and epigenetic target regions, e.g., hypermethylated variable target regions and / or fragmented variable target regions. A step of amplifying the DNA in the second pool may be performed prior to this capturing step. In some embodiments, capturing the second plurality of sets of target regions from the second pool includes contacting the DNA of the first pool with a second set of capture probes, the second set of capture probes including target binding probes specific for sequence variable target regions and target binding probes specific for epigenetic target regions. In some embodiments, the first set of target regions and the second set of target regions are not identical. For example, the first set of target regions may include one or more target regions that are not present in the second set of target regions. Alternatively or additionally, the second set of target regions may include one or more target regions not present in the first set of target regions. In some embodiments, at least one hypermethylated variable target region is captured from the second pool but not from the first pool. In some embodiments, a plurality of hypermethylated variable target regions is captured from the second pool but not from the first pool. In some embodiments, the first set of target regions includes sequence variable target regions and / or the second set of target regions includes epigenetic target regions. In some embodiments, the first set of target regions includes sequence variable target regions and fragmentation variable target regions; and the second set of target regions includes epigenetic target regions, e.g., hypermethylated variable target regions and fragmentation variable target regions. In some embodiments, the first set of target regions includes sequence variable target regions, fragmentation variable target regions, and includes hypomethylated variable target regions; and the second set of target regions includes epigenetic target regions, e.g., hypermethylated variable target regions and fragmentation variable target regions.

[0259] In some embodiments, the first pool comprises a majority of the DNA from the hypomethylated fraction and a portion (e.g., about half) of the DNA from the hypermethylated fraction, and the second pool comprises a portion (e.g., about half) of the DNA from the hypermethylated fraction. In some such embodiments, the first set of target regions comprises sequence variable target regions and / or the second set of target regions comprises epigenetic target regions. The sequence variable target regions and / or epigenetic target regions may be as described in any of the embodiments described elsewhere herein.

[0260] In some embodiments, a fraction of DNA is desalted and enriched in preparation for the enzymatic steps of library preparation. Sequences containing structural mutations may tend to be hypomethylated. Thus, in some embodiments, the DNA contacted with a plurality of primers, including at least two primers that anneal in an antiparallel direction to the rearranged sequence of DNA, is derived from or comprises at least a portion of a hypomethylated fraction. The DNA derived from or comprises at least a portion of a hypomethylated fraction may or may not be combined with DNA derived from or comprises at least a portion of one or more other fractions, such as a moderate fraction or a hypermethylated fraction.

[0261] amplification Sample nucleic acids flanked by adaptors can be amplified by PCR and other amplification methods. Amplification is typically primed by primers that bind to primer binding sites in the adaptors that flank the DNA molecules to be amplified. Amplification methods can include cycles of denaturation, annealing and extension due to thermocycling, or can be isothermal, such as in the case of transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustaining sequence-based replication.

[0262] In some embodiments, the method performs dsDNA ligation with T-tail and C-tail adapters resulting in at least 50, 60, 70, or 80% amplification of double stranded nucleic acid. Preferably, the method increases the amount or number of amplified molecules by at least 10, 15, or 20% compared to a control method performed with T-tail adapters alone.

[0263] In some embodiments, the sample nucleic acid is amplified before sequencing. Amplification may be before and / or after the conversion step. Amplification may in some cases be before one or more capture steps. In some embodiments, ligation is performed before or simultaneously with amplification.

[0264] In some embodiments, amplification is primed by a primer binding to a primer binding site in an adapter oligonucleotide. The known modified nucleosides of the adapter may be present outside of the primer binding site, i.e., the known modified nucleosides used for quality control methods are not present in the primer binding site or are not bound by the amplification primers.

[0265] Capture Probes; Enrichment, Capture, and Use of Target Regions Nucleic acids in a sample may undergo a capture step where molecules with target sequences are captured for subsequent analysis. Capture may be performed using any suitable technique known in the art. Target capture may include the use of a bait set that includes a capture moiety, e.g., an oligonucleotide bait that is labeled with biotin or other examples described below. Probes may have sequences that are selected to juxtapose through a panel of regions such as genes. Such bait sets are combined with the sample under conditions that allow hybridization of target molecules with the baits. The captured molecules are then isolated using a capture moiety. For example, a biotin capture moiety is mediated by bead-based streptavidin. Such methods are further described, for example, in U.S. Pat. No. 9,850,523, issued Dec. 26, 2017, which is incorporated herein by reference.

[0266] Capture moieties include, but are not limited to, biotin, avidin, streptavidin, nucleic acids containing specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractable particles. Extraction moieties can be members of binding pairs, such as biotin / streptavidin or hapten / antibody. In some embodiments, the capture moiety attached to the analyte is captured by its binding pair attached to an isolatable moiety, such as a magnetically attractable particle or a larger particle that can be sedimented through centrifugation. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids with a capture moiety from nucleic acids that lack the capture moiety. An exemplary capture moiety is biotin, which allows affinity separation by binding to streptavidin that is linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation by binding to a complementary oligonucleotide that is linked or linkable to a solid phase.

[0267] In some embodiments, DNA is captured that includes a region that contains a type-specific epigenetic mutation. In some embodiments, the mutation is present in healthy cells, but not normally present in a sample type such as a blood sample. In some embodiments, the mutation is present in abnormal cells (e.g., hyperplastic, dysplastic, or neoplastic cells).

[0268] In some embodiments, the first set of captured epigenetic target regions captured from the sample or first subsample comprises hypermethylated variable target regions. In some embodiments, the hypermethylated variable target regions show type-specific hypermethylation in healthy cfDNA from one or more related cell or tissue types. Without wishing to be bound by any particular theory, the presence of cancer cells may increase the shedding of DNA (e.g., from the cancer and / or surrounding tissues) into the bloodstream. As such, the distribution of tissues of origin of cfDNA may change during carcinogenesis. Thus, increased levels of hypermethylated variable target regions in the first subsample may be indicative of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0269] In some embodiments, the method herein comprises capturing a second set of captured epigenetic target regions from the sample or the second subsample. In some embodiments, the second set of epigenetic target regions comprises hypomethylated variable target regions. 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 during carcinogenesis. Thus, an increased level of hypomethylated variable target regions in the second subsample may be indicative of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0270] In addition, the set of captured target regions may include DNA corresponding to a set of sequence variable target regions. The captured sets may be combined to provide a combined captured set.

[0271] In some embodiments, including combined captured sets, where a captured set includes DNA corresponding to a set of sequence variable target regions and a set of epigenetic target regions, as discussed above, the DNA corresponding to the set of sequence variable target regions is at a greater concentration than the DNA corresponding to the set of epigenetic target regions, e.g., 1.1-1.2 fold greater, 1.2-1.4 fold greater, 1.4-1.6 fold greater, 1.6-1.8 fold greater, 1.8-2.0 fold greater, 2.0-2.2 fold greater, 2.2-2.4 fold greater, 2.4-2.6 fold greater, 2.6-2.8 fold greater, 2.8-3.0 fold greater, 3.0-3.5 fold greater, 3.5-4.0 fold greater, 4.0-4.5 fold greater, 4.5-5.0 fold greater, 5.0-5.5 fold greater. degree, 5.5-6.0 times greater concentration, 6.0-6.5 times greater concentration, 6.5-7.0 times greater concentration, 7.0-7.5 times greater concentration, 7.5-8.0 times greater concentration, 8.0-8.5 times greater concentration, 8.5-9.0 times greater concentration, 9.0-9.5 times greater concentration, 9.5-10.0 times greater concentration, 10-11 times greater concentration, 11-12 times greater concentration, 12-13 times greater concentration, 13-14 times greater concentration, It may be present at 14-15 fold greater concentration, 15-16 fold greater concentration, 16-17 fold greater concentration, 17-18 fold greater concentration, 18-19 fold greater concentration, 19-20 fold greater concentration, 20-30 fold greater concentration, 30-40 fold greater concentration, 40-50 fold greater concentration, 50-60 fold greater concentration, 60-70 fold greater concentration, 70-80 fold greater concentration, 80-90 fold greater concentration, or 90-100 fold greater concentration. The degree of difference in concentration accounts for normalization to the footprint size of the target region as discussed in the definitions section.

[0272] In some embodiments, the captured DNA comprises an intron region. In some embodiments, the intron region comprises one or more introns that are likely to distinguish the DNA from neoplastic (e.g., tumor or cancer) cells and healthy cells, such as non-neoplastic circulating cells. For example, an intron containing a rearrangement known to be present in some neoplastic cells and absent in healthy cells can be used to distinguish the DNA from neoplastic (e.g., tumor or cancer) cells and healthy cells. In some embodiments, the rearrangement is a translocation.

[0273] In some embodiments, the captured intron region has 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 set of intron target regions have a footprint in the range of 30 bp to 1000 kb, e.g., 30 bp to 100 bp, 100 bp to 200 bp, 200 bp to 500 bp, 500 bp to lkb, 1 kb to 2 kb, 2 kb to 5 kb, 5 kb to 10 kb, 10 kb to 20 kb, 20 kb to 50 kb, 50 kb to 100 kb, 100 to 200 kb, 200 to 300 kb, 300 to 400 kb, 400 to 500 kb, 500 to 600 kb, 600 to 700 kb, 700 to 800 kb, 800 to 900 kb, and 900 to 1,000 kb.

[0274] Exemplary rearrangements, such as intronic rearrangements, that can be detected using the methods described herein include, but are not limited to, rearrangements in which at least one of the two genes involved in the rearrangement is a receptor tyrosine kinase. Exemplary rearrangement products are BCR-ABL fusions and fusions involving any of ALK, FGFR2, FGFR3, NTRK1, RET, or ROS1.

[0275] In some embodiments, the captured DNA comprises a target region with type-specific epigenetic mutations. In some embodiments, the set of epigenetic target regions consists of target regions with type-specific epigenetic mutations. In some embodiments, type-specific epigenetic mutations, such as differential methylation or type-specific fragmentation patterns, are likely to distinguish DNA from cells of one or more related cell types or tissue types from DNA from other cell types or tissue types present in the sample or subject.

[0276] In some embodiments, the nucleic acid captured or enriched using the methods described herein comprises captured DNA, e.g., a set of one or more captured DNAs. In some embodiments, the captured DNA comprises a target region that is differentially methylated in different immune cell types. In some embodiments, the immune cell types comprise rare or closely related immune cell types, e.g., activated and naive lymphocytes or myeloid cells at different stages of differentiation.

[0277] In some embodiments, the captured set of epigenetic target regions captured from the sample or first subsample comprises a hypermethylated variable target region. In some embodiments, the hypermethylated variable target region is differentially or exclusively hypermethylated in one or more related cell types or tissue types. In some embodiments, the hypermethylated variable target region is differentially or exclusively hypermethylated in one cell type or one immune cell type or one immune cell type in a cluster. In some embodiments, the hypermethylated variable target region is hypermethylated to a degree that is discernibly higher or exclusively present in one cell type or one immune cell type or one immune cell type in a cluster. Such hypermethylated variable target regions may be hypermethylated in other cell types or tissue types, but not to the degree observed in one or more related cell types or tissue types. In some embodiments, the hypermethylated variable target region exhibits lower methylation in healthy cfDNA than in at least one other tissue type. In some embodiments, the hypermethylated variable target region exhibits even higher methylation in cfDNA from diseased cells of one or more related cell or tissue types. In some embodiments, the target region comprises a hypermethylated region with abnormally high copy number. In some such embodiments, the target region is hypermethylated in healthy and diseased colon tissues and has abnormally high copy number in precancerous or cancerous colon tissues. Examples of such target regions are shown in Table 1 below.

[0278] [Table 1]

[0279] [Table 2]

[0280] In some embodiments, the captured epigenetic target region set captured from a sample or subsample comprises a hypomethylated variable target region. In some embodiments, the hypomethylated variable target region is exclusively hypomethylated in one or more associated cell types or tissue types. In some embodiments, the hypomethylated variable target region is exclusively hypomethylated in one cell type or one immune cell type, or one immune cell type in a cluster. In some embodiments, the hypomethylated variable target region is hypomethylated to the extent that it is exclusively present in one cell type or one immune cell type, or one immune cell type in a cluster. Such hypomethylated variable target regions may be hypomethylated in other cell types or tissue types, but not to the extent found in one or more cell types or tissue types. In some embodiments, the hypomethylated variable target region shows higher methylation in healthy cfDNA than in at least one other tissue type.

[0281] Without wishing to be bound by any particular theory, in individuals with cancer, proliferating or activated immune cells and / or dying cancer cells may shed more DNA into the bloodstream than immune cells and / or healthy cells of the same tissue type in healthy individuals, respectively. As such, the distribution of cell types and / or tissues of origin of cfDNA may change during carcinogenesis. Thus, the presence and / or level of cfDNA originating from a particular cell or tissue type may be indicative of disease. Variations in hypermethylation and / or hypomethylation may be indicative of disease. For example, increased levels of hypermethylated and / or hypomethylated variable target regions in subsamples after the splitting step may be indicative of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0282] Exemplary hypermethylated and hypomethylated variable target regions useful for distinguishing between various cell types, including, but not limited to, immune cell types, were identified by analyzing DNA obtained from various cell types via whole genome bisulfite sequencing, for example, as described in Scott, CA, Duryea, JD, 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.

[0283] In some embodiments, the first and second captured target region sets each include 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 the epigenetic target region set may have any of the features described for such sets in WO 2020 / 160414, which is incorporated by reference in its entirety. In some embodiments, the epigenetic target region set includes a hypermethylated variable target region set. In some embodiments, the epigenetic target region set includes a hypomethylated variable target region set. In some embodiments, the epigenetic target region set includes a CTCF binding region. In some embodiments, the epigenetic target region set includes a fragmented variable target region. In some embodiments, the epigenetic target region set includes a transcription start site. In some embodiments, the set of epigenetic target regions includes one or more of the following regions that may exhibit focal amplification in cancer: AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, the set of epigenetic target regions includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the targets.

[0284] In some embodiments, the set of sequence variable target regions includes a plurality of regions known to undergo somatic mutations in cancer. In some aspects, the set of sequence variable target regions targets a plurality of different genes or genomic regions ("panels") selected such that a determined proportion of subjects with cancer exhibits genetic variants or tumor markers in one or more of the different genes or genomic regions in the panel. The panel may be selected to restrict the region for sequencing to a certain number of base pairs. The panel may be selected to sequence a desired amount of DNA, for example, by adjusting the affinity and / or amount of 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 relative to the amount of base pairs sequenced. The panel may be selected to achieve a theoretical sensitivity, theoretical specificity, and / or theoretical accuracy for detecting one or more genetic variants in a sample.

[0285] The probe for detecting the panel of regions may include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be considered in the design of the probe, and / or the probe can be designed to maximize the possibility that a specific site (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation affected by nucleosome binding patterns and GC sequence composition. Regions as used herein may also include non-hotspot regions that are optimized based on nucleosome positions and GC models.

[0286] The probe for detecting the panel of regions may include those for detecting genomic regions of interest (hotspot regions). Information about chromatin structure can be considered in the design of the probe, and / or the probe can be designed to maximize the possibility that a specific site (e.g., KRAS codons 12 and 13) can be captured, and may be designed to optimize capture based on the analysis of cfDNA coverage and fragment size variation affected by nucleosome binding patterns and GC sequence composition. Regions as used herein may also include non-hotspot regions that are optimized based on nucleosome positions and GC models.

[0287] Examples of lists of genomic locations of interest can be found in Tables 3 and 4 of WO 2020 / 160414. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure includes 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 in Table 3 of WO 2020 / 160414. In some embodiments, the set of sequence variable target regions used in the methods of the present disclosure includes 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 in Table 4 of WO 2020 / 160414. Additionally or alternatively, suitable target region sets are available in the literature. For example, Gale et al., PLoS One 13:e0194630 (2018), 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, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0288] In some embodiments, the set of sequence variable target regions includes target regions from at least 10, 20, 30, or 35 cancer associated genes, such as the cancer associated genes listed above and in WO 2020 / 160414.

[0289] In some embodiments, the collection of capture probes includes capture probes used in the methods described herein, e.g., prepared by any method disclosed herein for doing so. In some embodiments, the collection of capture probes further includes target binding probes specific to a set of sequence variable target regions and / or target binding probes specific to a set of epigenetic target regions. In some embodiments, the capture yield of the target binding probes specific to a set of sequence variable target regions is higher (e.g., at least 2-fold higher) than the capture yield of the target binding probes specific to a set of epigenetic target regions. In some embodiments, the collection of capture probes is designed to have a capture yield specific to a set of sequence variable target regions that is higher (e.g., at least 2-fold higher) than its capture yield specific to a set of epigenetic target regions.

[0290] In some embodiments, the capture yield of target binding probes specific for the set of sequence variable target regions is at least 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x higher than the capture yield of target binding probes specific for the set of epigenetic target regions. In some embodiments, the capture yield of target binding probes specific for the set of sequence variable target regions is 1.25-1.5 times, 1.5-1.75 times, 1.75-2 times, 2-2.25 times, 2.25-2.5 times, 2.5-2.75 times, 2.75-3 times, 3-3.5 times, 3.5-4 times, 4-4.5 times, 4.5-5 times, 5-5.5 times, 5.5-6 times, 6-7 times, 7-8 times, 8-9 times, 9-10 times, 10-11 times, 11-12 times, 13-14 times, or 14-15 times higher than the capture yield of target binding probes specific for the set of epigenetic target regions.

[0291] In some embodiments, the collection of capture probes is designed to have a capture yield specific for a set of sequence variable target regions that is at least 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x higher than its capture yield for a set of epigenetic target regions. In some embodiments, the collection of capture probes is designed to have a capture yield specific for the set of sequence variable target regions that is 1.25-1.5 times, 1.5-1.75 times, 1.75-2 times, 2-2.25 times, 2.25-2.5 times, 2.5-2.75 times, 2.75-3 times, 3-3.5 times, 3.5-4 times, 4-4.5 times, 4.5-5 times, 5-5.5 times, 5.5-6 times, 6-7 times, 7-8 times, 8-9 times, 9-10 times, 10-11 times, 11-12 times, 13-14 times, or 14-15 times higher than its capture yield specific for the set of epigenetic target regions.

[0292] A collection of probes can be designed to provide higher capture yields for a set of sequence-variable target regions in a variety of ways, including concentration, different lengths and / or chemistries (e.g., affecting affinity), and combinations thereof. Affinity can be adjusted by adjusting probe length and / or including nucleotide modifications as discussed below.

[0293] In some embodiments, the capture probes specific for the set of sequence variable target regions are present in a higher concentration than the capture probes specific for the set of epigenetic target regions, in some embodiments, the concentration of target binding probes specific for the set of sequence variable target regions is at least 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.5x, 4x, 4.5x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x higher than the concentration of target binding probes specific for the set of epigenetic target regions. In some embodiments, the concentration of target binding probes specific to the set of sequence variable target regions is 1.25-1.5 times, 1.5-1.75 times, 1.75-2 times, 2-2.25 times, 2.25-2.5 times, 2.5-2.75 times, 2.75-3 times, 3-3.5 times, 3.5-4 times, 4-4.5 times, 4.5-5 times, 5-5.5 times, 5.5-6 times, 6-7 times, 7-8 times, 8-9 times, 9-10 times, 10-11 times, 11-12 times, 13-14 times, or 14-15 times higher than the concentration of target binding probes specific to the set of epigenetic target regions. In such embodiments, concentration may refer to the average mass per volume concentration of individual probes in each set.

[0294] In some embodiments, capture probes specific to a set of sequence-variable target regions have higher affinity to their targets than capture probes specific to a set of epigenetic target regions. Affinity can be adjusted in any manner known to those skilled in the art, for example, by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in certain sequence contexts), modifications that provide heteroatoms at the 2' sugar position, and LNA nucleotides, can increase the stability of double-stranded nucleic acids, and it has been shown that oligonucleotides with such modifications have relatively higher affinity to their complementary sequences. See, for example, Severin et al., Nucleic Acids Res. 39: 8740-8751 (2011); Freier et al., Nucleic Acids Res. 25: 4429-4443 (1997); U.S. Patent No. 9,738,894. Also, a longer sequence length will generally result in increased affinity. Other nucleotide modifications, such as substitution of the nucleobase hypoxanthine with guanine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, capture probes specific to a set of sequence variable target regions have modifications that increase their affinity for their targets. In some embodiments, alternatively or additionally, capture probes specific to a set of epigenetic target regions have modifications that reduce their affinity for their targets. In some embodiments, capture probes specific to a set of sequence variable target regions have a longer average length and / or a higher average melting temperature than capture probes specific to a set of epigenetic target regions. These embodiments may be combined with each other and / or with concentration differences as discussed above to achieve a desired fold change in capture yield, such as any of the fold changes or ranges described above.

[0295] In some embodiments, the capture probe comprises a capture moiety. The capture moiety may be any of the capture moieties described herein, e.g., biotin. In some embodiments, the capture probe is linked to a solid support, e.g., covalently or non-covalently, e.g., through interaction of a binding pair of the capture moiety. In some embodiments, the solid support is a bead, such as a magnetic bead.

[0296] In some embodiments, the capture probes specific to a set of sequence variable target regions and / or the capture probes specific to a set of epigenetic target regions are capture probe sets discussed above, e.g., probes that contain sequences that have been selected to align across a panel of regions such as capture moieties and genes.

[0297] In some embodiments, the capture probes are provided in a single composition, which may be in solution (liquid or frozen), or it may be lyophilized.

[0298] Alternatively, the capture probes can be provided as multiple compositions, including, for example, a first composition comprising probes specific to a set of epigenetic target regions and a second composition comprising probes specific to a set of sequence-variable target regions. These probes can be mixed in appropriate ratios to provide a combined probe composition with any of the aforementioned fold changes in concentration and / or capture yield. Alternatively, they can be used in separate capture procedures (e.g., with aliquots of a sample or with the same sample sequentially) to provide first and second compositions comprising captured epigenetic and sequence-variable target regions, respectively.

[0299] Epigenetic target region specific probes The probes for the set of epigenetic target regions may include probes specific for one or more types of target regions that are likely to distinguish DNA from neoplastic (e.g., tumor or cancer) cells from DNA from healthy cells, e.g., non-neoplastic circulating cells. Exemplary types of such regions are discussed in detail herein, e.g., in the section above relating to the captured set. The probes for the set of epigenetic target regions may also include probes for one or more control regions, e.g., as described herein.

[0300] In some embodiments, the probes for the set of epigenetic target regions 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 set of epigenetic target regions have 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 set of epigenetic target regions has a footprint of at least 20 Mbp.

[0301] Hypermethylated variable target regions In some embodiments, the probes for the set of epigenetic target regions include probes specific for one or more hypermethylated variable target regions. The hypermethylated variable target regions may be referred to herein as hypermethylated DMRs (differentially methylated regions). The hypermethylated variable target regions may be any of those described above. For example, in some embodiments, the probes specific for the hypermethylated variable target regions include probes specific for a plurality of the 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 some embodiments, the probes specific for the hypermethylated variable target regions include probes specific for a plurality of the 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 hypermethylated variable target regions include probes specific for a plurality of the loci listed in Table 1 or Table 2, for example, 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 hybridization sites that bind between the transcription start site of the gene and the stop codon (the last stop codon of an alternatively spliced ​​gene). In some embodiments, the one or more probes bind within 300 bp, e.g., within 200 or 100 bp, of the listed position. In some embodiments, the probes have hybridization sites that overlap with the positions listed above. In some embodiments, the probes specific for hypermethylated target regions include probes specific for a subset of one, two, three, four, or five of the hypermethylated target regions that collectively indicate hypermethylation in one, two, three, four, or five of the following cancers: breast cancer, colon cancer, kidney cancer, liver cancer, and lung cancer.

[0302] Hypomethylated variable target regions In some embodiments, the probes for the epigenetic target region set include probes specific for one or more hypomethylated variable target regions. The hypomethylated variable target region may be referred to herein as hypomethylated DMR (differentially methylated region). The hypomethylated variable target region may be any of those described above. For example, the probes specific for one or more hypomethylated variable target regions may include probes for regions such as repetitive sequences, e.g., LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and satellite DNA, and intergenic regions that are normally methylated in healthy cells may show reduced methylation in tumor cells.

[0303] In some embodiments, the probes specific for the hypomethylated variable target region comprise probes specific for repetitive sequences and / or intergenic regions, hi some embodiments, the probes specific for repetitive sequences comprise probes specific for one, two, three, four, or five of the following: a LINE1 element, an Alu element, a centromeric tandem repeat, a pericentromeric tandem repeat, and / or satellite DNA.

[0304] Exemplary probes specific for genomic regions exhibiting cancer-associated hypomethylation include probes specific for nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1. In some embodiments, probes specific for hypomethylated variable target regions include probes specific for regions overlapping with or including nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1.

[0305] CTCF binding region In some embodiments, the probes for the set of epigenetic target regions include probes specific for CTCF binding regions. In some embodiments, the probes specific for CTCF binding regions include 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, such as those in the CTCFBSDB or one or more of the Cuddapah et al., Martin et al., or Rhee et al. articles described above or cited above. In some embodiments, the probes for the set of epigenetic target regions include 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 CTCF binding site.

[0306] Transcription start site In some embodiments, the probes for the set of epigenetic target regions include probes specific for transcription start sites. In some embodiments, the probes specific for transcription start sites include probes specific for at least 10, 20, 50, 100, 200, or 500 transcription start sites, or 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 transcription start sites, such as those listed in the DBTSS. In some embodiments, the probes for the set of epigenetic target regions include 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 transcription start site.

[0307] Focal amplification As mentioned above, focal amplifications are somatic mutations, but they can be detected by sequencing based on read frequency in a manner similar to the method for detecting certain epigenetic changes, such as changes in methylation. As such, regions that may show focal amplifications in cancer can be included in the epigenetic target region set, as discussed above. In some embodiments, the probes specific to the epigenetic target region set include probes specific to focal amplifications. In some embodiments, the probes specific to focal amplifications include probes specific to one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, focal amplification specific probes 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 aforementioned targets.

[0308] Control Region To facilitate data validation, it may be useful to include control regions. In some embodiments, the probes specific for the set of epigenetic target regions 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 set of epigenetic target regions include probes specific for control hypomethylated regions that are expected to be hypomethylated in essentially all samples.

[0309] Probes specific to sequence-variable target regions The probes for the sequence variable target region set may include probes specific to multiple regions known to undergo somatic mutation in cancer. The probes may be specific to any sequence variable target region set described herein. Exemplary sequence variable target region sets are discussed in detail herein, for example in the section above related to the captured set.

[0310] 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 epigenetic 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, 900-1,000 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.

[0311] In some embodiments, the probes specific for the set of sequence variable target regions include 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 least 70 of the genes of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include 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, or 70 of the SNVs of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include 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, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least one, at least two, or three indels of Table 3. In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for at least a portion of at least five, 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, the probes specific for the set of sequence variable target regions include probes specific for at least five, 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, the probes specific for the set of sequence variable target regions include probes specific for at least one, at least two, at least three, at least four, at least five, or six of the fusions in Table 4.In some embodiments, the probes specific for the set of sequence variable target regions include 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, the probes specific for the set of sequence variable target regions include 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.

[0312] [Table 3]

[0313] [Table 4]

[0314] [Table 5-1] [Table 5-2] [Table 5-3]

[0315] In some embodiments, the probes specific for the set of sequence variable target regions include probes specific for target regions from at least 10, 20, 30, or 35 cancer-associated genes, such as AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AFl.

[0316] Sequencing Generally, the sample nucleic acid flanked by the adaptor can be subjected to sequencing, with or without prior amplification. Sequencing methods include, for example, Sanger sequencing, high-throughput sequencing, pyrosequencing, sequencing by synthesis, single molecule sequencing, nanopore sequencing, semiconductor sequencing, sequencing by ligation, sequencing by hybridization, digital gene expression (Helicos), next generation sequencing (NGS), single molecule sequencing by synthesis (SMSS) (Helicos), massively parallel sequencing, clonal single molecule array (Solexa), shotgun sequencing, Ion Torrent, Oxford Nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking, and sequencing using PacBio, SOLiD, Ion Torrent, or Nanopore platforms. Sequencing reactions can be performed in a variety of sample processing units, which may include multiple lanes, multiple channels, multiple wells, or other means of processing multiple sample sets substantially simultaneously. The sample processing unit may also contain multiple sample chambers allowing for the processing of multiple runs simultaneously.

[0317] In some embodiments, sequence coverage of the genome may be less than, for example, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100%. In some embodiments, the sequence reaction may provide, for example, sequence coverage of at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of the genome. Sequence coverage may be performed, for example, for at least 5, 10, 20, 70, 100, 200, or 500 different genes, or up to, for example, 5000, 2500, 1000, 500, or 100 different genes.

[0318] Simultaneous sequencing reactions can be carried out using multiplex sequencing.In some cases, cell-free nucleic acid can be sequenced at least, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions.In other cases, cell-free nucleic acid can be sequenced, for example, less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions.Sequencing reactions can be carried out consecutively or simultaneously.Subsequent data analysis can be carried out on all or part of sequencing reactions. In some cases, data analysis can be performed on at least, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other cases, data analysis can be performed on less than, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. Exemplary read depths are 1000-50000, or 1000-10000, or 1000-20000 reads per locus (base).

[0319] In general, sequencing of epigenetic target regions, for example to analyze modified nucleoside profiles of DNA, requires a lower depth of sequencing than sequencing of sequence variable target regions, for example to analyze mutations. Thus, the lower sequencing depths described herein may in some cases be sufficient for the methods described herein.

[0320] analysis Detection of false negatives involving a conversion procedure to convert modified nucleosides In these embodiments, the adapter sequences used in the disclosed methods include quality control nucleosides with known modifications. When modified quality control nucleosides are used in combination with a conversion procedure that alters the base pair specificity of the modified nucleoside, it is expected that the conversion procedure applied will result in altering the base pair specificity of the quality control nucleoside. Therefore, whether a conversion procedure was effective for a particular known modified nucleoside is determined simply by reading how many modified nucleotides are sequenced. For example, if an adapter molecule containing mC in the quality control nucleoside is subjected to the TAPS procedure and is decoded when sequenced as T, the conversion was effective. If the known mC in the quality control nucleoside is decoded as C, the conversion was ineffective. Therefore, it is possible to identify (individual / single) adapted DNA molecules with suboptimal conversion of quality conversion nucleosides in the adapter. If an adaptor (or multiple adaptors, one at each end) contains multiple known modified quality control nucleosides, some of the modified quality control nucleosides may be correctly converted and others may not. Thus, it is possible to determine the conversion rate of a particular molecule, which is equal to the number of correctly converted known modified quality control nucleosides divided by the total number of known modified nucleosides in the quality control nucleosides of the molecular adaptor. Similarly, the conversion rate of an entire sample (or a fraction / subsample thereof) can be determined by dividing the total number of correctly converted known modified quality control nucleosides in the uniquely identified molecules of the sample by the total number of known modified nucleosides in the same set of uniquely identified sample molecules. In other words, the conversion rate of a sample or molecule containing nucleosides with unknown modification states is inferred from the conversion rate of known modified nucleosides in the adaptor sequence.

[0321] The conversion rate can be used as a quality control measure of the conversion rate at the sample or molecular level. In some cases, individual molecules or samples with suboptimal conversion rates, e.g., conversion rates below a certain threshold, can be excluded from further analysis. In other cases, the conversion rate can be partial or suboptimal, but can still be used in the analysis, taking into account known incomplete conversion of modified nucleosides in the molecule or sample. For example, a score or weight can be applied to each molecule and / or sample depending on the conversion rate determined. Molecules or samples with higher conversion rates are assigned higher weights, i.e., more significance is ascribed in downstream analysis or determination of the modified nucleoside profile of the sample DNA. Molecules or samples with lower relative conversion rates are assigned lower weights, i.e., less significance is ascribed in downstream analysis. For example, DNA may be obtained from a subject suspected of having cancer. The analysis includes determining the modified nucleoside profile of multiple epigenetic target regions if the altered modified nucleoside profile is associated with cancer. In the disclosed method, different conversion rates can be determined in DNA molecules from each of the target regions.The diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with lower conversion rates is reduced compared to the diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with higher conversion rates.Therefore, the lower conversion rate region is assigned lower weight when the modified nucleoside profile analysis from all of the regions is typically combined with other indicators such as the detection of cancer-related sequence mutations, and an overall diagnostic determination is provided, for example as a sum of risk scores.

[0322] Detection of false negatives involving conversion procedures that convert unmodified nucleosides When modified quality control nucleosides are used in combination with a conversion procedure that alters the base pair specificity of unmodified nucleosides, it is typically not expected to alter the base pair specificity of the quality control nucleoside as a result of the conversion procedure being applied. Therefore, whether a conversion procedure was effective for a particular known modified nucleoside is determined simply by reading how much of the modified nucleotide is sequenced. For example, if an adapter molecule containing mC in the quality control nucleoside has been subjected to a bisulfite procedure and is read as C when sequenced, the conversion procedure will not erroneously convert the mC and the known mC will be correctly identified as methylated. If the known mC in the quality control nucleoside is read as T, the conversion procedure will erroneously convert the mC and the known mC will be incorrectly identified as unmethylated (i.e., a false negative). Therefore, it is possible to identify (individually / single) adapted DNA molecules with incorrect conversion of a quality conversion nucleoside in the adapter. If an adaptor (or multiple adaptors, one at each end) contains multiple known modified nucleosides, some of the modified nucleosides may be misconverted and others may not. It is therefore possible to determine the misconversion rate of a particular molecule, which is equal to the number of known modified nucleosides that are misconverted divided by the total number of known modified nucleosides in the quality control nucleosides of the molecular adaptor. Similarly, the misconversion rate of an entire sample (or a fraction / subsample thereof) can be determined by dividing the total number of known modified nucleosides that are misconverted in the uniquely identified molecules of the sample by the total number of known modified nucleosides in the same set of uniquely identified sample molecules. In other words, the misconversion rate of an entire sample or molecule, including nucleosides with unknown modification states, is inferred from the misconversion rate of known modified nucleosides in the adaptor sequence.

[0323] The misconversion rate can be used as a quality control measure of the misconversion rate at the sample or molecular level. In some cases, individual molecules or samples with high misconversion rates, e.g., conversion rates above a predefined threshold, can be excluded from further analysis. In other cases, the misconversion rate can be above a predefined threshold, but can still be used in the analysis, taking into account known misconversions of modified nucleosides in the molecule or sample. For example, a score or weight can be applied to each molecule and / or sample depending on the determined misconversion rate. Molecules or samples with higher misconversion rates are assigned lower weights, i.e., less significance is ascribed in downstream analysis or determination of the modified nucleoside profile of the sample DNA. Molecules or samples with lower relative misconversion rates are assigned higher weights, i.e., more significance is ascribed in downstream analysis. For example, DNA may be obtained from a subject suspected of having cancer. The analysis includes determining the modified nucleoside profile of multiple epigenetic target regions, if the altered modified nucleoside profile is associated with cancer. The disclosed method can determine different misconversion rates in DNA molecules from each of the target regions.The diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with higher misconversion rates is reduced compared to the diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with lower misconversion rates.Therefore, the higher misconversion rate regions are assigned lower weights when the modified nucleoside profile analysis from all of the regions is typically combined with other indicators such as the detection of cancer-related sequence mutations, and an overall diagnostic determination is provided, for example as a sum of risk scores.

[0324] Detection of false positives involving conversion procedures that convert modified nucleosides When unmodified quality control nucleosides are used in combination with a conversion procedure that alters the base pair specificity of the modified nucleoside, it is not expected to alter the base pair specificity of the quality control nucleoside as a result of the conversion procedure being applied. Therefore, whether or not a conversion procedure was effective for a particular known unmodified nucleoside is determined simply by reading how much of the unmodified nucleotide is sequenced. For example, if an adapter molecule containing an unmodified C in the quality control nucleoside is subjected to the TAPS procedure and is read as C when sequenced, the conversion procedure will not erroneously convert the unmodified C and the known unmodified C will be correctly identified as unmethylated. If a known unmodified C in the quality control nucleoside is read as T, the conversion procedure will erroneously convert the unmodified C and the known unmodified C will be incorrectly identified as methylated (i.e., a false positive). Therefore, it is possible to identify (individually / single) adapted DNA molecules with incorrect conversion of a quality conversion nucleoside in the adapter. If an adaptor (or multiple adaptors, one at each end) contains multiple known unmodified nucleosides, some of the unmodified nucleosides may be misconverted and others may not. Thus, it is possible to determine the misconversion rate of a particular molecule, which is equal to the number of known unmodified nucleosides that are misconverted divided by the total number of known unmodified nucleosides in the quality control nucleosides of the molecular adaptor. Similarly, the misconversion rate of an entire sample (or a fraction / subsample thereof) can be determined by dividing the total number of known unmodified nucleosides that are misconverted in the uniquely identified molecules of the sample by the total number of known unmodified nucleosides in the same set of uniquely identified sample molecules. In other words, the misconversion rate of an entire sample or molecule, including nucleosides with unknown modification states, is inferred from the misconversion rate of known unmodified nucleosides in the adaptor sequence.

[0325] The misconversion rate can be used as a quality control measure of the misconversion rate at the sample or molecular level. In some cases, individual molecules or samples with high misconversion rates, e.g., conversion rates above a predefined threshold, can be excluded from further analysis. In other cases, the misconversion rate can be above a predefined threshold, but can still be used in the analysis, taking into account known misconversions of modified nucleosides in the molecule or sample. For example, a score or weight can be applied to each molecule and / or sample depending on the determined misconversion rate. Molecules or samples with higher misconversion rates are assigned lower weights, i.e., less significance is ascribed in downstream analysis or determination of the modified nucleoside profile of the sample DNA. Molecules or samples with lower relative misconversion rates are assigned higher weights, i.e., more significance is ascribed in downstream analysis. For example, DNA may be obtained from a subject suspected of having cancer. The analysis includes determining the modified nucleoside profile of multiple epigenetic target regions, if the altered modified nucleoside profile is associated with cancer. The disclosed method can determine different misconversion rates in DNA molecules from each of the target regions.The diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with higher misconversion rates is reduced compared to the diagnostic value of the modified nucleoside profile determined by sequencing the molecules in the region with lower misconversion rates.Therefore, the higher misconversion rate regions are assigned lower weights when the modified nucleoside profile analysis from all of the regions is typically combined with other indicators such as the detection of cancer-related sequence mutations, and an overall diagnostic determination is provided, for example as a sum of risk scores.

[0326] Detection of false positives involving conversion procedures that convert unmodified nucleosides When unmodified quality control nucleosides are used in combination with a conversion procedure that alters the base pair specificity of the unmodified nucleoside, it is not expected to alter the base pair specificity of the quality control nucleoside as a result of the conversion procedure being applied. Therefore, whether a conversion procedure was effective for a particular known unmodified nucleoside is determined simply by reading how much of the unmodified nucleotide is sequenced. For example, if an adapter molecule containing an unmodified C in the quality control nucleoside is subjected to the bisulfite procedure and is read upon sequencing as a T, the conversion was effective. If a known unmodified C in the quality control nucleoside is read as a C, the conversion was invalid. In such a case, this would normally lead to the identification of the unmodified C as modified (i.e., a false positive). Therefore, it is possible to identify (individually / single) adapted DNA molecules with suboptimal conversion of the quality conversion nucleoside in the adapter. If an adaptor (or multiple adaptors, one at each end) contains multiple known unmodified nucleosides, some of the unmodified nucleosides may be correctly converted and others may not. Thus, it is possible to determine the conversion rate of a particular molecule, which is equal to the number of correctly converted known unmodified nucleosides divided by the total number of known unmodified nucleosides in the quality control nucleosides of the molecular adaptor. Similarly, the conversion rate of an entire sample (or a fraction / subsample thereof) can be determined by dividing the total number of correctly converted known unmodified nucleosides in the uniquely identified molecules of the sample by the total number of known unmodified nucleosides in the same set of uniquely identified sample molecules. In other words, the conversion rate of an entire sample or molecule, including nucleosides with unknown modification states, is inferred from the conversion rate of known unmodified nucleosides in the adaptor sequence. The conversion rate can be used as a quality control measure of the conversion rate at the sample level or molecule level. In some cases, individual molecules or samples with suboptimal conversion rates, for example, conversion rates below a certain threshold, can be excluded from further analysis.In other cases, the conversion rate may be partial or suboptimal, but may still be used in the analysis, taking into account known incomplete conversion of unmodified nucleosides in the molecule or sample. For example, a score or weighting may be applied to each molecule and / or sample depending on the conversion rate determined. A molecule or sample with a higher conversion rate is assigned a higher weight, i.e., a higher significance is ascribed in downstream analysis or determination of the modified nucleoside profile of the sample DNA. A molecule or sample with a lower relative conversion rate is assigned a lower weight, i.e., a lower significance is ascribed in downstream analysis. For example, DNA may be obtained from a subject suspected of having cancer. The analysis includes determining the modified nucleoside profile of multiple epigenetic target regions, where the altered modified nucleoside profile is associated with cancer. In the disclosed method, a different conversion rate in DNA molecules from each of the target regions may be determined. The diagnostic value of a modified nucleoside profile determined by sequencing molecules in regions with lower conversion rates is reduced compared to the diagnostic value of a modified nucleoside profile determined by sequencing molecules in regions with higher conversion rates. Thus, the lower conversion rate regions are assigned lower weights when modified nucleoside profile analysis from all of the regions is typically combined with other indicators, such as detection of cancer-associated sequence mutations, to provide an overall diagnostic call, for example as a sum of risk scores.

[0327] Exemplary Applications The methods presented herein may be used as part of any method that benefits from obtaining an accurate modified nucleoside profile of DNA in any sample.

[0328] sample The sample may be any biological sample that has been isolated from a subject. The sample may be a bodily sample. The sample may include bodily tissues or fluids, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid, synovial fluid, lymphatic fluid, peritoneal fluid, interstitial or extracellular fluid, fluid in the space between cells, gingival crevicular fluid, bone marrow, pleural fluid, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, and urine. The sample is preferably a bodily fluid, in particular blood and its fractions, cerebrospinal fluid, pleural fluid, saliva, sputum, or urine. The sample may be in the form in which it is originally isolated from the subject, or may have undergone further processing to remove or add components such as cells, or to enrich one component for another. Thus, the preferred bodily fluid for analysis is plasma or serum, which contains cell-free nucleic acids.

[0329] In some embodiments, the population of nucleic acids is obtained from serum, plasma or blood samples from subjects suspected of having or previously diagnosed with a neoplasm, tumor, precancer or cancer. The population includes nucleic acids with different levels of sequence diversity, epigenetic mutations, and / or post-replicative or transcriptional modifications. Post-replicative modifications include modifications of cytosine, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, particularly at the 5-position of the nucleobase.

[0330] The sample can be isolated or obtained from the subject and transported to a location for sample analysis. The sample can be stored and transported at a desired temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. The sample can be isolated or obtained from the subject at the location for sample analysis. The subject can be a human, mammal, animal, companion animal, service animal, or pet. The subject can have cancer, pre-cancer, infection, transplant rejection, or other disease or disorder associated with alterations in the immune system. The subject may not have cancer or detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, e.g., any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may or may not have been diagnosed with cancer or susceptible to any cancer-related genetic mutation / disorder.

[0331] In some embodiments, the sample comprises plasma. The volume of plasma obtained may depend on the desired read depth of the sequenced region. Exemplary volumes are 0.4-40 mL, 5-20 mL, 10-20 mL. For example, the volume may be 0.5 mL, 1 mL, 5 mL, 10 mL, 20 mL, 30 mL, or 40 mL. The volume of plasma collected may be 5-20 mL.

[0332] A sample may contain nucleic acids containing various amounts of genome equivalents. For example, a sample of about 30 ng of DNA may contain about 10,000 (10 4 ) haploid human genome equivalents, and for cfDNA, approximately 200 billion (2 × 10 11 Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, about 600 billion individual molecules.

[0333] The sample may include nucleic acids from different sources, e.g., from cells and acellular of the same subject, from cells and acellular of different subjects. The sample may include nucleic acids having mutations. For example, the sample may include DNA having germline mutations and / or somatic mutations. A germline mutation refers to a mutation present in the germline DNA of a subject. A somatic mutation refers to a mutation that originates within the somatic cells of a subject, e.g., within a cancer cell. The sample may include DNA having a cancer-associated mutation (e.g., a cancer-associated somatic mutation). The sample may include epigenetic variants (i.e., chemical or protein modifications), where the epigenetic variants are associated with the presence of a genetic variant, such as a cancer-associated mutation. In some embodiments, the sample includes epigenetic variants associated with the presence of a genetic variant, where the sample does not include a genetic variant.

[0334] Exemplary amounts of cell-free nucleic acid in a sample prior to amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can include obtaining between 1 femtogram (fg) and 200 ng of cell-free nucleic acid molecules.

[0335] Cell-free DNA refers to DNA that is not contained within cells at the time of its isolation from a subject. For example, cfDNA can be isolated from a sample as DNA remaining in the sample after removing intact cells without lysing the cells or otherwise extracting intracellular DNA. Cell-free nucleic acids include DNA, RNA, and hybrids thereof, such as genomic DNA, mitochondrial DNA, siRNA, miRNA, circular RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-translated RNA (long ncRNA), or fragments of any of these. Cell-free nucleic acids can be double-stranded, single-stranded, or hybrids thereof. Cell-free nucleic acids can be released into bodily fluids through secretion or cell death processes, such as cell necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluids from cancer cells, including, for example, circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell-free nucleic acids are produced by tumor cells. In some embodiments, the cell-free nucleic acid is produced by a mixture of tumor cells and non-tumor cells.

[0336] Cell-free nucleic acids have a typical size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of the molecules, with a mode at about 168 nucleotides and a second minor peak in the range of 240-440 nucleotides.

[0337] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation or partitioning step, where the cell-free nucleic acids found in solution are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, the cells in the bodily fluid can be lysed and the cell-free and cellular nucleic acids can be processed together. Generally, after addition of buffer and washing steps, the nucleic acids can be precipitated with alcohol. Additionally, clean-up steps, such as silica-based columns, can be used to remove contaminants or salts. Non-specific bulk carrier nucleic acids, such as herring sperm DNA, can be added throughout the reaction to optimize certain aspects of the procedure, such as yield.

[0338] After such processing, the sample may contain various forms of nucleic acid, including double-stranded DNA, single-stranded DNA, and single-stranded RNA, hi some embodiments, single-stranded DNA and RNA may be converted to double-stranded form for inclusion in subsequent processing and analysis steps.

[0339] The double-stranded DNA molecules in the sample and the single-stranded nucleic acid molecules that have been converted to double-stranded DNA molecules can be ligated to adapters at either one or both ends. Typically, the double-stranded molecules are blunt-ended by treatment with a polymerase that has a 5'-3' polymerase and a 3'-5' exonuclease (or proofreading function) in the presence of all four standard nucleotides. Klenow large fragment and T4 polymerase are examples of suitable polymerases. The blunt-ended DNA molecules can be ligated, at least in part, with double-stranded adapters (e.g., Y-shaped or bell-shaped adapters). Alternatively, complementary nucleotides can be added to the blunt ends of the sample nucleic acid and the adapter to facilitate ligation. Contemplated herein are both blunt-end ligation and sticky-end ligation. In blunt-end ligation, both the nucleic acid molecule and the adapter tag have blunt ends. In sticky end ligation, typically the nucleic acid molecule has an "A" overhang and the adaptor has a "T" overhang.

[0340] Amplification of adapter-containing DNA The sample nucleic acids flanked by the adaptors can be amplified by PCR and other amplification methods. Such amplification can be used to increase the amount of DNA available for subsequent steps such as sequencing. For example, this type of amplification can be performed as part of library preparation and / or after preparation of a targeted library. Amplification can be performed after a conversion procedure. Amplification can be stimulated by primers that bind to primer binding sites in the adaptors flanking the DNA molecules to be amplified. Amplification methods can include cycles of denaturation, annealing and extension resulting from thermocycling, or can be isothermal, such as in the case of transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustaining sequence-based replication.

[0341] In some embodiments, the method performs dsDNA ligation with T-tail and C-tail adapters resulting in at least 50, 60, 70, or 80% amplification of double stranded nucleic acid prior to ligation to the adapters. Preferably, the method increases the amount or number of amplified molecules by at least 10, 15, or 20% compared to a control method performed with T-tail adapters alone.

[0342] Applicable One important exemplary application of the disclosed methods is the use of modified nucleoside profiles in the diagnosis and prognosis of cancer or other genetic diseases or conditions.

[0343] Thus, in some embodiments, the methods described herein include identifying or predicting the presence or absence of DNA being produced by a tumor (or neoplastic cell, or cancer cell), determining the probability that a test subject has a tumor or cancer, and / or characterizing a tumor, neoplastic cell, or cancer as described herein.

[0344] The method can be used to diagnose the presence or absence of a condition, particularly cancer, in a subject, characterize the condition (e.g., stage the cancer or determine the heterogeneity of the cancer), monitor the response to treatment of the condition, and provide a prognostic risk of developing the condition or the course of the condition thereafter. The present disclosure can also be useful in determining the effectiveness of a particular treatment option. If the treatment is successful, as more cancers may die and shed DNA, the success of the treatment option may increase the amount of copy number variation or rare mutations detected in the blood of the subject. In other examples, this may not occur. In another example, perhaps a particular treatment option may correlate with the genetic profile of the cancer over time. This correlation may be useful in selecting a treatment. In some embodiments, hypermethylated variable epigenetic target regions are analyzed to determine whether they show hypermethylation characteristic of tumor cells or cells that do not normally contribute significantly to cfDNA, and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they show hypomethylation characteristic of tumor cells or cells that do not normally contribute significantly to cfDNA.

[0345] In some embodiments, the method is used in a method for screening for or screening for cancer. For example, the sample can be obtained from a subject who has not been previously diagnosed with cancer. In some embodiments, the subject can have or not have cancer. In some embodiments, the subject can have or not have early stage cancer. In some embodiments, the subject has one or more risk factors for cancer, such as tobacco use (e.g., smoking), being overweight or obese, having a high body mass index (BMI), being elderly, malnutrition, high alcohol consumption, or a family history of cancer.

[0346] In some embodiments, the subject has been using tobacco for, e.g., at least 1, 5, 10, or 15 years. In some embodiments, the subject has a high BMI, e.g., a BMI of 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more. In some embodiments, the subject is at least 40, 45, 50, 55, 60, 65, 70, 75, or 80 years old. In some embodiments, the subject has a nutritional deficiency, e.g., high consumption of one or more of red and / or processed meats, trans fats, saturated fats, and refined sugars, and / or low consumption of fruits and vegetables, complex carbohydrates, and / or unsaturated fats. High and low consumption can be defined, for example, as above or below the recommendations in the Dietary Guidelines for Americans 2020-2025, available at: www.dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020-2025.pdf, respectively. In some embodiments, the subject has high alcohol consumption, for example, averaging at least 3, 4, or 5 drinks per day (where a drink is about 1 ounce or 30 mL of 80-proof hard liquor or equivalent). In some embodiments, the subject has a family history of cancer, for example, at least one, two, or three blood relatives previously diagnosed with cancer. In some embodiments, the relative is at least a third-degree relative (e.g., a great-grandparent, a great-aunt or great-uncle, a first cousin), at least a second-degree relative (e.g., a grandparent, an aunt or uncle, or a half-sibling), or a first-degree relative (e.g., a parent or full sibling).

[0347] In addition, where a cancer is found to be in remission following treatment, the methods can be used to monitor for residual disease or recurrence of the disease.

[0348] Typically, the disease under consideration is, for example, any type of cancer referred to herein. The types and number of cancers that may be detected may include blood cancer, brain cancer, lung cancer, skin cancer, nasal cancer, pharyngeal cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, renal cancer, oral cancer, stomach cancer, solid tumors, heterogeneous tumors, homogeneous tumors, etc. Specific examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, brain cancer, glioma, astrocytoma, breast cancer, metaplastic carcinoma, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolypoid colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, intraocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, transitional cell carcinoma, urothelial carcinoma, Wilms' tumor, leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CM) L), chronic myelomonocytic leukemia (CMML), liver cancer, liver cancer, liver tumor, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, B cell lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, mantle cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma, multiple myeloma, nasopharyngeal carcinoma (NPC), neuroblastoma, oropharyngeal carcinoma, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, gastric cancer (stomach Examples of cancer include gastric cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer, and uterine sarcoma.

[0349] The type and / or stage of cancer can be detected from genetic variations including mutations, rare mutations, indels, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosomal fusions, gene truncations, gene amplifications, gene duplications, chromosomal lesions, DNA lesions, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns, and abnormal changes in nucleic acid 5-methylcytosine. Thus, the method can be used in some cases in combination with methods used to detect other genetic / epigenetic variations, for example in the methods for detecting or characterizing cancer or other methods described herein.

[0350] In some embodiments, the methods described herein include identifying the presence of a target region and / or DNA produced by a tumor (or neoplastic or cancerous cell) or by a pre-cancerous cell. In some embodiments, the methods described herein include determining the level of the target region and / or identifying the presence of DNA produced by the tumor (or neoplastic or cancerous cell) or by a pre-cancerous cell. In some embodiments, determining the level of the target region includes determining either an increased or decreased level of the target region, where the increased or decreased level of the target region is determined by comparing the level of the target region to a threshold level / value.

[0351] Genetic and / or epigenetic data can also be used to characterize specific forms of cancer. Cancers are often heterogeneous in both composition and staging. Genetic and / or epigenetic profile data can allow characterization of specific subtypes of cancer, which may be important in the diagnosis or treatment of that specific subtype. This information may also provide clues to the subject or practitioner regarding the prognosis of a particular type of cancer, allowing either the subject or practitioner to adapt treatment options as the disease progresses. Some cancers may progress and become more aggressive and genetically unstable. Other cancers may remain benign, inactive, or dormant. The systems and methods of the present disclosure can be useful in determining disease progression.

[0352] Furthermore, the methods of the present disclosure can be used to characterize heterogeneity of abnormal conditions in a subject. Such methods can include, for example, creating a genetic and / or epigenetic profile of extracellular polynucleotides from a subject, where the genetic and / or epigenetic profile includes a plurality of data obtained from copy number variation and rare mutation analysis. In some embodiments, the abnormal condition is, for example, a cancer as described herein. In some embodiments, the abnormal condition can be one that results in a heterogeneous genomic population. In the example of cancer, it is known that some tumors contain tumor cells that are at different stages of cancer. In other examples, the heterogeneity can include multiple foci of disease. Again, in the example of cancer, there can be multiple tumor foci, where perhaps one or more foci are the result of metastases spreading from a primary site.

[0353] The methods can be used to generate a profile, fingerprint or dataset that is the sum of genetic and / or epigenetic information obtained from different cells in a heterogeneous disease, which may include copy number variation, epigenetic variation, and mutation analysis, either alone or in combination.

[0354] The methods can be used to diagnose, prognose, monitor or observe cancer or other diseases. In some embodiments, the methods herein do not include diagnosing, prognosing or monitoring a fetus and therefore are not directed to non-invasive prenatal testing. In other embodiments, the methods can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in fetal subjects where DNA and other polynucleotides may co-circulate with maternal molecules.

[0355] Non-limiting examples of other gene-based diseases, disorders, or conditions that may optionally be evaluated using the methods and systems disclosed herein include achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth (CMT), Cri du Chat, Crohn's disease, cystic fibrosis, Dercum's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome. These include: Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland anomaly, porphyria, progeria, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, spinal muscular atrophy, Tay-Sachs syndrome, thalassemia, trimethylaminuria, Turner syndrome, palatocardiofacial syndrome, WAGR syndrome, and Wilson's disease.

[0356] In some embodiments, the methods provided herein are methods of determining the risk of cancer recurrence in a subject. In some embodiments, the methods provided herein are methods of classifying a subject as a candidate for a subsequent cancer treatment.

[0357] Any of these methods may include collecting a sample from a subject who has been diagnosed with cancer at one or more preselected time points after one or more previous cancer treatments for the subject. The subject may be any of the subjects described herein. The sample may include DNA, e.g., cfDNA. The DNA may be obtained from a tissue sample or a liquid sample.

[0358] Any of these methods may include contacting the sample or a subsample thereof with a plurality of primers to generate capture probes to capture and detect the presence or level of at least one structural variation according to any of the embodiments described herein. In some embodiments, the method may include contacting the sample or a subsample thereof with a plurality of capture probes specific to members of a set of epigenetic target regions according to any of the embodiments described herein. In some embodiments, the capture probes include capture probes that have been generated using a sample obtained from the same subject at an earlier time point. The method may further include capturing a set of target regions from DNA from the subject, where the set of target regions includes a set of sequence-variable target regions and / or a set of epigenetic target regions, thereby generating a captured set of DNA molecules. The one or more capture steps may be performed according to any of the embodiments described elsewhere herein. Any of these methods may include sequencing the captured DNA molecules, thereby generating a set of sequence information. The captured DNA molecules of the set of sequence-variable target regions may be sequenced to a greater depth of sequencing than the captured DNA molecules of the set of epigenetic target regions. Any of these methods may include using the set of sequence information to detect the presence or absence of DNA originating from or derived from the tumor cell at a preselected time point. Detecting the presence or absence of DNA originating from or derived from the tumor cell may be performed according to any of the embodiments thereof described elsewhere herein.

[0359] In any of these methods, the previous cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0360] The method for determining the risk of cancer recurrence in a subject may include determining a cancer recurrence score, which refers to the presence or absence, or amount, of at least one rearranged DNA sequence, type-specific target region, and / or DNA originating from or derived from a tumor cell in a subject. The cancer recurrence score can further be used to determine a cancer recurrence status. The cancer recurrence status can be, for example, a risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. The cancer recurrence status can be, for example, a low or lower risk of cancer recurrence when the cancer recurrence score is above a predetermined threshold. In certain embodiments, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence status that is either a risk of cancer recurrence, or a low or lower risk of cancer recurrence.

[0361] A method of classifying a subject as a candidate for a subsequent cancer treatment may include comparing the subject's cancer recurrence score to a predefined cancer recurrence threshold, thereby classifying the subject as a candidate for the subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold, or as not a candidate for the treatment when the cancer recurrence score is below the cancer recurrence threshold. In certain embodiments, a cancer recurrence score equal to the cancer recurrence threshold may result in classification as either a candidate for the subsequent cancer treatment or as not a candidate for the treatment. In some embodiments, the subsequent cancer treatment comprises administration of a chemotherapy or a therapeutic composition.

[0362] Any of these methods may include determining a disease-free survival (DFS) period for the subject based on the cancer recurrence score; for example, the DFS period may be 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.

[0363] In some embodiments, the set of sequence information comprises a sequence variable target region sequence, and determining the cancer recurrence score may comprise determining at least a first subscore signifying the level of a particular immune cell type, SNV, insertion / deletion, CNV and / or fusion present within the sequence variable target region sequence.

[0364] In some embodiments, the number of mutations in the sequence variable target region selected from 1, 2, 3, 4, or 5 is sufficient to result in a cancer recurrence score in which the first subscore is classified as positive for cancer recurrence. In some embodiments, the number of mutations is selected from 1, 2, or 3.

[0365] In some embodiments, the set of sequence information includes epigenetic target region sequences, and determining the cancer recurrence score includes determining a second subscore that represents the amount of molecules (obtained from the epigenetic target region sequences) that represent an epigenetic state that differs from the DNA found in a corresponding sample from a healthy subject (e.g., cfDNA found in a blood sample from the healthy subject, or DNA found in a tissue sample from the healthy subject if the tissue sample is of the same type of tissue as that obtained from the test subject). These abnormal molecules (i.e., molecules that have an epigenetic state that differs from the DNA found in the corresponding sample from the healthy subject) may correspond to epigenetic changes associated with cancer, such as methylation of a hypermethylated variable target region and / or perturbed fragmentation of a fragmented variable target region, where "perturbed" means different from the DNA found in the corresponding sample from the healthy subject.

[0366] In some embodiments, a percentage of molecules corresponding to the set of hypermethylated variable target regions and / or the set of fragmented variable target regions that exhibit hypermethylation in the set of hypermethylated variable target regions and / or aberrant fragmentation in the set of fragmented variable target regions equal to or greater than a value in the range of 0.001% to 10% is sufficient for the second subscore to be classified as positive for cancer recurrence. The range may be 0.001% to 1%, 0.005% to 1%, 0.01% to 5%, 0.01% to 2%, or 0.01% to 1%.

[0367] In some embodiments, any of these methods may include determining the fraction of tumor DNA from the fraction of molecules in the set of sequence information that exhibit one or more characteristics indicative of origin from tumor cells. This can be done for molecules that correspond to some or all of the epigenetic target regions, including, for example, one or both of the hypermethylated variable target regions and the fragmented variable target regions (hypermethylation of the hypermethylated variable target regions and / or aberrant fragmentation of the fragmented variable target regions can be considered indicative of origin from tumor cells). This can be done for molecules that correspond to sequence variable target regions, for example, molecules that include alterations consistent with cancer, such as SNVs, indels, CNVs, and / or fusions. The fraction of tumor DNA can be determined based on a combination of molecules that correspond to epigenetic target regions and molecules that correspond to sequence variable target regions.

[0368] The determination of the cancer recurrence score may be based, at least in part, on a fraction of tumor DNA, where 10 -11 ~1 or 10 -10 A fraction of tumor DNA greater than a threshold in the range of 10 to 1 is sufficient for the Cancer Recurrence Score to be classified as positive for cancer recurrence. -10 ~10 -9 , 10 -9 ~10 -8 , 10 -8 ~10 -7 , 10 -7 ~10 -6 , 10 -6 ~10 -5 , 10-5 ~10 -4 , 10 -5 ~10 -3 , 10 -5 ~10 -2 , or 10 -2 ~10 -1 In some embodiments, a fraction of tumor DNA equal to or greater than a threshold in the range of at least 10 is sufficient for the Cancer Recurrence Score to be classified as positive for cancer recurrence. -7 A tumor DNA fraction greater than the threshold value is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. The determination that the tumor DNA fraction is greater than a threshold value, for example, a threshold value corresponding to any of the above-mentioned embodiments, may be based on a cumulative probability. For example, a sample was considered positive if the cumulative probability that the tumor fraction was greater than a threshold value in any of the above-mentioned ranges exceeds a probability threshold of at least 0.5, 0.75, 0.9, 0.95, 0.98, 0.99, 0.995, or 0.999. In some embodiments, the probability threshold is at least 0.95, for example 0.99.

[0369] In some embodiments, the set of sequence information includes sequence variable target region sequences and epigenetic target region sequences, and determining the cancer recurrence score includes determining a first subscore that indicates the amount of SNVs, insertions / deletions, CNVs and / or fusions present in the sequence variable target region sequences and a second subscore that indicates the amount of abnormal molecules in the epigenetic target region sequences, and combining the first subscore and the second subscore to provide a cancer recurrence score. When the first subscore and the second subscore are combined, they can be combined by applying a threshold value to each subscore independently (e.g., greater than a predetermined number of mutations (e.g., >1) in the sequence variable target region and greater than a predetermined fraction of abnormal molecules (i.e., molecules with an epigenetic state different from the DNA found in the corresponding sample from a healthy subject; e.g., tumor) in the epigenetic target region), or by training a machine learning classifier to determine the status based on multiple positive and negative training samples.

[0370] In some embodiments, a Combined Score value within the range of -4 to 2 or -3 to 1 is sufficient for the Cancer Recurrence Score to be classified as positive for cancer recurrence.

[0371] In either embodiment, if the Cancer Recurrence Score is classified as positive for cancer recurrence, the subject's cancer recurrence status may be at risk for cancer recurrence and / or the subject may be classified as a candidate for subsequent cancer treatment.

[0372] In some embodiments, the cancer is any one of the types of cancer described elsewhere herein, for example, colorectal cancer.

[0373] In certain embodiments, the methods disclosed herein relate to identifying and administering a customized therapy to a patient. In some embodiments, the determination of the level of a particular nucleic acid facilitates the selection of an appropriate therapy. In some embodiments, the patient or subject has a given disease, disorder, or condition. Essentially any cancer therapy (e.g., surgical therapy, radiation therapy, chemotherapy, etc.) may be included as part of these methods. In certain embodiments, the therapy administered to the subject includes at least one chemotherapeutic agent. In some embodiments, the chemotherapeutic agent may include alkylating agents (e.g., but not limited to, chlorambucil, cyclophosphamide, cisplatin, and carboplatin), nitrosoureas (e.g., but not limited to, carmustine and lomustine), antimetabolites (e.g., but not limited to, Fluorauracil, methotrexate, and fludarabine), plant alkaloids and natural products (e.g., but not limited to, vincristine, paclitaxel, and topotecan), antitumor antibiotics (e.g., but not limited to, bleomycin, doxorubicin, and mitoxantrone), hormonal agents (e.g., but not limited to, prednisone, dexamethasone, tamoxifen, and leuprolide), and biological response modifiers (e.g., but not limited to, herceptin and avastin, erbitux, and rituxan). In some embodiments, the chemotherapy administered to the subject may include FOLFOX or FOLFIRI. In certain embodiments, a therapy comprising at least one PARP inhibitor may be administered to the subject. In certain embodiments, the PARP inhibitor may include, among others, olaparib, talazoparib, rucaparib, niraparib (trade name Zejula). Typically, the therapy comprises at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method of enhancing immune response against a given cancer type. In certain embodiments, immunotherapy refers to a method of enhancing T cell response against tumor or cancer.

[0374] In some embodiments, treatments are customized based on the status of the nucleic acid variant, such as somatic or germline in origin. In some embodiments, essentially any cancer therapy (e.g., surgery, radiation, chemotherapy, etc.) may be included as part of these methods.

[0375] In some embodiments, the immunotherapy or immunotherapeutic agent targets immune checkpoint molecules. Certain tumors can evade the immune system by exploiting immune checkpoint pathways. Thus, targeting immune checkpoints has been recognized as an effective approach to combat the tumor's ability to evade the immune system and activate anti-tumor immunity against certain cancers. Pardoll, Nature Reviews Cancer, 2012, 12:252-264.

[0376] In certain embodiments, the immune checkpoint molecule is an inhibitory molecule that reduces signals involved in T cell responses to antigens. For example, CTLA4 is expressed on T cells and exerts a role in downregulating T cell activation by binding to CD80 (also known as B7.1) or CD86 (also known as B7.2) on antigen presenting cells. PD-1 is another inhibitory checkpoint molecule expressed on T cells. PD-1 limits the activity of T cells in peripheral tissues during inflammatory responses. Furthermore, ligands for PD-1 (PD-L1 or PD-L2) are commonly upregulated on the surface of many types of tumors, resulting in downregulation of anti-tumor immune responses in the tumor microenvironment. In certain embodiments, the inhibitory immune checkpoint molecule is CTLA4 or PD-1. In other embodiments, the inhibitory immune checkpoint molecule is a ligand for PD-1, such as PD-L1 or PD-L2. In other embodiments, the inhibitory immune checkpoint molecule is a ligand for CTLA4, such as CD80 or CD86. In other embodiments, the inhibitory immune checkpoint molecule is lymphocyte activation gene 3 (LAG3), killer cell immunoglobulin-like receptor (KIR), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), or adenosine A2a receptor (A2aR).

[0377] Antagonists targeting these immune checkpoint molecules can be used to enhance antigen-specific T cell responses to certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist of an inhibitory immune checkpoint molecule. In certain embodiments, the inhibitory immune checkpoint molecule is PD-1. In certain embodiments, the inhibitory immune checkpoint molecule is PD-L1. In certain embodiments, the antagonist of an inhibitory immune checkpoint molecule is an antibody (e.g., a monoclonal antibody). In certain embodiments, the antibody or monoclonal antibody is an anti-CTLA4, anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody. In certain embodiments, the antibody is a monoclonal anti-PD-1 antibody. In some embodiments, the antibody is a monoclonal anti-PD-L1 antibody. In certain embodiments, the monoclonal antibody is a combination of an anti-CTLA4 antibody and an anti-PD-1 antibody, an anti-CTLA4 antibody and an anti-PD-L1 antibody, or an anti-PD-L1 antibody and an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody is one or more of pembrolizumab (KEYTRUDA®) or nivolumab (OPDIVO®). In certain embodiments, the anti-CTLA4 antibody is ipilimumab (YERVOY®). In certain embodiments, the anti-PD-L1 antibody is one or more of atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), or durvalumab (IMFINZI®).

[0378] In certain embodiments, the immunotherapy or immunotherapeutic agent is an antagonist (e.g., an antibody) against CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In other embodiments, the antagonist is a soluble version of an inhibitory immune checkpoint molecule, such as a soluble fusion protein comprising the extracellular domain of an inhibitory immune checkpoint molecule and the Fc domain of an antibody. In certain embodiments, the soluble fusion protein comprises the extracellular domain of CTLA4, PD-1, PD-L1, or PD-L2. In some embodiments, the soluble fusion protein comprises the extracellular domain of CD80, CD86, LAG3, KIR, TIM3, GAL9, or A2aR. In one embodiment, the soluble fusion protein comprises the extracellular domain of PD-L2 or LAG3.

[0379] In certain embodiments, the immune checkpoint molecule is a costimulatory molecule that amplifies signals involved in T cell responses to antigens. For example, CD28 is a costimulatory receptor expressed on T cells. When a T cell binds to an antigen through its T cell receptor, CD28 binds to CD80 (also known as B7.1) or CD86 (also known as B7.2) on an antigen-presenting cell, amplifying T cell receptor signaling and promoting T cell activation. Because CD28 binds to the same ligands (CD80 and CD86) as CTLA4, CTLA4 can oppose or regulate the costimulatory signaling mediated by CD28. In certain embodiments, the immune checkpoint molecule is a costimulatory molecule selected from CD28, inducible T cell costimulator (ICOS), CD137, OX40, or CD27. In other embodiments, the immune checkpoint molecule is a ligand for a costimulatory molecule, including, for example, CD80, CD86, B7RP1, B7-H3, B7-H4, CD137L, OX40L, or CD70.

[0380] Agonists that target these costimulatory checkpoint molecules can be used to enhance antigen-specific T cell responses to certain cancers. Thus, in certain embodiments, the immunotherapy or immunotherapeutic agent is an agonist of a costimulatory checkpoint molecule. In certain embodiments, the agonist of a costimulatory checkpoint molecule is an agonist antibody, preferably a monoclonal antibody. In certain embodiments, the agonist antibody or monoclonal antibody is an anti-CD28 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-ICOS, anti-CD137, anti-OX40, or anti-CD27 antibody. In other embodiments, the agonist antibody or monoclonal antibody is an anti-CD80, anti-CD86, anti-B7RP1, anti-B7-H3, anti-B7-H4, anti-CD137L, anti-OX40L, or anti-CD70 antibody.

[0381] In certain embodiments, the status of nucleic acid variants from a sample from a subject, of somatic or germline origin, can be compared to a database of comparator results from a reference population to identify customized or targeted therapies for the subject. Typically, the reference population includes subjects who have undergone or have undergone the same therapy as the subject and / or patients with the same cancer or disease type as the patient. Customized or targeted therapies (or therapies) can be identified when the nucleic acid variants and the comparator results meet (e.g., are substantially or substantially identical to) certain classification criteria.

[0382] In certain embodiments, the customized therapy described herein is typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing immunotherapeutic agents are typically administered intravenously. Certain therapeutic agents are administered orally. However, the customized therapy (e.g., immunotherapeutic agents, etc.) may also be administered by any method known in the art, such as buccal, sublingual, rectal, vaginal, urethral, ​​topical, intraocular, intranasal, and / or intraauricular, and administration may include tablets, capsules, gran...

Claims

1. 1. A quality control method for monitoring false negative and / or false positive detection of modified nucleosides in DNA in a sample, comprising: (a) ligating the DNA to an oligonucleotide adaptor, the adaptor comprising quality control nucleosides, the adaptor quality control nucleosides comprising a modified first quality control nucleoside and an unmodified second quality control nucleoside, the quality control nucleosides having the same nucleoside identity and the same or different modification state relative to a modified nucleoside to be detected in the DNA, the modification state of the quality control nucleoside being known; (b) applying to the adapted DNA, or a subsample thereof, a conversion procedure that alters the base pair specificity of the quality control nucleoside or does not alter the base pair specificity of the quality control nucleoside depending on the modification state of the nucleoside, (i) altering the base pair specificity of adapted DNA nucleosides that have the same nucleoside identity and modification state as a quality control nucleoside in the adapter, and not altering the base pair specificity of adapted DNA nucleosides that have the same nucleoside identity but a different modification state as a quality control nucleoside in the adapter; and / or (ii) not altering the base-pairing specificity of adapted DNA nucleosides that have the same nucleoside identity and modification state as the quality control nucleosides in the adapter, and altering the base-pairing specificity of adapted DNA nucleosides that have the same nucleoside identity but a different modification state as the quality control nucleosides in the adapter. and applying a transformation procedure selected so as to (c) sequencing the adapted DNA after the conversion step (b); (d) using the sequence data obtained in step (c) to determine the base pair specificity conversion of said quality control nucleosides within said adapter; (e) using the base pair specificity conversion of the quality control nucleosides in the adapter as a quality control measure for conversion step (b), wherein suboptimal conversion of adapter quality control nucleosides after the conversion procedure of step (b)(i) and / or erroneous conversion of adapter quality control nucleosides after the conversion procedure of step (b)(ii) predicts false negative and / or false positive detection of modified nucleosides in the DNA sample. Quality control methods.

2. The method of claim 1, wherein the conversion procedure is selected to change the base pair specificity of quality control nucleosides in the adapter but not to change the base pair specificity of nucleosides of a DNA sample having the same nucleoside identity and a different modification state; and suboptimal conversion of the quality control nucleoside predicts false positive detection of a DNA sample nucleoside having the different modification state, for example, the quality control nucleoside in the adapter comprises cytosine and / or the conversion procedure comprises bisulfite conversion.

3. A method according to claim 1 or claim 2, wherein the first quality control nucleoside is a modified cytosine and the second quality control nucleoside is an unmodified cytosine, for example, the first quality control nucleoside is 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).

4. The conversion procedure: (i) selected to alter the base pair specificity of the first quality control nucleoside but not the second quality control nucleoside; (ii) is selected to alter the base pair specificity of the second quality control nucleoside but not the first quality control nucleoside; or (iii) selected to alter the base pair specificity of modified quality control nucleosides within the adapter but not alter the base pair specificity of DNA sample nucleosides having the same nucleoside identity but a different modification state and / or no modification; wherein suboptimal conversion of the modified quality control nucleoside predicts false negative detection of DNA sample nucleosides having the same nucleoside identity and modification state, or a different modification state and the same change in base pair specificity, as the quality control nucleoside upon exposure to the conversion procedure.

5. using the sequence data obtained in step (c), (i) identifying adapted DNA molecules with suboptimal or incorrect conversion of quality control nucleosides within the adapter sequence; (ii) predicting suboptimal or erroneous conversions of nucleosides with the same nucleoside identity and modification state in the full-length molecule identified in step (i); and The method of claim 1 further comprising:

6. determining the conversion rate of quality control nucleosides in the adapted DNA or individual adapted DNA molecules; (i) a weight depending on the conversion rate, (A) the DNA sample; or (B) Individual, adapted DNA molecules applying the modified nucleoside to the assay for detection; (ii) from further analysis to detect modified nucleosides (A) suboptimal conversion of adapter quality control nucleosides or conversion rates of adapter quality control nucleosides below a predetermined quality control threshold; and / or (B) Incorrect conversion of adapter quality control nucleosides or conversion rates of adapter quality control nucleosides above a predetermined quality control threshold. and / or excluding DNA samples having (iii) from further analysis to detect modified nucleosides. (A) suboptimal conversion of adapter quality control nucleosides or conversion rates of adapter quality control nucleosides below a predetermined quality control threshold; and / or (B) Incorrect conversion of adapter quality control nucleosides or conversion rates of adapter quality control nucleosides above a predetermined quality control threshold. and excluding adapted DNA molecules having The method of claim 1 further comprising:

7. 2. The method of claim 1, further comprising enriching the DNA by capturing a set of target regions from the sample, wherein the capturing step occurs before, after, or during the ligating step (a) and the converting step (b).

8. (i) the sequence data obtained in step (c), (A) a predetermined reference sequence; and / or (B) comparing with sequence data obtained by sequencing a subsample of said DNA that was not subjected to said conversion procedure; (ii) identifying point differences between the converted DNA sequence and the reference sequence (A) or unconverted DNA sequence data (B) as nucleosides having a modification state that allows for a change in base pair specificity upon exposure to the conversion procedure; The method of claim 1 further comprising: (i) the DNA comprises cell-free DNA (cfDNA) obtained from a test subject, and optionally, the test subject is a patient having or suspected of having cancer. (ii) the method further comprises using the detection of modified nucleosides in the DNA sample to determine or predict the presence of DNA produced by a cancer cell or tumor, to determine the probability that a test subject has a tumor or cancer, or to characterize a cancer or tumor in the subject. (iii) the subsamples of DNA are not subjected to the conversion procedure prior to sequencing, the converted subsamples and the unconverted subsamples have different adapter sequences, and the converted subsamples and the unconverted subsamples are recombined for sequencing step (c). (iv) the method further comprises analyzing the DNA to detect copy number variations, single base mutations, insertions, deletions, methylations, and / or fusions. (v) the method further comprises capturing an epigenetic target region from the adaptor-ligated DNA; and amplifying and sequencing the epigenetic target region; wherein the captured epigenetic target regions form an epigenetic target region set. For example, the set of epigenetic target regions comprises a plurality of type-specific epigenetic target regions, and the type-specific epigenetic target regions are type-specific differentially methylated regions and / or type-specific fragments; Further optionally, the plurality of type-specific epigenetic target regions comprises type-specific hypomethylated regions or type-specific hypermethylated regions; and / or (vi) The method of claim 1, wherein the sample is a blood sample. (i) the type-specific epigenetic target region comprises a cell type-specific, tissue type-specific, and / or cancer type-specific epigenetic target region; and / or 10. The method of claim 9, wherein (ii) the blood sample is fractionated prior to capturing at least the set of epigenetic target regions of DNA.

11. Dividing the sample or aliquot thereof into a plurality of divided subsamples, including a first divided subsample and a second divided subsample; contacting the second divided subsample with a methylation-dependent nuclease, thereby degrading non-specifically divided DNA in the second subsample and generating a treated second subsample, and optionally contacting the first divided subsample with a methylation-sensitive endonuclease, thereby degrading non-specifically divided DNA in the first divided subsample and generating a treated first subsample; wherein the first subsample comprises DNA having a greater proportion of cytosine modifications than the second subsample; wherein an epigenetic target region is captured from said first sub-sample or at least a portion of said processed first sub-sample; Optionally, the DNA from the subject that is contacted with the one or more capture probes comprises DNA from the first divided sub-sample, the processed first sub-sample, the second divided sub-sample, and / or the processed second sub-sample. (i) the cytosine modification is methylation, and optionally, the cytosine modification is methylation at the 5-position of cytosine. (ii) the first subsample is contacted with a methylation-sensitive endonuclease, e.g., the methylation-sensitive endonuclease cleaves unmethylated CpG sequences; and / or (iii) the methylation-sensitive endonuclease is one or more of AatII, AccII, AciI, Aor13HI, Aor15HI, BspT104I, BssHII, BstUI, Cfr10I, ClaI, CpoI, Eco52I, HaeII, HapII, HhaI, Hin6I, HpaII, HpyCH4IV, MluI, NaeI, NotI, NruI, NsbI, PmaCI, Psp1406I, PvuI, SacII, SalI, SmaI, and SnaBI.

13. 2. The method of claim 1, wherein the conversion procedure comprises bisulfite conversion; protection of 5hmC; Tet-assisted bisulfite conversion; optionally, Tet-assisted conversion with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; protection of hmC followed by Tet-assisted conversion, optionally, with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; protection of hmC followed by deamination of mC and / or C; optionally, chemically assisted conversion with a substituted borane reducing agent, which is 2-picoline borane, borane pyridine, tert-butylamine borane, or ammonia borane; or enzymatic protection of a modified cytosine followed by deamination of the unprotected cytosine to uracil.

14. 14. The method of claim 13, wherein the deamination of mC and / or C comprises treatment with an AID / APOBEC family DNA deaminase enzyme, or the protection of hmC comprises glycosylation of hmC. (i) the oligonucleotide adapter comprises a sequencing primer binding site, and the quality control nucleoside is located downstream of the sequencing primer binding site; (ii) the method further comprises amplifying the DNA using primers targeted to the adapter, the amplification step occurring between the converting step (b) and the sequencing step (c); and / or 10. The method of claim 1, wherein (iii) the results of the method are used as input to generate a report, for example (a) the report is in paper or electronic format, (b) false positive and / or false negative detections, or information derived therefrom, are presented directly in the report, and / or (c) diagnostic information or treatment recommendations based at least in part on the method are included in the report.

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