Methods for detecting and reducing sample preparation-induced methylation artifacts

Abstract

The present disclosure relates to improved sequencing reactions. In particular, the present disclosure provides methods that allow for the identification of regions in DNA molecules that are synthesized during end repair and / or A-tailing reactions. Sequence data derived from such a synthesized region may not represent a corresponding region in the original DNA molecule, for example, the synthesized region may contain an artificial methylation state. Thus, the identification of these synthetic regions allows such potential human data to be identified and filtered accordingly.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 391,213 filed on July 21, 2022 and U.S. Provisional Patent Application No. 63 / 513,250 filed on July 12, 2023, each of which is incorporated herein by reference in its entirety for all purposes. Field of the Invention

[0003] The present disclosure relates to methods for identifying regions in sequenced DNA that are synthesized during the end repair process, which is typically present in sequencing workflows. Such methods are important for accurately detecting methylation states and variations present in DNA, which in turn may be important for inferring information about the cells and subjects from which the DNA samples were derived.

[0004] background

[0005] DNA end repair is a common step in the sequencing workflow to prepare DNA for adapter ligation. Sample DNA molecules typically contain a mixture of blunt ends, 3' terminal overhangs, and 5' terminal overhangs. The end repair step standardizes these ends by removing the 3' overhangs and filling the 5' overhangs to produce blunt-ended DNA, which can then be subjected to blunt-ended adapter ligation or A-tailing before sticky end ligation. Such end repair steps are commonly used in sequencing workflows, such as those used for mutation analysis and / or those used to determine the methylation state of nucleotides at single-base resolution.

[0006] For example, single nucleotide resolution assays for detecting nucleoside methylation typically require conversion of modified nucleosides or corresponding unmodified nucleosides to alter their base pairing 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 TAPSβ conversion, ACE-seq, and SEM-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, E. K. et al.; Vaisvila et al., Genome Research 2021 31(7): 1280-1289; and Vaisvila et al., 2023. Discovery of novel DNA cytosine deaminase activities enables a nondestructive single-enzyme methylation sequencing method for base resolution high-coverage methylome mapping of cell-free and ultra-low input DNA. bioRxiv doi:10.1101 / 2023.06.29.547047(doi.org / 10.1101 / 2023.06.29.547047).

[0007] Bisulfite-based EM-seq and SEM-seq methylation assays convert unmethylated cytosine to uracil, which is amplified by PCR and read as thymine in the sequencing reaction. In other methods, the epigenetic conversion is the opposite, that is, modified nucleosides are converted instead of unmodified nucleosides. For example, in the TAPS method, methylated cytosine (5mC and 5hmC) is converted to dihydrouridine (DHU), which is amplified by PCR and read as thymine in the sequencing reaction. Single-molecule sequencing technologies, such as nanopore-based sequencing and single-molecule real-time sequencing (SMRT) can be optionally used to detect the modification state of nucleosides. These single-molecule technologies can directly detect the specific modification state of nucleosides, and therefore do not rely on the conversion of modified nucleosides or corresponding unmodified nucleosides to change their base pairing specificity.

[0008] During the end repair step, repair and synthesis of the region of DNA molecule can occur, which means that the modification state of the nucleosides in these regions of the DNA of the end repair may not accurately reflect the modification state of the corresponding nucleosides in the original DNA fragment. Similarly, the templated nature of the end repair may mean that it eradicates any mispairing (such as the result of single-stranded mutation) between the complementary chains. This may mistakenly cause the detected sequence to have a conclusion of double-stranded support evidence (doublestranded support) (that is, there is sequence data from two chains of the original DNA molecule supporting the sequence). Therefore, using the end repair may cause artificial information that does not represent the original DNA fragment, which in turn may cause incorrect inferences related to the DNA sample and the experimenter from which the DNA sample is obtained.

[0009] Given the issues surrounding artifacts in these assays, there exists a need for improved methods that allow for the identification of sequencing data that are known to accurately reflect the original DNA fragments.

[0010] Overview

[0011] The present disclosure provides an embodiment of a method comprising allowing to distinguish sequence data from the original DNA molecule from sequence data from the region ("synthetic region") synthesized during the end repair reaction in the DNA molecule. Therefore, the method can be used to avoid using sequence data from these synthetic regions to determine the properties of the original DNA molecule. For example, if the method is used to determine the methylation state of cytosine in the original DNA molecule, the methylation state observed in the synthetic region may not accurately reflect the methylation state of the corresponding nucleoside in the original DNA molecule. Therefore, the method can be used to use the data known to be derived from the original DNA molecule (that is, not the synthetic region) to determine the modification state of nucleosides, and thereby accurately represent the methylation state of nucleosides in the original DNA molecule. Similarly, the identification of such a synthetic region can be used to explain the sequencing data relevant to variation detection. For example, because the synthetic region is templated from complementary strands, it will eliminate any base mismatches present in the region in the original DNA molecule. Therefore, any variation detected in the synthetic region can not be reliably classified as having double-stranded support evidence (that is, sequence data from two chains of the original DNA molecule supporting the variation).

[0012] The disclosed method achieves this by using at least one type of dNTP containing a modified base (e.g., methylated deoxycytidine triphosphate, such as deoxycytidine triphosphate containing 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC)) in the end repair reaction. During the end repair reaction, the methylated deoxycytidine triphosphate will be incorporated into the synthesized region regardless of the sequence environment. This will result in methylated cytosine in non-CpG positions, which are very rare in nature. Therefore, these methylated non-CpG cytosines can be used as markers for identifying the synthesized region in the DNA molecule of the end repair. Similarly, other types of dNTPs containing modified bases that are uncommon or non-existent in nature can be used. The identification of such modified bases can be performed using modification-sensitive sequencing, and the regions containing these modifications can be interpreted as defined regions synthesized in the end repair reaction.

[0013] Then, the subsequent sequence analysis of the DNA sample can be focused on the regions in the original DNA molecule that are known not to be synthesized during the end repair reaction, and thus accurately reflect the properties (e.g., modification status) of the original DNA molecule. Therefore, the method provides sequencing data with improved accuracy because it allows subsequent sequence analysis to be focused on regions that do not contain artificial information (e.g., methylation status) related to the regions synthesized during the end repair reaction in the DNA. The method can also be used to quantify the level of DNA damage in the original DNA molecule by quantitative analysis of the synthesized regions.

[0014] In one aspect, the present disclosure provides a method comprising: (a) subjecting a DNA sample to end-repair to produce end-repaired DNA, wherein the end-repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base; (b) performing a ligation reaction to ligate an adaptor to the end-repaired DNA to produce an adapted DNA, wherein the ligation reaction also seals a nick present in the end-repaired DNA; (c) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; and (d) analyzing the sequence data obtained in step (c) to identify one or more regions synthesized during the end-repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a).

[0015] In some embodiments, the end repair is performed with a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand displacement DNA polymerase. In some embodiments, the DNA polymerase is T4 DNA polymerase, T7 DNA polymerase or Klenow fragment. In some embodiments, the end repair is performed with a DNA polymerase that has 5'-3' exonuclease activity and / or is a strand displacement DNA polymerase. In some embodiments, at least one type of dNTP comprising a modified base includes a dNTP comprising 4-methylcytosine (4mC), a dNTP comprising 5-methylcytosine (5mC), a dNTP comprising 5-hydroxymethyl-cytosine (5hmC), a dNTP comprising N6-methyladenosine (6mA), a dNTP comprising bromodeoxyuridine (BrdU) and / or a dNTP comprising 8-oxoguanine (8oxoG). In some embodiments, the method further comprises performing an A-tailing reaction between steps (a) and (b).

[0016] In some embodiments, the end repair and A-tailing reactions are performed in a single tube. In some embodiments, A-tailing is performed using a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand displacement DNA polymerase, optionally, the DNA polymerase is HemoKlen Taq. In some embodiments, A-tailing is performed using Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, large fragment or Tth DNA polymerase.

[0017] In some embodiments, the end repair and A-tailing reactions are performed as separate reactions, wherein a reaction cleaning step is performed after the end repair and before the A-tailing reaction. In some embodiments, A-tailing is performed using a DNA polymerase that does not have 3'-5' exonuclease activity, such as a Klenow fragment that lacks 3'-5' exonuclease activity. In some embodiments, A-tailing is performed using a DNA polymerase that has 5'-3' exonuclease activity and / or is a strand displacement DNA polymerase. In some embodiments, the ligation reaction is a blunt end ligation reaction. In some embodiments, the ligation reaction is a sticky end ligation reaction.

[0018] In some embodiments, modification-sensitive sequencing includes a conversion program that changes the base pairing specificity of the base or does not change the base pairing specificity of the base according to the modification state of the base. In some embodiments, the modified base is a methylated cytosine, and wherein the conversion program converts the methylated cytosine, such as the conversion program is a Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-picolineborane, pyridine borane, tert-butylamine borane or ammonia borane. In some embodiments, the modified base is a methylated cytosine, and the conversion program converts unmethylated cytosine, such as the conversion program is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC coupled epigenetic (ACE) conversion, enzymatic methyl sequencing (EM-seq) or single enzyme 5-methylcytosine sequencing (SEM-seq). In some embodiments, modification-sensitive sequencing includes sequencing based on nanopores or single molecule real-time (SMRT) sequencing. In some embodiments, modification-sensitive sequencing comprises nanopore-based sequencing, and the at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC (4-methyldeoxycytidine), a dNTP comprising 5mC (5-methyldeoxycytidine), a dNTP comprising 5hmC (5-hydroxymethyldeoxycytidine), a dNTP comprising 6mA (6-methyldeoxyadenosine), a dNTP comprising BrdU (5-bromodeoxyuridine), dUTP, a dNTP comprising FldU (5-fluorodeoxyuridine), a dNTP comprising IdU (5-iododeoxyuridine), a dNTP comprising IdU (5-iododeoxyuridine), and / or a dNTP comprising EdU (5-ethynyldeoxyuridine). In some embodiments, modification-sensitive sequencing comprises single-molecule real-time (SMRT) sequencing, and at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, and / or a dNTP comprising 8oxoG (8-oxodeoxyguanosine). In some embodiments, at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, a dUTP, a dNTP comprising FldU, a dNTP comprising IdU, a dNTP comprising EdU, and / or a dNTP comprising 8oxoG.

[0019] In some embodiments, the modified base is 5mC or 5hmC.

[0020] The sequence data obtained in the analysis step (c) includes identifying regions containing modified bases (e.g., 5mC or 5hmC) in a non-CpG sequence environment and classifying these regions as regions synthesized during end repair. A modified base appears in a non-CpG sequence environment when a base other than G is adjacent to the 3' of the modified base. In some embodiments, the modified base is not 5mC or 5hmC, and the region in one or more regions is defined as: (i) a sequence between two non-modified bases spanning a modified base, wherein the two non-modified bases have the same identity as a modified base present in at least one type of dNTP; and / or (ii) a sequence between a non-modified base and the end of a sequence read, wherein there is no additional non-modified base between the non-modified base and the end of the sequence read, wherein the additional non-modified base has the same identity as a modified base present in the at least one type of dNTP.

[0021] In some embodiments, the modified base is a methylated cytosine, such as 5mC or 5hmC, and the region in one or more regions is defined as: (i) a sequence between two non-methylated cytosines spanning a methylated non-CpG cytosine; and / or (ii) a sequence between a non-methylated cytosine and the end of a sequence read, wherein there is no additional non-methylated cytosine between the non-methylated cytosine and the end of the sequence read.

[0022] In some embodiments, the method further comprises: (i) filtering out sequence data from one or more regions identified as synthesized during end repair so that these sequence data are not used for subsequent analysis; or (ii) marking sequence data from one or more regions identified as synthesized during end repair as potentially containing artifactual sequence data that may not represent the DNA sample. In some embodiments, the method further comprises analyzing at least some of the sequence data corresponding to regions not identified as synthesized during end repair to detect the presence or absence of base modifications or mutations in the DNA sample. In some embodiments, the method is used to detect the methylation state of cytosine in a DNA sample, and wherein analyzing the sequence data comprises filtering out one or more regions of the end-repaired DNA identified as synthesized during end repair so that the one or more regions are not used to determine the methylation state of cytosine in the DNA sample. In some embodiments, the method is used to detect single nucleotide variations (SNVs) in a DNA sample, and wherein analyzing the sequence data comprises classifying all base calls in one or more regions as having no double-stranded support evidence.

[0023] In some embodiments, the method also includes quantifying DNA damage in the DNA sample by identifying one or more regions synthesized during the end repair in the DNA of the end repair. In some embodiments, the level of DNA damage is used to predict whether a portion of the DNA in the DNA sample is derived from cancer cells. In some embodiments, the DNA sample comprises cell-free DNA (cfDNA), and the method also includes analyzing the sequence data obtained in step (c) to determine the level of artificial artifacts (artifacts) measured in cfDNA. In some embodiments, the method also compares the level of artificial artifacts measured with one or more reference levels. In some embodiments, the method also includes (i) using the level of artificial artifacts measured and / or comparing the level of artificial artifacts measured with one or more reference levels to predict whether a portion of cfDNA is derived from cancer cells, or (ii) using the level of artificial artifacts measured and / or comparing the level of artificial artifacts measured with one or more reference levels to determine the possibility that a portion of cfDNA is derived from cancer cells. In some embodiments, the DNA sample comprises cell-free DNA or DNA from formalin-fixed paraffin-embedded samples.

[0024] On the other hand, the present disclosure provides a method comprising: (a) subjecting the DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs) comprising 5mCTP; (b) performing a ligation reaction to connect an adaptor to the end-repaired DNA to produce an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA; (c) subjecting the adapted DNA to bisulfite sequencing to obtain sequencing data derived from the DNA sample; (d) analyzing the sequence data obtained in step (c) to identify one or more regions of the end-repaired DNA synthesized during end repair by the presence of 5mC at non-CpG positions; and (e) optionally also comprising analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during end repair to detect the presence or absence of cytosine methylation in the DNA sample.

[0025] In another aspect, the present disclosure provides a method comprising: (a) subjecting a DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base; (b) performing a ligation reaction to ligate an adaptor to the end-repaired DNA to produce an adapted DNA, wherein the ligation reaction also seals a nick present in the end-repaired DNA; (c) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP, wherein the modification-sensitive sequencing is nanopore sequencing, single-molecule real-time sequencing, or Tet-assisted pyridine borane sequencing (TAPS); (d) analyzing the sequence data obtained in step (c) to identify one or more regions synthesized during the end repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a); and (e) further comprising analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during the end repair to detect the presence or absence of mutations present in the DNA sample.

[0026] In another aspect, the present disclosure provides a method comprising: (a) subjecting the DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, wherein the end repair is performed with a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, such as T4 DNA polymerase, T7 (b) performing an A-tailing reaction as a single tube reaction with end repair, optionally wherein the A-tailing is performed using a DNA polymerase that does not have 3'-5' exonuclease activity and / or is not a strand displacement DNA polymerase; (c) performing a ligation reaction to ligate an adaptor to the end-repaired A-tailed DNA to produce an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA; (d) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; (e) analyzing the sequence data obtained in step (d) to identify one or more regions synthesized during the end repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a); and (f) optionally also comprising analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations in the DNA sample.

[0027] In another aspect, the present disclosure provides a method comprising: (a) subjecting the DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, wherein the end repair is performed with a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, such as T4 DNA polymerase, T7 (b) performing a reaction cleaning step on the end-repaired DNA and then performing an A-tailing reaction to produce A-tailed DNA; (c) performing a ligation reaction to connect an adaptor to the A-tailed DNA to produce an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA; (d) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; (e) analyzing the sequence data obtained in step (d) to identify one or more regions synthesized during the end-repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a); and (f) optionally also including analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during the end-repair to detect the presence or absence of base modifications or mutations in the DNA sample.

[0028] In another aspect, the present disclosure provides a method comprising: (a) subjecting the DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base, wherein the end repair is performed with a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, such as T4 DNA polymerase, T7 (b) performing a blunt-end ligation reaction to connect an adaptor to the end-repaired DNA to produce an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA; (c) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; (d) analyzing the sequence data obtained in step (c) to identify one or more regions synthesized during the end-repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a); and (e) optionally also including analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during the end-repair to detect the presence or absence of base modifications or mutations in the DNA sample.

[0029] In some embodiments, a kit is provided that includes a) a first reagent for end repair to produce end-repaired DNA, wherein the first reagent includes at least one type of dNTP that includes a modified base, b) a second reagent for ligating adaptors to the end-repaired DNA to produce adapted DNA, wherein the second reagent also seals nicks present in the end-repaired DNA, c) reagents for modification-sensitive sequencing that are capable of identifying base modifications in at least one type of dNTP, and / or a DNA polymerase for incorporating the first reagent into DNA during end repair, and d) library adaptors with different molecular barcodes.

[0030] In some embodiments, the kit further comprises more than one oligonucleotide probe and / or primer for sequencing. In some embodiments, the first reagent of the kit comprises at least one type of dNTPs comprising a modified base selected from the following: dNTPs comprising 4-methylcytosine (4mC), dNTPs comprising 5-methylcytosine (5mC), dNTPs comprising 5-hydroxymethylcytosine (5hmC), dNTPs comprising N6-methyladenosine (6mA), dNTPs comprising bromodeoxyuridine (BrdU), dNTPs comprising 8-oxoguanine (8oxoG), dNTPs, dNTPs comprising fluorodeoxyuridine (FldU), dNTPs comprising iododeoxyuridine (IdU) and / or dNTPs comprising ethynyl deoxyuridine (EdU). In some embodiments, the DNA polymerase of the kit does not have 5'-3' exonuclease activity and / or is not a strand displacement DNA polymerase. In some embodiments, the DNA polymerase of the test kit is T4 DNA polymerase, T7 DNA polymerase or Klenow fragment. In some embodiments, the DNA polymerase of the test kit has 5'-3' exonuclease activity and / or is a strand displacement DNA polymerase. In some embodiments, the test kit also includes a reagent for performing an A-tailing reaction. In some embodiments, the reagent for performing an A-tailing reaction includes a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand displacement DNA polymerase, and optionally, the reagent for performing an A-tailing reaction is HemoKlen Taq. In some embodiments, the reagent for performing an A-tailing reaction includes Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, large fragment or Tth DNA polymerase. In some embodiments, the reagent for performing an A-tailing reaction includes a DNA polymerase that does not have 3'-5' exonuclease activity, and optionally wherein the reagent for performing an A-tailing reaction is a Klenow fragment lacking 3'-5' exonuclease activity. In some embodiments, the reagents used to perform the A-tailing reaction include a DNA polymerase having 5'-3' exonuclease activity and / or a strand-displacing DNA polymerase.

[0031] In some embodiments, the kit comprises: a) more than one oligonucleotide probe that selectively hybridizes to at least 5, 6, 7, 8, 9, 10, 20, 30, 40, or all of the genes selected from: ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZH2 , FGFRl, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PRO C, PTPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRK1, b) library adapters that do not contain flow cell sequences or sequences that allow formation of hairpin loops for sequencing, c) library adapters are blunt-ended and Y-shaped, and / or d) the length of the library adapters is less than or equal to 40 nucleic acid bases. In some embodiments, the kit also includes instructions for performing any of the methods described herein.

[0032] Embodiments described herein include, but are not limited to, the following.

[0033] Embodiment 1 is a method comprising:

[0034] (a) subjecting a DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base;

[0035] (b) performing a ligation reaction to connect the adaptor to the end-repaired DNA to generate an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA;

[0036] (c) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; and

[0037] (d) analyzing the sequence data obtained in step (c) to identify one or more regions synthesized during the end repair in the end-repaired DNA by the presence of base modifications in at least one type of dNTP used in step (a).

[0038] Embodiment 2 is the method of embodiment 1, wherein the end repair is performed using a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase.

[0039] Embodiment 3 is the method of embodiment 2, wherein the DNA polymerase is T4 DNA polymerase, T7 DNA polymerase, or Klenow fragment.

[0040] Embodiment 4 is the method of embodiment 1, wherein the end repair is performed using a DNA polymerase having 5'-3' exonuclease activity and / or a strand-displacing DNA polymerase.

[0041] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the at least one type of dNTP containing a modified base includes dNTPs containing 4-methylcytosine (4mC), dNTPs containing 5-methylcytosine (5mC), dNTPs containing 5-hydroxymethylcytosine (5hmC), dNTPs containing N6-methyladenosine (6mA), dNTPs containing bromodeoxyuridine (BrdU) and / or dNTPs containing 8-oxoguanine (8oxoG).

[0042] Embodiment 6 is the method of any one of embodiments 1 to 5, further comprising performing an A-tailing reaction between steps (a) and (b).

[0043] Embodiment 7 is the method of embodiment 6, wherein the end repair and A-tailing reactions are performed in the same reaction mixture, optionally wherein the end repair and A-tailing reactions are performed in a single tube and / or optionally wherein the end repair and A-tailing reactions are performed without an intermediate cleanup step.

[0044] Embodiment 8 is the method of embodiment 7, wherein A-tailing is performed using a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

[0045] Embodiment 9 is the method of embodiment 7, wherein A-tailing is performed using a thermostable DNA polymerase.

[0046] Embodiment 10 is the method of embodiment 7, wherein A-tailing is performed using Taq DNA polymerase, Tfl DNA polymerase, BstDNA polymerase, Large Fragment, or Tth DNA polymerase.

[0047] Embodiment 11 is the method of embodiment 6, wherein the end repair and A-tailing reactions are performed as separate reactions, wherein a reaction cleanup step is performed after the end repair and before the A-tailing reaction.

[0048] Embodiment 12 is the method of embodiment 11, wherein the reaction cleaning step removes unincorporated dNTPs.

[0049] Embodiment 13 is the method of embodiment 11 or 12, wherein A-tailing is performed using a DNA polymerase lacking 3'-5' exonuclease activity, optionally wherein the DNA polymerase is a Klenow fragment lacking 3'-5' exonuclease activity.

[0050] Embodiment 14 is the method of embodiment 7 or embodiment 11, wherein the A-tailing is performed using a DNA polymerase having 5'-3' exonuclease activity and / or a strand-displacing DNA polymerase.

[0051] Embodiment 15 is the method of any of embodiments 6-14, wherein the A-tailing reaction is performed at a higher temperature than the end repair, optionally wherein the end repair is performed at about 15°C-35°C and / or the A-tailing is performed at a temperature greater than about 60°C, and optionally wherein the temperature greater than 60°C is about 60°C-75°C.

[0052] Embodiment 16 is the method of any one of embodiments 1-5, wherein the ligation reaction is a blunt end ligation reaction.

[0053] Embodiment 17 is the method of any one of embodiments 6-15, wherein the ligation reaction is a sticky end ligation reaction.

[0054] Embodiment 18 is the method of any one of embodiments 1-17, wherein modification-sensitive sequencing comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base according to the modification state of the base.

[0055] Embodiment 19 is the method of embodiment 18, wherein the modified base is a methylated cytosine, and wherein the conversion procedure converts the methylated cytosine, for example, wherein the conversion procedure is a Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane or ammonia borane.

[0056] Embodiment 20 is the method of embodiment 18, wherein the modified base is a methylated cytosine, and wherein the conversion procedure converts unmethylated cytosine, for example, wherein the conversion procedure is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymatic methyl sequencing (EM-seq) or single enzyme 5-methylcytosine sequencing (SEM-seq).

[0057] Embodiment 21 is the method of any one of embodiments 1-17, wherein the modification-sensitive sequencing comprises nanopore-based sequencing or single-molecule real-time (SMRT) sequencing.

[0058] Embodiment 22 is the method described in embodiment 21, wherein modification-sensitive sequencing comprises nanopore-based sequencing, and the at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, a dUTP, a dNTP comprising FldU, a dNTP comprising IdU and / or a dNTP comprising EdU.

[0059] Embodiment 23 is the method described in embodiment 21, wherein modification-sensitive sequencing includes single-molecule real-time (SMRT) sequencing, and the at least one type of dNTP containing a modified base includes a dNTP containing 4mC, a dNTP containing 5mC, a dNTP containing 5hmC, a dNTP containing 6mA and / or a dNTP containing 8oxoG.

[0060] Embodiment 24 is the method of any one of embodiments 1-23, wherein the modified base is 5mC.

[0061] Embodiment 25 is the method of any one of embodiments 1-23, wherein the modified base is 5hmC.

[0062] Embodiment 26 is a method as described in any of embodiments 1-25, wherein analyzing the sequence data obtained in step (c) comprises identifying regions containing modified bases in a non-CpG sequence environment and classifying these regions as regions synthesized during end repair.

[0063] Embodiment 27 is the method of any one of embodiments 1-23, wherein the modified base is not 5mC or 5hmC, and a region in the one or more regions is defined as:

[0064] (i) a sequence between two non-modified bases spanning a modified base, wherein the two non-modified bases have the same identity as a modified base present in at least one type of dNTP; and / or

[0065] (ii) a sequence between a non-modified base and an end of the sequence read, wherein there are no additional non-modified bases between the non-modified base and said end of the sequence read, wherein the additional non-modified bases have the same identity as a modified base present in at least one type of dNTP.

[0066] Embodiment 28 is the method of any one of embodiments 1-23, wherein the modified base is a methylated cytosine, such as 5mC or 5hmC, and the region in the one or more regions is defined as:

[0067] (i) a sequence between two non-methylated cytosines spanning a methylated non-CpG cytosine; and / or

[0068] (ii) a sequence between an unmethylated cytosine and an end of a sequence read, wherein no additional unmethylated cytosines are present between said unmethylated cytosine and said end of said sequence read.

[0069] Embodiment 29 is the method of any one of embodiments 1-28, further comprising: (i) filtering out sequence data from one or more regions identified as synthesized during end repair so that such sequence data are not used for subsequent analysis; or (ii) marking sequence data from one or more regions identified as synthesized during end repair as potentially containing artifactual sequence data that may not be representative of the DNA sample.

[0070] Embodiment 30 is the method of any one of embodiments 1-29, further comprising analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during end repair to detect the presence or absence of base modifications or mutations in the DNA sample.

[0071] Embodiment 31 is a method described in any one of embodiments 1-30, wherein the method is used to detect the methylation state of cytosine in a DNA sample, and wherein analyzing the sequence data includes filtering out one or more regions in the end-repaired DNA that are identified as being synthesized during the end-repair, so that the one or more regions are not used to determine the methylation state of cytosine in the DNA sample.

[0072] Embodiment 32 is the method of any one of embodiments 1-31, wherein the method is used to detect single nucleotide variations (SNVs) in a DNA sample, and wherein analyzing the sequence data comprises classifying all base calls within one or more regions as having no double-stranded supporting evidence.

[0073] Embodiment 33 is the method of any one of embodiments 1-32, wherein the method further comprises quantifying DNA damage in the DNA sample by identifying one or more regions in the end-repaired DNA that are synthesized during the end-repair.

[0074] Embodiment 34 is the method of embodiment 33, wherein the level of DNA damage is used to predict whether a portion of the DNA in the DNA sample originates from a cancer cell.

[0075] Embodiment 35 is a method as described in any one of embodiments 1-34, wherein the DNA sample comprises cell-free DNA (cfDNA), and the method further comprises analyzing the sequence data obtained in step (c) to determine the level of artifacts measured in the cfDNA.

[0076] Embodiment 36 is the method of embodiment 35, further comprising comparing the measured level of the artifact to one or more reference levels.

[0077] Embodiment 37 is the method described in embodiment 35 or 36, further comprising (i) using the measured level of the artifact and / or comparing the measured level of the artifact to one or more reference levels to predict whether a portion of the cfDNA is derived from cancer cells, or (ii) using the measured level of the artifact and / or comparing the measured level of the artifact to one or more reference levels to determine the likelihood that a portion of the cfDNA is derived from cancer cells.

[0078] Embodiment 38 is the method of any one of embodiments 1-34, wherein the DNA sample comprises cell-free DNA or DNA from a formalin-fixed paraffin-embedded sample.

[0079] Embodiment 39 is the method of the preceding embodiment, wherein the DNA sample comprises cell-free DNA.

[0080] Embodiment 40 is a method as described in any of the preceding embodiments, wherein the sample is from a subject, and the method further comprises determining the presence or absence of cancer in the subject based at least in part on the sequencing data.

[0081] Embodiment 41 is the method of any of the preceding embodiments, further comprising at least one DNA amplification step, optionally wherein the DNA amplification step is performed after step (b) and before step (c).

[0082] Embodiment 42 is the method of the preceding embodiment, wherein the DNA amplification step comprises PCR.

[0083] Embodiment 43 is a method as described in any of the preceding embodiments, wherein the adaptor comprises a molecular barcode.

[0084] Embodiment 44 is the method of any of the preceding embodiments, further comprising enriching more than one target region of DNA prior to step (c).

[0085] Embodiment 45 is the method of embodiment 44, wherein the more than one target regions include epigenetic target regions.

[0086] Embodiment 46 is the method described in embodiment 45, wherein the epigenetic target region includes a hypermethylated variable target region.

[0087] Embodiment 47 is the method of embodiment 45 or 46, wherein the epigenetic target region comprises a hypomethylated variable target region.

[0088] Embodiment 48 is the method of any one of embodiments 44-47, wherein the more than one target regions include sequence variable target regions.

[0089] Embodiment 49 is a kit comprising:

[0090] a) a first reagent for end repair to produce end-repaired DNA, wherein the first reagent comprises at least one type of dNTP, the dNTP comprising a modified base;

[0091] b) a second reagent for ligating an adaptor to the end-repaired DNA to generate adapted DNA, wherein the second reagent also seals the nick present in the end-repaired DNA;

[0092] c) reagents for modification-sensitive sequencing capable of identifying base modifications in at least one type of dNTP, and / or a DNA polymerase for incorporating a first reagent into DNA during end repair; and

[0093] d) Library adapters with different molecular barcodes.

[0094] Embodiment 50 is the kit of embodiment 49, further comprising more than one oligonucleotide probe and / or primer for sequencing.

[0095] Embodiment 51 is a kit as described in embodiment 49 or 50, wherein the first reagent comprises at least one type of dNTP containing a modified base selected from the following: dNTP containing 4-methylcytosine (4mC), dNTP containing 5-methylcytosine (5mC), dNTP containing 5-hydroxymethylcytosine (5hmC), dNTP containing N6-methyladenosine (6mA), dNTP containing bromodeoxyuridine (BrdU), dNTP containing 8-oxoguanine (8oxoG), dUTP, dNTP containing fluorodeoxyuridine (FldU), dNTP containing iododeoxyuridine (IdU) and / or dNTP containing ethynyldeoxyuridine (EdU).

[0096] Embodiment 52 is the kit of any one of embodiments 49-51, wherein the DNA polymerase does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase.

[0097] Embodiment 53 is the kit of any one of embodiments 49-52, wherein the DNA polymerase is T4 DNA polymerase, T7 DNA polymerase, or Klenow fragment.

[0098] Embodiment 54 is the kit of embodiment 49, wherein the DNA polymerase has 5'-3' exonuclease activity and / or is a strand-displacing DNA polymerase.

[0099] Embodiment 55 is the kit of any one of embodiments 49-54, further comprising reagents for performing an A-tailing reaction.

[0100] Embodiment 56 is the kit of the previous embodiment, further comprising a reagent for a clean-up step after the A-tailing reaction, optionally wherein the reagent for the clean-up step is used to remove unincorporated dNTPs.

[0101] Embodiment 57 is the kit of embodiment 55 or 56, wherein the reagent for performing the A-tailing reaction comprises a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, optionally wherein the reagent for performing the A-tailing reaction is HemoKlen Taq.

[0102] Embodiment 58 is a kit according to any one of embodiments 55-57, wherein the reagents for performing the A-tailing reaction include Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, Large Fragment or Tth DNA polymerase.

[0103] Embodiment 59 is the kit of any one of embodiments 55-58, wherein the reagents for performing the A-tailing reaction comprise a thermostable DNA polymerase.

[0104] Embodiment 60 is the kit of any one of embodiments 55-56, wherein the reagent for performing the A-tailing reaction comprises a DNA polymerase without 3'-5' exonuclease activity, optionally wherein the reagent for performing the A-tailing reaction is a Klenow fragment lacking 3'-5' exonuclease activity.

[0105] Embodiment 61 is the kit of any one of embodiments 55-56, wherein the reagents for performing the A-tailing reaction include a DNA polymerase having 5'-3' exonuclease activity and / or a strand-displacing DNA polymerase.

[0106] Embodiment 62 is the kit of any one of embodiments 49-61, wherein:

[0107] a) more than one oligonucleotide probe selectively hybridizes to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZH2, FGFR1, F GFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, PT PN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRKl;

[0108] b) the library adapters do not contain flow cell sequences or sequences that allow the formation of hairpin loops for sequencing;

[0109] c) the library adaptors are blunt ended and Y-shaped; and / or

[0110] d) The length of the library adapter is less than or equal to 40 nucleic acid bases.

[0111] Embodiment 63 is the kit of any one of embodiments 49-62, further comprising instructions for performing the method of any one of embodiments 1-48.

[0112] In some embodiments, the results of the methods disclosed herein are used as input to generate a report. The report can be in paper or electronic format. For example, the true methylation state of cytosine or variation obtained by the methods disclosed herein or information derived therefrom can be directly displayed in such a report. Alternatively or additionally, diagnostic information or treatment recommendations based at least in part on the methods disclosed herein can be included in the report.

[0113] The various steps of the methods disclosed herein can be performed at the same or different times, in the same or different geographic locations, such as countries, and / or by the same or different persons.

[0114] 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

[0116] exist Figure 1-Figure 6 In the figure, the solid mushroom shapes represent methylated CpG sites, and the hollow mushroom shapes represent unmethylated CpG sites. The solid thick lines represent regions of the DNA molecule that existed in the original DNA molecule (i.e., before end repair). The dotted lines represent synthetic regions synthesized during end repair, including regions added to the 3' end of the DNA molecule at the 5' overhang during end repair, and internal synthetic regions generated by gap filling and nick translation. The solid thin lines represent synthetic regions synthesized during A-tailing. The cross-hatched circles represent polymerases used in end repair. The hollow circles represent polymerases used in A-tailing. The triangles represent ligation sites. The stars represent 5mCpH sites.

[0117] Figure 1 is a schematic diagram showing the effect of a combined end repair and A-tailing reaction on the methylation state of cytosine in a DNA molecule when unmodified dCTP is used in the end repair.

[0118] Figure 2 is a schematic diagram showing the effect of separate end-repair and A-tailing reactions on the methylation status of intact, nicked, and gapped DNA molecules when unmodified dCTP is used in the end-repair.

[0119] Figure 3is a schematic diagram showing the effect of end-repair followed by blunt-end ligation on the methylation status of intact, nicked, and gapped DNA molecules when unmodified dCTP is used in end-repair.

[0120] Figure 4 is a schematic diagram showing the effect of combined end-repair and A-tailing reactions on the methylation status of intact, nicked, and gapped DNA molecules when methylated CTP is used in both the end-repair and A-tailing reactions.

[0121] Figure 5 is a schematic diagram showing the effect of separate end repair and A-tailing reactions on the methylation status of intact, nicked, and gapped DNA molecules when methylated CTP is used in the end repair.

[0122] Figure 6 is a schematic diagram showing the effect of end-repair followed by blunt-end ligation on the methylation status of intact, nicked, and gapped DNA molecules when methylated CTP is used in end-repair.

[0123] Fig. 7A is a graph showing the estimated level of end repair in paired strands of DNA molecules. Figure 7B is a sequence diagram showing the forward strand (upper two sequences) and reverse strand (lower two sequences) of an exemplary alignment. shows the aligned reads, where "." represents any non-cytosine base, "o" and "M" represent unmethylated and methylated cytosine, respectively.

[0124] Figure 8 A series of M-bias plots are shown showing the percentage of methylation levels at CpG sites (upper row) and non-CpG sites (lower row) throughout the sequence reads. The two left columns represent experiments using a single-tube end repair / A-tailing reaction with dCTP containing unmodified cytosine, while the two right columns represent experiments using a single-tube end repair / A-tailing reaction with dCTP containing 5-methylcytosine (5mCTP).

[0125] Fig. 9 A series of M-bias plots are shown showing the percentage of methylation levels at CpG sites (upper row) and non-CpG sites (lower row) throughout the sequence reads. The two left columns represent experiments using dCTP containing unmodified cytosine for end repair followed by blunt end ligation. The three right columns represent experiments using dCTP containing unmodified cytosine for single-tube end repair / A-tailing reactions followed by sticky end ligation.

[0126] Fig.10A series of M-bias plots are shown showing the percentage of methylation levels at CpG sites (upper row) and non-CpG sites (lower row) throughout the sequence reads. The three left columns represent experiments using dCTP containing 5-methylcytosine (5mCTP) for end repair followed by blunt end ligation. The three right columns represent experiments using dCTP containing 5-methylcytosine (5mCTP) for single-tube end repair / A-tailing reactions followed by sticky end ligation.

[0127] Figure 11A-11B Shown is the incorporation of mCTP compared to that of CTP in the positive (A) and negative (B) control regions after end repair / A-tailing.

[0128] Figures 12A-12D Shown is a comparison of mCTP end trimming and CTP end trimming after end repair / A-tailing.

[0129] Fig.13 is a schematic diagram of an example of a system suitable for use with some embodiments of the present disclosure.

[0130] Details

[0131] Some embodiments of the present disclosure will now be mentioned in detail. Although the present disclosure will be described in conjunction with such embodiments, it should be understood that they are not intended that the present disclosure be limited to these embodiments. On the contrary, it is intended that the present disclosure cover all substitutions, modifications and equivalents, which may be included in the present disclosure limited by the appended claims.

[0132] Before describing this teaching 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 referents unless the context clearly dictates otherwise. Thus, for example, reference to "nucleic acid" includes more than one nucleic acid.

[0133] Numerical ranges are inclusive of the numbers defining the range. Measured and measurable values ​​should be understood to be approximate, taking into account significant digits and errors associated with the measurements.

[0134] Unless otherwise specifically noted in the foregoing description, embodiments reciting in the description "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the components.

[0135] The section headings used herein are for organizational purposes and are not to be construed as limiting the disclosed subject matter in any way.

[0136] 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. If different versions of a publication, website, etc. are published at different times, the version most recently published as of the effective filing date of this application is intended, unless otherwise indicated.

[0137] definition

[0138] "Reaction cleaning" refers to the removal of contaminants such as salts, enzymes, unincorporated dNTPs, primers, ethidium bromide, and other impurities that may interfere with downstream analysis. For example, when reaction cleaning is performed between end repair and A-tailing reactions, it removes unincorporated dNTPs so that the A-tailing reaction can be performed only in the presence of dATP (i.e., not dCTP, dGTP, and dCTP used in the end-tailing reaction). Reaction cleaning can be performed using commercially available kits such as the MinElute reaction cleaning kit (Qiagen).

[0139] "Synthesized regions", also referred to as "regions in the end-repaired DNA that are synthesized during the end-repair", refer to regions in the DNA that were not present in the DNA prior to the end-repair and A-tailing reactions. If synthesized regions exist, they are regions synthesized by the polymerase used in the end-repair and / or A-tailing reactions (if any). In the case where the A-tailing and end-repair reactions are performed in the same tube, all four types of dNTPs will be present, and thus the polymerase used for A-tailing can generate synthesized regions, for example, by nick translation. In the case where the A-tailing is performed separately from the end-repair reaction, and these steps are separated by reaction cleaning, only dATP will be present in the A-tailing reaction, and thus the polymerase used for A-tailing will generally not generate synthesized regions because not all of the dNTP components are present in the A-tailing reaction mixture.

[0140] As used herein, "base pairing specificity" refers to the standard DNA base (A, C, G, or T) to which a particular base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G), while uracil and cytosine have different base pairing specificities, as uracil has a base pairing specificity for A, while cytosine has a base pairing specificity for G. For example, the ability of uracil to form a wobble pair with G is irrelevant, as uracil still most preferentially pairs with A of the four standard DNA bases.

[0141] "Type of dNTP" refers to a dNTP containing a specific base, including A, T, G, or C. Therefore, when an end repair reaction is performed with dNTPs, wherein at least one type of dNTP contains a modified base, the end repair reaction can be performed using dCTP containing 5mC and dATP, dTTP, and dGTP, all of which contain unmodified bases.

[0142] "Capable of identifying base modifications in at least one type of dNTP" refers to the ability of a modification-sensitive sequencing method to detect the presence or absence of base modifications in at least one type of dNTP containing modified bases for end repair. This detection of base modifications can be direct, such as in nanopore sequencing or single-molecule real-time sequencing, where the sequencing data itself indicates the presence or absence of base modifications. Alternatively, the detection of base modifications can be indirect, such as where the method involves a conversion procedure that changes base pairing specificity according to the base modification state. It is these changes in base pairing specificity that can be detected by sequencing methods, such as by comparing sequencing data with a reference sequence. In addition, regardless of whether the modification-sensitive sequencing method is able to distinguish one base modification from all other base modifications, the method is able to identify base modifications in at least one type of dNTP. For example, one form of modification-sensitive sequencing is sequencing after bisulfite conversion. The method is able to distinguish 5hmC from 5mC from unmethylated cytosine, but cannot distinguish 5hmC from 5mC.

[0143] A base of "same identity" refers to the same base regardless of the modification state of the base. For example, cytosine is considered to be the "same identity" as 5-methylcytosine (5mC) and / or 5-hydroxymethyl-cytosine (5hmC), even though they have different modification states.

[0144] "Capturing" one or more target nucleic acids means preferentially isolating or separating the one or more target nucleic acids from non-target nucleic acids.

[0145] "Cell-free DNA," "cfDNA molecules," or simply "cfDNA" include DNA molecules that naturally exist in an extracellular form in a subject (e.g., in blood, serum, plasma, or other body fluids such as lymph, cerebrospinal fluid, urine, or sputum). Although cfDNA is initially present in one or more cells in a large complex biological organism (e.g., a mammal), cfDNA undergoes release from the cells into a fluid present in the organism and can be obtained from a sample of the fluid without the need for an in vitro cell lysis step.

[0146] As used herein, "cellular nucleic acid" means nucleic acid that is within one or more cells from which the nucleic acid originates, at least at the time a sample is obtained or collected from a subject, even if the nucleic acid is subsequently removed (e.g., by cell lysis) as part of a particular analytical procedure.

[0147] DNA is "derived from cancer cells" if it is derived from tumor cells. Cell-free DNA derived from cancer cells includes ctDNA or circulating tumor DNA. Tumor cells are neoplastic cells derived from a tumor, whether they remain in the tumor or become separated from the tumor (as in the case of, for example, metastatic cancer cells and circulating tumor cells).

[0148] The term "methylation" or "DNA methylation" refers to the addition of methyl groups to nucleotide bases in nucleic acid molecules. In some embodiments, methylation refers to the addition of methyl groups to cytosine at CpG sites (cytosine-phosphate-guanine sites (i.e., cytosine followed by guanine in the 5'→3' direction of the nucleic acid sequence)). In some embodiments, DNA methylation refers to the addition of methyl groups to adenine, such as in N6-methyladenine (6mA). In some embodiments, DNA methylation is 5-methylation (modification of the carbon at the 5th position of the cytosine ring). In some embodiments, 5-methylation refers to the addition of methyl groups 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-carboxylcytosine (5-caC). In some embodiments, DNA methylation is 3C methylation (modification of the carbon at the 3rd position of the cytosine ring). In some embodiments, 3C methylation includes adding 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 change the activity of methylated DNA regions. For example, when the DNA in the promoter region is methylated, the transcription of the gene can be inhibited. DNA methylation is essential for normal development, and the abnormality of methylation can destroy epigenetic regulation. Destruction in epigenetic regulation, such as inhibition, can cause diseases, such as cancer. DNA methylation of promoters can indicate cancer.

[0149] "Modified nucleoside spectrum of DNA" means the position and identity of nucleosides in the DNA sequence and the modification state of the nucleosides, such as methylation. As described above, different modification-sensitive sequencing methods can be used to detect such modifications. This includes methods involving conversion and then sequencing to detect one or more different types of modified or unmodified nucleosides. For example, the TAPS method detects but does not distinguish between 5-methylcytosine (5mC) and 5-hydroxymethyl-cytosine (5hmC). Therefore, the method for the modified nucleoside spectrum of DNA in the analysis sample generally means identifying a specific modification or a modified group, such as 5mC and / or 5hmC. According to the specific method / conversion program used as described above, the modified nucleosides are identified. This generally involves comparing the sequence data obtained from the DNA that has undergone the conversion program with the reference sequence. Generally, the method comprises (i) comparing sequence data to (A) one or more predetermined reference sequences or (B) sequence data obtained by sequencing a subsample of DNA that has not undergone a transformation procedure, e.g., a subsample isolated prior to subjecting the individual subsamples to a transformation 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 altered base pairing specificity upon exposure to the transformation procedure.

[0150] As used herein, a modification or other feature is present in a "greater proportion" in a first sample or population of nucleic acids than in a second sample or population when the fraction of nucleotides having the modification or other feature is higher in the first sample or population than in the second sample or population. For example, if in the first sample, one tenth of the nucleotides are mC, and in the second sample, one twentieth of the nucleotides are mC, then the first sample contains a greater proportion of 5-methylated cytosine modifications than the second sample.

[0151] As used herein, "does not substantially alter the base pairing specificity of a particular nucleobase" means that the majority of molecules comprising the nucleobase that can be sequenced do not have an alteration in the base pairing specificity of a second nucleobase relative to the base pairing specificity of the nucleobase in the sample from which it was originally isolated. In some embodiments, 75%, 90%, 95%, or 99% of the molecules comprising the nucleobase that can be sequenced do not have an alteration in the base pairing specificity of a second nucleobase relative to the base pairing specificity of the nucleobase in the sample from which it was originally isolated.

[0152] As used herein, "base pairing specificity" refers to the standard DNA base (A, C, G, or T) to which a particular base most preferentially pairs. Thus, for example, unmodified cytosine and 5-methylcytosine have the same base pairing specificity (i.e., specificity for G), while uracil and cytosine have different base pairing specificities, as uracil has a base pairing specificity for A, while cytosine has a base pairing specificity for G. The ability of uracil to form a wobble pair with G is irrelevant, as uracil still most preferentially pairs with A of the four standard DNA bases.

[0153] As used herein, "modified cytosine" refers to a cytosine in which at least one position of the cytosine has been substituted with a chemical moiety such as a methyl or hydroxymethyl group that is different from the substituent at that position in the unmodified cytosine. For the avoidance of doubt, "modified cytosine" does not include unmodified cytosine.

[0154] As used herein, a "combination" comprising more than one member refers to a single composition or a group of contiguous compositions comprising the members, for example, in separate containers or compartments within a larger container, such as in a multiwell plate, tube rack, refrigerator, freezer, incubator, water bath, ice bucket, machine or other storage form.

[0155] The "capture yield" of a probe set for a particular target set refers to the amount of nucleic acid corresponding to the target set captured by the probe set under typical conditions (e.g., relative to the amount or absolute amount of another target set). Exemplary typical capture conditions are incubation of sample nucleic acid with probes at 65°C for 10-18 hours in a small reaction volume (about 20 μL) containing stringent hybridization buffer. Capture yields can be expressed in absolute values, or in relative values ​​for more than one probe set. When the capture yields of more than one target area set are compared, they are normalized for the footprint size of the target area set (e.g., on a per kilobase basis). Thus, for example, if the footprint sizes of the first target area and the second target area are 50 kb and 500 kb, respectively (providing a normalization factor of 0.1), when the mass / volume concentration of the captured DNA corresponding to the first target area set is more than 0.1 times the mass / volume concentration of the captured DNA corresponding to the second target area set, the DNA corresponding to the first target area set is captured at a higher yield than the DNA corresponding to the second target area set. As another example, using the same footprint size, if the captured DNA corresponding to the first target area group has a mass / volume concentration that is 0.2 times the mass / volume concentration of the captured DNA corresponding to the second target area group, the DNA corresponding to the first target area group is captured with a capture yield that is twice as great as the DNA corresponding to the second target area group.

[0156] "Capturing" one or more target nucleic acids means preferentially isolating or separating the one or more target nucleic acids from non-target nucleic acids.

[0157] A "captured set" of nucleic acids refers to nucleic acids that have undergone capture.

[0158] "Target-region set" or "set of target regions" refers to more than one genomic loci that are targeted for capture and / or targeted by a probe set (eg, by sequence complementarity).

[0159] "Corresponding to a target region set" means that a nucleic acid, such as cfDNA, is derived from a locus in the target region set or specifically binds to one or more probes for the target region set.

[0160] As used herein, "differentially methylated region" (DMR) refers to a region of DNA with a detectable different degree of methylation in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type; or a region of DNA with a detectable different degree of methylation in at least one cell or tissue type obtained from a subject with a disease or disorder relative to the degree of methylation in the same region of DNA in the same cell or tissue type obtained from a healthy subject. In some embodiments, the DMR has a detectable higher degree of methylation (e.g., a hypermethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type of a healthy subject. In some embodiments, the DMR has a detectable lower degree of methylation (e.g., a hypomethylated region) in at least one cell or tissue type relative to the degree of methylation in the same region of DNA from at least one other cell or tissue type or from the same cell or tissue type of a healthy subject.

[0161] "Specific binding" 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 minimizing the formation of stable probe: non-target hybrids. Thus, the probe hybridizes to a target sequence or a copy thereof to a greater extent than to a non-target sequence to achieve capture or detection of the target sequence. Appropriate hybridization conditions are well known in the art and can be predicted based on sequence composition or can be determined by use of routine testing procedures (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57, in particular §§9.50-9.51, 11.12-11.13, 11.45-11.47 and 11.55-11.57, incorporated herein by reference).

[0162] A "set of sequence variable target regions" refers to a set of target regions that may exhibit sequence changes (such as nucleotide substitutions (ie, single nucleotide variations), insertions, deletions, or gene fusions or transpositions) in neoplastic cells (eg, tumor cells and cancer cells).

[0163] "Epigenetic target group" refers to a target group that may show sequence-independent changes in neoplastic cells (e.g., tumor cells and cancer cells) or in cfDNA from subjects with cancer relative to cfDNA from healthy subjects. Examples of sequence-independent changes include, but are not limited to, changes in methylation levels (increase or decrease), nucleosome distribution, CTCF binding, transcription start sites, and regulatory protein binding regions. For the purposes of the present invention, loci susceptible to focal amplification and / or gene fusion associated with neoplasms, tumors, or cancers may also be included in the epigenetic target group, because detection of changes in copy number by sequencing or detection of fusion sequences mapped to more than one locus in a reference genome tends to be more similar to detection of the exemplary epigenetic changes discussed above than detection of nucleotide substitutions, insertions, or deletions, for example, in the following aspects: Focused amplification and / or gene fusions can be detected with relatively shallow sequencing depths because their detection does not rely on the accuracy of base determinations (calls) at one or a few single positions.

[0164] The term "hypermethylation" refers to an increased level or degree of methylation of a nucleic acid molecule relative to other nucleic acid molecules within a nucleic acid molecule population (e.g., a sample). In some embodiments, hypermethylated DNA can include a DNA molecule comprising at least 1 methylated residue, at least 2 methylated residues, at least 3 methylated residues, at least 5 methylated residues, or at least 10 methylated residues.

[0165] The term "hypomethylation" refers to the methylation level or degree of the reduction of a nucleic acid molecule relative to other nucleic acid molecules in a nucleic acid molecule population (e.g., a sample). In some embodiments, hypomethylated DNA includes unmethylated DNA molecules. In some embodiments, hypomethylated DNA can include DNA molecules containing 0 methylated residues, at most 1 methylated residue, at most 2 methylated residues, at most 3 methylated residues, at most 4 methylated residues, or at most 5 methylated residues.

[0166] As used herein, "methylation state" can refer to the presence or absence of a methyl group on a DNA base (e.g., cytosine) at a specific genomic position in a nucleic acid molecule. It can also refer to the degree of methylation in a nucleic acid sequence (e.g., a highly methylated, hypomethylated, moderately methylated, or unmethylated nucleic acid molecule). Methylation state can also refer to the number of methylated nucleotides in a particular nucleic acid molecule.

[0167] As used herein, "mutation" refers to a variation from a known reference sequence, and includes mutations such as, for example, single nucleotide variations (SNVs) and insertions / deletions. Mutations can be germline mutations or somatic mutations. In some embodiments, the reference sequence for comparison purposes is the wild-type genomic sequence of the species of the subject providing the test sample, typically the human genome.

[0168] As used herein, the terms "neoplasm" and "tumor" are used interchangeably. They refer to abnormal growths of cells in a subject's body. A neoplasm or tumor can be benign, potentially malignant, or malignant. Malignant tumors are called cancer or cancerous tumors.

[0169] As used herein, "next generation sequencing" or "NGS" refers to a sequencing technology with increased throughput compared to traditional Sanger and capillary electrophoresis-based methods, e.g., the ability to generate hundreds of thousands of relatively small sequencing reads at a time. Some examples of next generation sequencing technologies include, but are not limited to, synthetic sequencing, ligation sequencing, and hybridization sequencing. In some embodiments, next generation sequencing includes the use of an instrument capable of sequencing a single molecule. Examples of commercially available instruments for performing next generation sequencing include, but are not limited to, NextSeq, HiSeq, NovaSeq, MiSeq, Ion PGM, and IonGeneStudio S5.

[0170] As used herein, "nucleic acid tag" refers to a short nucleic acid (e.g., less than about 500 nucleotides, about 100 nucleotides, about 50 nucleotides, or about 10 nucleotides in length) used to distinguish nucleic acids from different samples (e.g., presented as a sample index, distinguish nucleic acids from different partitions (e.g., presented as a partition tag), or different nucleic acid molecules of different types or different treatments in the same sample (e.g., presented as a molecular barcode). The nucleic acid tag comprises a predetermined, fixed, non-random, random, or semi-random oligonucleotide sequence. Such a nucleic acid tag can be used to label different nucleic acid molecules or different nucleic acid samples or subsamples. The nucleic acid tag can be single-stranded, double-stranded, or at least partially double-stranded. The nucleic acid tags optionally have the same length or different lengths. The nucleic acid tag also Can include double-stranded molecules with one or more blunt ends, include 5' or 3' single-stranded regions (e.g., overhangs), and / or include one or more other single-stranded regions at other locations within a given molecule. Nucleic acid tags can be attached to one or both ends of other nucleic acids (e.g., sample nucleic acids to be amplified and / or sequenced). Nucleic acid tags can be decoded to reveal information, such as the sample source, form, or treatment of a given nucleic acid. For example, nucleic acid tags can also be used to enable the pooling and / or parallel processing of more than one sample containing nucleic acids with different molecular barcodes and / or sample indexes, where the nucleic acids are subsequently deconvoluted by detecting (e.g., reading) the nucleic acid tags. Nucleic acid tags can also be used to detect (e.g., read) nucleic acid tags. It is called an identifier (e.g., a molecular identifier, a sample identifier). Additionally or alternatively, a nucleic acid tag can be used as a molecular identifier (e.g., to distinguish different molecules or amplicons of different parent molecules in the same sample or subsample). For example, this includes uniquely labeling different nucleic acid molecules in a given sample, or non-uniquely labeling these molecules. In the case of non-unique labeling applications, a limited number of tags (i.e., molecular barcodes) can be used to label each nucleic acid molecule so that different molecules can be identified based on their endogenous sequence information (e.g., the start and / or end position mapped to a selected reference genome, a subsequence at one or both ends of the sequence, and / or the length of the sequence) with at least Typically, a sufficient number of different molecular barcodes are used so that the probability that any two molecules may have the same endogenous sequence information (e.g., start and / or end position, subsequence of one or both ends of the sequence, and / or length) and also have the same molecular barcode is low (e.g., less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%). Terms such as "library adapters with different molecular barcodes" encompass library adapters used to uniquely tag or non-uniquely tag molecules, because different barcodes will be present in the adapter population regardless of whether the adapter is used for unique tagging or non-unique tagging.

[0171] As used herein, "unfixed" or "free in solution" DNA refers to DNA that is not covalently or non-covalently bound to a solid support (such as a bead). During any step (such as all steps) of the disclosed methods, such DNA may be free in solution.

[0172] As used herein, "partitioning" refers to physically separating or grading a mixture of nucleic acid molecules in a sample based on the characteristics of nucleic acid molecules. Partitioning can be a physical partitioning of molecules. Partitioning can include dividing nucleic acid molecules into groups (groups) or sets based on epigenetic features (e.g., methylation) levels. For example, nucleic acid molecules can be partitioned based on the methylation levels of nucleic acid molecules. In some embodiments, methods and systems for partitioning are found in PCT patent application No. PCT / US2017 / 068329, which is incorporated herein by reference in its entirety.

[0173] As used herein, "partitioned set" or "partition" refers to a nucleic acid molecule group that is partitioned into a set or group based on the different binding affinities of a nucleic acid molecule or a protein associated with a nucleic acid molecule and a binding agent. A partition group may also be referred to as a subsample. A binding agent preferentially binds to a nucleic acid molecule comprising a nucleotide with an epigenetic modification. For example, if the epigenetic modification is methylation, the binding agent may be a methyl binding domain (MBD) protein. In some embodiments, a partition group may include nucleic acid molecules belonging to a specific epigenetic feature (e.g., methylation) level or degree. For example, nucleic acid molecules may be partitioned into three groups--one group is a highly methylated nucleic acid molecule (a first subsample, a high partition, a high partition group, or a high methylation partition group), a second group is a low methylated nucleic acid molecule (a second subsample, a low partition, a low partition group, or a low methylation partition group), and a third group is a medium methylated nucleic acid molecule (a third subsample, a medium partition group, a medium methylation partition group, a residual partition group, or a residual partition). In another example, nucleic acid molecules can be partitioned based on the number of methylated nucleotides - one partition group can have nucleic acid molecules with 9 methylated nucleotides and another partition group can have unmethylated nucleic acid molecules (zero methylated nucleotides).

[0174] As used herein, "polynucleotide", "nucleic acid", "nucleic acid molecule" or "oligonucleotide" refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides or their analogs) connected by internucleoside bonds. Typically, a polynucleotide contains at least three nucleosides. The size of an oligonucleotide generally ranges from a few monomer units, for example, 3-4 to several hundred monomer units. When a polynucleotide is represented by a letter sequence, such as "ATGCCTG", the nucleotides are presented from left to right in a 5'→3' order, and in the case of DNA, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents deoxythymidine, unless otherwise indicated. The letters A, C, G and T can be used to refer to a base itself, a nucleoside, or a nucleotide comprising a base.

[0175] As used herein, "processing" refers to a set of steps for generating a nucleic acid library suitable for sequencing. This set of steps may include, but is not limited to, partitioning of nucleic acids, end repair, addition of sequencing adapters, tagging, and / or PCR amplification.

[0176] As used herein, "quantitative measure" refers to an absolute measure or a relative measure. A quantitative measure can be, but is not limited to, a number, a statistical measure (e.g., frequency, mean, median, standard deviation, or quantile) or a degree or relative amount (e.g., high, medium, and low). A quantitative measure can be the ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative metric can be a normalized metric.

[0177] As used herein, "reference sequence" refers to a known sequence for the purpose of comparison with an experimentally determined sequence. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. A reference sequence can be aligned to a single continuous sequence of a genome or chromosome or chromosome arm, or can include non-continuous segments aligned to different regions of a genome or chromosome. Examples of reference sequences include, for example, human genomes, such as, hg19 and hg38.

[0178] As used herein, "sample" means anything that is capable of being analyzed by the methods and / or systems disclosed herein.

[0179] As used herein, "sequencing" refers to any of a number of techniques used to determine the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, a nucleic acid such as DNA or RNA. Examples of sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturing temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired-end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as, for example, by a genetic analyzer commercially available from Illumina, Inc, Pacific Biosciences, Inc, or Applied Biosystems / Thermo Fisher Scientific, among others.

[0180] As used herein, "sequence information" in the context of a nucleic acid polymer refers to the order and identity of the monomeric units (eg, nucleotides, etc.) in the polymer.

[0181] As used herein, a "sequence variable target region set" refers to a set of target regions that may exhibit sequence changes (such as nucleotide substitutions, insertions, deletions, or gene fusions or transpositions) in neoplastic cells (eg, tumor cells and cancer cells).

[0182] As used herein, the terms "somatic mutation" or "somatic variation" are used interchangeably. They refer to mutations in the genome that occur after conception. Somatic mutations can occur in any cell of the body except germ cells and are therefore not passed on to future generations.

[0183] As used herein, "subject" refers to an animal, such as a mammalian species (e.g., human), or a poultry (e.g., bird) species, or other organisms, such as a plant. More specifically, a subject can be a vertebrate, such as a mammal such as a mouse, a primate, an ape or a human. Animals include farm animals (e.g., production cattle, dairy cows, poultry, horses, pigs, etc.), sports animals and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual with or suspected of having a disease or a tendency to suffer from the disease, or an individual who needs treatment or suspects that treatment is needed. The term "individual" or "patient" is intended to be interchangeable with "subject". For example, a subject can be an individual who has been diagnosed with cancer, will receive cancer treatment and / or has received at least one cancer treatment. A subject can be in the remission of cancer. As another example, a subject can be an individual diagnosed with an autoimmune disease. As another example, a subject can be a female individual who is pregnant or planning to be pregnant, who may have been diagnosed or suspected of having a disease, such as cancer, an autoimmune disease.

[0184] As used herein, "target-region set" or "set of target regions" or "target region" or "target region of interest" or "genomic region of interest" refers to more than one genomic locus or more than one genomic region that is targeted for capture and / or targeted by a probe set (e.g., by sequence complementarity).

[0185] As used herein, "tumor fraction" refers to the proportion of cfDNA molecules that originate from tumor cells for a given sample or sample-region pair.

[0186] As used herein, an "asymmetric adapter" is a double-stranded adapter in which the two strands are not completely complementary or otherwise distinguishable, such that synthesis of the complementary sequence of one strand of the adapter produces a sequence that is distinguishable from the sequence of the other strand of the adapter. Examples of asymmetric adapters are Y-shaped adapters and bubble adapters.

[0187] As used herein, "Y-shaped adapter" refers to an adapter comprising two DNA chains, wherein the two DNA chains comprise complementary and non-complementary portions, wherein the non-complementary portions form single-stranded arms. The adapter can, for example, be attached to a sample or inserted into a DNA molecule by connection so that the complementary (double-stranded) portion of the adapter is close to the sample or inserted into the DNA molecule. Prior to attachment, the double-stranded portion of the Y-shaped adapter can have, for example, a flat end or overhang of one to three nucleotides. The single-stranded arms may or may not have the same length.

[0188] As used herein, "bubble adapter" refers to an adapter comprising a non-complementary portion flanked by complementary portions, such that the adapter has two DNA chains with a single-stranded region between the double-stranded regions. The adapter can be attached to a sample or insert DNA molecule, for example, by connection, so that one of the complementary (double-stranded) portions of the adapter is close to the sample or insert DNA molecule. Prior to attachment, the double-stranded portion of the bubble adapter to be attached to the insert or sample molecule can have, for example, a flat end or overhang of one to three nucleotides. The single-stranded portions of the two chains may or may not have the same length.

[0189] As used herein, the terms "or a combination thereof" and "or combinations thereof" refer to any and all permutations and combinations of the terms listed preceding that term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if order is important in the particular context, BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0190] "Or" is used in an inclusive sense, ie, equivalent to "and / or", unless the context requires otherwise.

[0191] Samples and subjects

[0192] The present disclosure relates to a method for distinguishing artificial information from real information in a workflow involving sequencing of a DNA sample. In some cases, the DNA sample is obtained from a subject or has been obtained from a subject. In some embodiments, the DNA sample may include or consist of DNA from a biological sample obtained from a subject. The subject may be a human, mammal, animal, primate, rodent (including mice and rats) or other common laboratory, domestic, companion, service or agricultural animal, such as a rabbit, dog, cat, horse, cow, sheep, goat or pig. Preferably, the DNA sample is from a human. In some cases, the subject may suffer from or be suspected of having cancer, a tumor or a neoplasm. In other cases, the subject may not suffer from cancer or have detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, for example, any one or more of chemotherapy, antibodies, vaccines or biologics. The subject may be in remission from, for example, a tumor, cancer or neoplasia (for example, after treatment such as chemotherapy, surgical resection, radiation or a combination thereof). The subject may be diagnosed or may not be diagnosed as susceptible to cancer or any cancer-related genetic mutation / disorder. In some embodiments, the sample is a DNA sample obtained from a tumor tissue biopsy.Cancer, tumor or neoplasm can generally be any type, such as a cancer tumor or neoplasm of the lung, colon, rectum (or colorectal), kidney, breast, prostate or liver or other types of cancer as described herein.In some embodiments, the sample is obtained from a subject who is in remission (for example, after chemotherapy, surgical resection, radiation or a combination thereof) from a tumor, cancer or neoplasm.In any of the foregoing embodiments, precancer, cancer, tumor or neoplasm or suspected precancer, cancer, tumor or neoplasm can be bladder, head and neck, lung, colon, rectum, kidney, breast, prostate, skin or liver.In some embodiments, precancer, cancer, tumor or neoplasm or suspected precancer, cancer, tumor or neoplasm is lung.In some embodiments, precancer, cancer, tumor or neoplasm or suspected precancer, cancer, tumor or neoplasm is colon or rectum.In some embodiments, precancer, cancer, tumor or neoplasm or suspected precancer, cancer, tumor or neoplasm is breast. In some embodiments, the precancerous lesion, cancer, tumor or neoplasm or suspected precancerous lesion, cancer, tumor or neoplasm is prostatic. In any of the aforementioned embodiments, the subject can be a human subject. In some embodiments, the sample is obtained from a subject suffering from Phase I cancer, Phase II cancer, Phase III cancer or Phase IV cancer.

[0193] In some embodiments, the subject may suffer from infection, transplant rejection, or other diseases or disorders associated with changes in the immune system. The subject may be free of cancer or detectable symptoms of cancer. The subject may have been treated with one or more cancer therapies, for example, any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may or may not be diagnosed as susceptible to cancer or any cancer-related genetic mutation / disorder.

[0194] Biological sample can be any biological sample separated from a subject. Biological sample can include body tissue, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, feces, red blood cells, white blood cells or leukocytes, endothelial cells, tissue biopsy, cerebrospinal fluid, synovial fluid, lymphatic fluid, ascites, interstitial fluid or extracellular fluid, fluid in the gap between cells (including gingival sulcus fluid), bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine. In some embodiments, biological sample is body fluid, particularly blood and its components, or urine. Sample can be the form initially separated from the subject, or can be further processed to remove or add components, such as cells, or enrich a component relative to other components. Sample can be separated or obtained from a subject, and transported to a sample analysis site. Sample can be stored and transported at a desired temperature, such as room temperature, 4 ° C, -20 ° C and / or -80 ° C. Sample can be separated or obtained from a subject at a sample analysis site.

[0195] In a preferred embodiment, the DNA sample comprises cell-free DNA. In another preferred embodiment, the DNA sample is a DNA sample from a formalin-fixed paraffin-embedded (FFPE) sample.

[0196] In some embodiments, nucleic acid colonies are obtained from serum, plasma or blood samples of subjects suspected of suffering from vegetation, tumor, precancerous lesion or cancer or previously diagnosed as suffering from vegetation, tumor, precancerous lesion or cancer. Colonies include nucleic acids with different levels of sequence variation, epigenetic variation and / or replication or transcription modification. Replication and modification include modification of cytosine, particularly at the 5-position of nucleobase, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine.

[0197] In some embodiments, the DNA sample is from plasma. The volume of plasma used to obtain the DNA sample can depend on the required reading depth of the sequencing region. Example volumes are 0.4-40ml, 5-20ml, 10-20ml. For example, the volume can be 0.5mL, 1mL, 5mL, 10mL, 20mL, 30mL or 40mL. The volume of the plasma sampled can be 5ml to 20ml.

[0198] The sample may contain various amounts of DNA containing genome equivalents. For example, a sample of about 30 ng of DNA may contain about 10,000 (10 4 ) haploid human genome equivalents, and in the case of cell-free DNA (cfDNA), can contain approximately 200 billion (2×10 11 ) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA may contain about 30,000 haploid human genome equivalents, and in the case of cfDNA, about 600 billion individual molecules.

[0199] The sample may include nucleic acids from different sources, for example, nucleic acids and cell-free nucleic acids from the same subject, and nucleic acids from cells and cell-free nucleic acids from different subjects. In some embodiments, nucleic acids may be DNA. The sample may include DNA carrying mutations. For example, the sample may include DNA carrying germline mutations and / or somatic mutations. Germline mutations refer to mutations present in the germline DNA of a subject. Somatic mutations refer to mutations of somatic cells derived from a subject, for example, cancer cells. The sample may include DNA carrying cancer-associated mutations (e.g., cancer-associated somatic mutations). The sample may include epigenetic variations, wherein the epigenetic variations are associated with the presence of genetic variations (such as cancer-associated mutations). In some embodiments, the sample includes epigenetic variations associated with the presence of genetic variations, wherein the sample does not include the genetic variations.

[0200] The DNA sample can be or include cell-free nucleic acid or cfDNA. cfDNA can be obtained from a test subject, such as described above. For example, the sample for analysis can be plasma or serum containing cell-free nucleic acid. "Cell-free DNA", "cfDNA molecules" or "cfDNA", for example, include DNA molecules naturally present in an extracellular form (e.g., in blood, serum, plasma or other body fluids such as lymph, cerebrospinal fluid, urine or sputum) in a subject. Although cfDNA is initially present in one or more cells in a large complex biological organism (e.g., mammal), cfDNA has undergone release from in vitro cells into a fluid present in an organism, and can be obtained by obtaining a sample of a fluid without the need for an in vitro cell lysis step. In other words, cell-free nucleic acid or cfDNA is a nucleic acid or DNA that is not contained in a cell or otherwise combined with a cell, or a nucleic acid or DNA that is retained in a sample after removing intact cells. Cell-free nucleic acids include DNA, RNA and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA) and / 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 body fluids by secretion or cell death programs such as cell necrosis and apoptosis. Some cell-free nucleic acids are released into body fluids from cancer cells, such as circulating tumor DNA (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA). In some embodiments, cell-free nucleic acids are produced by tumor cells. In some embodiments, cell-free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.

[0201] Exemplary amounts of cell-free nucleic acid (e.g., cfDNA) in a sample prior to amplification range from about 1 fg to about 1 μg, such as 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 1 femtogram (fg) to 200 ng of cell-free nucleic acid molecules from a sample.

[0202] The cell-free nucleic acids have an example size distribution of about 100-500 nucleotides, with molecules of 110 to about 230 nucleotides representing about 90% of the molecules, with a mode of about 168 nucleotides, and a second minor peak ranging between 240 and 440 nucleotides.

[0203] Cell-free nucleic acid can be separated from body fluid by a classification or partitioning step, in which the cell-free nucleic acid present in the solution is separated from intact cells and other insoluble components in the body fluid. Partitioning can include techniques such as centrifugation or filtration. Alternatively, cells in the body fluid can be lysed, and the cell-free nucleic acid and cellular nucleic acid are processed together. Typically, after adding a buffer and washing steps, the nucleic acid can be precipitated with alcohol. Further cleaning steps such as silica-based columns can be used to remove contaminants or salts. Non-specific bulk carrier nucleic acids, DNA or proteins for sequencing (e.g., bisulfite sequencing), hybridization and / or connection can be added throughout the reaction to optimize certain aspects of the procedure, such as yield.

[0204] After such treatment, the sample may include various forms of nucleic acids, including double-stranded DNA, single-stranded DNA, and single-stranded RNA. In some embodiments, single-stranded DNA and RNA may be converted to double-stranded forms so they are included in subsequent processing and analysis steps.

[0205] Method disclosed herein is also particularly useful for analyzing DNA from paraffin-embedded (FFPE) tissue samples fixed by formalin. Although the formalin fixation process fully preserves the ultrastructure of tissue, it causes various types of damage to DNA in the tissue, such as the nicks in the DNA. As explained elsewhere herein, these nicks may lead to the synthesis of the region of the DNA molecule in the end repair process. Method disclosed herein allows identification of these regions and correspondingly explains sequence data.

[0206] End repair and A-tailing

[0207] End repair refers to a method of repairing DNA by converting non-blunt-ended DNA into blunt-ended DNA. Sequencing workflows often use end repair to make the ends of DNA molecules compatible with adapters, which are then ligated to the DNA. Fragmented and / or damaged DNA (e.g., cfDNA or DNA from FFPE samples) often contains non-blunt ends that contain 3' overhangs and / or 5' overhangs. A 3' overhang refers to a DNA strand where the 3' end extends beyond the 5' end of the paired strand, resulting in one or more unpaired nucleotides at the 3' end of the DNA strand. Conversely, a 5' overhang refers to a DNA strand where the 5' end extends beyond the 3' end of the paired strand, resulting in one or more unpaired nucleotides at the 5' end of the DNA strand.

[0208] The process of end repair involves converting the double-stranded DNA with 3' overhang and / or 5' overhang into a double-stranded DNA without overhang. This can be accomplished using enzymes such as T4 DNA polymerase and / or Klenow fragment. The 3' to 5' exonuclease activity of these enzymes removes the 3' end at the 3' overhang, and the 5' to 3' polymerase activity of these enzymes extends the 3' end at the 5' overhang to remove the 5' overhang, thereby producing a flat-end DNA molecule. In order to fill these 5' overhangs, end repair is performed in the presence of dATP, dCTP, dGTP and dTTP. End repair can also include a second step, which involves adding a phosphate group to the 5' end of DNA by an enzyme such as a polynucleotide kinase. This makes the 5' end of the DNA molecule of the end repair compatible with the subsequent action of DNA polymerase and DNA ligase.

[0209] As used herein, the term "A-tailing" refers to adding a single deoxyadenosine residue to the end of a flat-ended double-stranded DNA fragment to form a 3' deoxyadenosine single base overhang. Such an A-tailing reaction is performed with a polymerase that has the ability to add a non-templated A to the 3' end of a flat double-stranded DNA molecule. A polymerase capable of A-tailing generally does not have 3'-5' exonuclease activity. When A-tailing is performed as a separate reaction for end repair, it is generally performed in the presence of dATP but not in the presence of dCTP, dTTP, and dGTP. A-tail fragments are incompatible with self-ligation (i.e., self-cyclization and connection of DNA), but they are compatible with 3' T-overhangs that can be used on adapters. Compared with flat-ended connections, methods including end repair, A-tailing, and connection to adapters with 3' T-overhangs can result in more efficient connections because flat-ended connections can result in self-ligation of adapters and / or DNA molecules.

[0210] In some cases, the methods disclosed herein include end repair of DNA molecules followed by blunt end ligation of adaptors. In other cases, the methods disclosed herein include end repair of DNA molecules followed by A-tailing of T-tailed adaptors and sticky end ligation. When the methods disclosed herein include an A-tailing step, it can be performed separately from the end repair with an intermediate reaction cleanup step, or it can be performed in the same reaction as the end repair (e.g., using overtake TM II End Repair / dA-tailing Module (E7546). In cases where the A-tailing reaction is performed in the same reaction as the end repair, sticky end ligation can be performed with a mixture of T-tailed adapters and C-tailed adapters.

[0211] The inventors have studied the effects of the end repair and A-tailing steps on DNA molecules and their effects on the corresponding sequence data. Figure 1-Figure 10 As shown in (discussed in detail below), end repair and A-tailing reactions can have different effects on the composition of DNA molecules, depending on the exact workflow and reaction components used. These reactions can result in the synthesis of regions at the 3' end of the DNA strand, but can also synthesize internal regions by nick translation and by gap filling followed by ligation. The impact of this synthesis is discussed in the context of its effect on cytosine methylation. Figure 1-Figure 10 Shown in.

[0212] Figure 1The schematic diagram of the influence of the combined end repair and A-tailing step on complete double-stranded DNA, nicked double-stranded DNA and gapped double-stranded DNA is shown. In all types of DNA molecules shown, end repair can lead to 3' filling of unmethylated cytosine, which may not reflect the true methylation state of this position in the DNA molecule before producing 5' overhang. Inferring the methylation state from the DNA molecule of this end repair may lead to incorrect methylation data at this position. In the DNA with nicks, the polymerase containing 5' to 3' exonuclease activity and / or strand displacement activity can lead to the internal region of the synthetic DNA molecule by nick translation. If the end repair reaction is carried out with non-methylated deoxycytidine triphosphate (dCTP), the synthetic region will potentially incorporate non-methylated dCTP at the position that originally contained methylated cytosine. Therefore, using the sequence data from these synthetic regions to infer the methylation state of the original DNA molecule may lead to inaccurate data. In the DNA with nicks, the DNA polymerase used in the end repair and the DNA polymerase used in the A-tailing can both lead to the generation of the synthetic region. The gap can be filled with the DNA polymerase used in the end repair reaction, whether they have 5' to 3' exonuclease activity or strand displacement activity. After the gap is filled, there will still be a nick between the synthetic region and the original DNA molecule region that is positioned at the 3' of the gap. Then, the A-tailing enzyme can be introduced into the further synthetic region by nick translation, as described for the DNA with nicks. The synthetic region can extend to the 3' end of DNA molecule. As previously mentioned, using these synthetic regions to infer the methylation state of the original DNA molecule may cause incorrect results.

[0213] In some embodiments, the end repair and A-tailing reactions are carried out in a single tube. In such a case, the A-tailing reaction can be carried out at a higher temperature than the end repair. Optionally, the end repair is carried out at ambient temperature (e.g., 15°C-35°C), and the A-tailing is carried out at a temperature exceeding 60°C. A-tailing reactions can be carried out using a thermostable polymerase (e.g., Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, large fragment, or Tth DNA polymerase), and the method also includes raising the temperature of the sample after the end repair to inactivate the polymerase (e.g., T4 DNA polymerase or Klenow fragment) used in the end repair. In some embodiments, A-tailing is carried out using the following DNA polymerases: (i) not having 5'-3' exonuclease activity; and / or (ii) not being a strand displacement DNA polymerase. These characteristics reduce the ability of DNA polymerases to extend from the nick. This reduces the level of synthesis that may occur during the end repair and A-tailing reactions, thereby reducing the proportion of sequencing data that may be filtered out that potentially contains artificial data. Therefore, in some embodiments, A-tailing is performed using a DNA polymerase that cannot extend from a nick in the DNA (such as HemoKlen Taq). In other embodiments, A-tailing is performed using Taq DNA polymerase. In other embodiments, A-tailing is performed using Tfl polymerase, Bst DNA polymerase, large fragment, or Tth polymerase.

[0214] Figure 2 A schematic diagram showing the potential effects of separate end repair and A-tailing steps on intact double-stranded DNA, nicked double-stranded DNA, and gapped double-stranded DNA. To reduce the level of synthesized regions, a DNA polymerase lacking 5' to 3' exonuclease activity and / or strand displacement activity (e.g., T4 DNA polymerase or Klenow fragment) can be used for the end repair reaction. This approach means that nick translation is reduced, allowing a greater portion of sequencing data to be used in the disclosed methylation analysis method, as less sequencing data will come from synthesized regions.

[0215] exist Figure 2In all types of DNA molecules shown in, end repair can lead to 3' filling of unmethylated cytosine, which may not reflect the true methylation state of the position in the DNA molecule before the 5' overhang is generated. Inferring the methylation state from the DNA molecule of the end repair may cause the data of the position to be incorrect. In the DNA with nicks and gapped DNA, by using a polymerase lacking 5' to 3' exonuclease activity and / or strand displacement activity, nick translation is reduced in the end repair. Separating the end repair and A-tailing reaction by reaction cleaning means that only dATP (not dCTP, dTTP or dGTP) is present in the A-tailing reaction. This means that effective nick translation cannot occur in the A-tailing reaction because three of the four nucleotide components are not present in the reaction mixture. In the DNA with gaps, the gap can be filled with the DNA polymerase used in the end repair reaction, regardless of whether they have 5' to 3' exonuclease activity or strand displacement activity. These filled gaps thus produce synthetic regions. As previously mentioned, using these synthetic regions to infer the methylation state of the original DNA molecule may lead to incorrect results. However, the extent of the synthetic area in such a workflow is relatively small compared to Figure 1 The workflow shown in Figure 1 is greatly reduced as the end repair and A-tailing reactions are performed in a single reaction.

[0216] Thus, in preferred embodiments, the methods disclosed herein use a polymerase that lacks 5' to 3' exonuclease activity and / or strand displacement activity to perform end repair. In some cases, the polymerase used in the end repair reaction can be High-fidelity DNA polymerase, Hot start high fidelity DNA polymerase, High-fidelity DNA polymerase, Hemo KlenTaq, Phi29 DNA polymerase, T7 DNA polymerase, DNA polymerase I (E. coli), DNA polymerase I Large (Klenow) fragment ("Klenow fragment"), or T4 DNA polymerase. In some embodiments, the polymerase used in end repair is T4 DNA polymerase or Klenow fragment.

[0217] In some embodiments, the methods disclosed herein include an A-tailing reaction after the end repair and before the ligation reaction, wherein the end repair and A-tailing reactions are separated by a reaction cleanup. The A-tailing reaction is typically performed in the presence of dATP but in the absence of dCTP, dTTP, and dGTP. In some embodiments, the A-tailing reaction is performed using a Klenow fragment lacking 3'-5' exonuclease activity.

[0218] Figure 3A schematic diagram showing the potential effects of end repair and blunt end ligation on intact double-stranded DNA, nicked double-stranded DNA, and gapped double-stranded DNA. To reduce the level of synthesized regions, a DNA polymerase lacking 5' to 3' exonuclease activity and / or strand displacement activity (e.g., T4 DNA polymerase or Klenow fragment) can be used for end repair reactions. This approach means that nick translation is reduced, allowing a greater portion of sequencing data to be used in the disclosed methylation analysis method, as less sequencing data will come from synthesized regions.

[0219] exist Figure 3 In all types of DNA molecules shown in , end repair can result in 3' filling of unmethylated cytosines, which may not reflect the true methylation state of that position in the DNA molecule before the 5' overhang was generated. Inferring the methylation state from this end-repaired DNA molecule may result in incorrect data for that position. In nicked DNA and gapped DNA, nick translation is reduced in end repair by using polymerases that lack 5' to 3' exonuclease activity and / or strand displacement activity. In gapped DNA, the gaps can be filled with DNA polymerases used in the end repair reaction, regardless of whether they have 5' to 3' exonuclease activity or strand displacement activity. These filled gaps thereby generate synthetic regions. As previously described, using these synthetic regions to infer the methylation state of the original DNA molecule may result in incorrect results. However, the extent of the regions synthesized in such a workflow is relative to Figure 1 The workflow shown in Figure 1 is greatly reduced as the end repair and A-tailing reactions are performed in a single reaction.

[0220] Figure 4 yes Figure 1 The schematic diagram shown in is repeated, but a dNTP containing a modified base is used in the combined end repair and A-tailing reaction. In this embodiment, the modified base is a methylated cytosine (5mC). The results show that the methylated cytosine is incorporated into the synthesized region at both the CpG site and the CpH site (i.e., CpA, CpC, and CpT sites). Although methylation of cytosine in a non-CpG environment has been described, it is believed that it accounts for 0.02% of the total methylcytosine in differentiated somatic cells (Jang et al. Genes (Basel). 2017Jun; 8(6): 148). Therefore, the presence of methylated cytosine in a non-CpG environment in the end-repaired DNA can be explained as being introduced during the end repair and / or A-tailing reaction. Therefore, the use of dNTPs containing modified bases can be used to effectively mark the region of synthesis of the end-repaired DNA.

[0221] Figure 5 yes Figure 2, but using dNTPs containing a modified base in the end repair, in this example, a methylated cytosine (5mC). Figure 4 As shown, it shows that methylated cytosine is incorporated into the synthetic region at both CpG sites and CpH sites (i.e., CpA, CpC, and CpT sites). Therefore, the use of dNTPs containing modified bases can be used to effectively label the synthetic region of end-repaired DNA.

[0222] Figure 6 yes Figure 3 , but using dNTPs containing a modified base in the end repair, in this example, a methylated cytosine (5mC). Figure 4 As shown, it shows that methylated cytosine is incorporated into the synthetic region at both CpG sites and CpH sites (i.e., CpA, CpC, and CpT sites). Therefore, the use of dNTPs containing modified bases can be used to effectively label the synthetic region of end-repaired DNA.

[0223] The dNTPs comprising modified bases can comprise any modified bases, wherein the presence or absence of the modification can be detected by a type of modification-sensitive sequencing. The modified bases can be 4-methylcytosine (4mC), 5-methylcytosine (5mC), 5-hydroxymethyl-cytosine (5hmC), N6-methyladenosine (6mA), bromodeoxyuridine (BrdU), 5-fluorodeoxyuridine (FldU), 5-iododeoxyuridine (IdU), 5-ethynyldeoxyuridine (EdU) and / or 8-oxoguanine (8oxoG).

[0224] When using dNTPs containing modified bases, they can be used in the end repair reaction instead of the equivalent unmodified base. For example, if a dCTP containing 5mC is used in the end repair reaction, there may be no dCTP containing unmodified cytosine. This will ensure that the dCTP incorporated into the DNA molecule during the end repair reaction contains 5mC. In some embodiments, multiple types of dNTPs containing modified bases are used in the end repair. For example, dATP containing 6mA and dCTP containing 5mC can be used in the end repair reaction to replace dATP containing unmodified adenine and dCTP containing unmodified cytosine. It is advantageous to use multiple types of dNTPs containing modified bases because it provides increased resolution in defining the region that has been synthesized during the end repair reaction in the DNA molecule of the end repair. This is because, in this example, the end of the synthesized region can be defined as the first unmodified adenine or unmodified cytosine following a stretch containing 6mAs and / or 5mCs, rather than relying on the detection of only unmodified adenines or only unmodified cytosines.

[0225] The modification-sensitive sequencing method used will depend on the type of modified base used in the end repair reaction, so that a specific modification can be detected. Exemplary conversion-based methods and the base modifications they can detect are described above. In addition, nanopore-based sequencing can be used to detect 4mC, 5mC, 5hmC, 6mA, BrdU, FdU, IdU and EdU, and single molecule real-time (SMRT) sequencing from Pacific Biosciences can be used to detect 4mC, 5mC, 5hmC, 6mA and 8oxoG.

[0226] Ligation with adapters

[0227] Once the DNA has been end-repaired, the DNA can be subjected to blunt end ligation with a blunt-ended adapter without A-tailing, or, when tailing is performed, the DNA can be subjected to sticky end ligation with a T-tailed adapter. The DNA molecule can be connected to an adapter at one or both ends. The DNA molecule can be connected to an at least partially double-stranded adapter (e.g., a Y-shaped or bell-shaped adapter). In embodiments where modification-sensitive sequencing includes a conversion procedure, the ligation step can be performed before or after the conversion step. In some embodiments, the ligation step is performed after the conversion step.

[0228] DNA ligase and adapter are added to connect the DNA molecules in the sample with the adapters on one or both ends, i.e., to form an adapted DNA. As used herein, "adapter" refers to a short nucleic acid (e.g., a length of less than about 500, less than about 100, or less than about 50 nucleotides, or 20-30, 20-40, 30-50, 30-60, 40-60, 40-70, 50-60, 50-70, 20-500, or 30-100 bases from end to end), which is usually at least partially double-stranded and can be connected to the end of a given sample DNA molecule. In some cases, two adapters can be connected to a single sample DNA molecule, wherein each end of the sample nucleic acid molecule is connected to an adapter.

[0229] In some embodiments, the ligase used in the ligation reaction can act on both single-stranded DNA nicks and double-stranded DNA ends. In some cases, the ligase is T4 DNA ligase or T3 DNA ligase. The adapter can include a primer binding site that allows amplification of the sample DNA molecule that is an adapter at both end flanks, and / or a sequencing primer binding site, including a primer binding site for sequencing applications such as various next-generation sequencing (NGS) applications. The adapter can include a sequence for hybridization with a solid support, for example, a flow cell sequence. The adapter can also include a binding site for a capture probe, such as an oligonucleotide attached to a flow cell support, etc. The adapter can also include a sample index and / or a molecular barcode. These are usually positioned relative to amplification primers and sequencing primer binding sites so that sample index and / or molecular barcode are included in the amplicon and sequencing reads of a given DNA molecule. The adapter of the same or different sequence can be connected to the corresponding end of the sample DNA molecule. In some cases, the adapter of the same or different sequence is connected to the corresponding end of the DNA molecule, except that the sequence of the sample index and / or molecular barcode is different. In some embodiments, the adapter is a Y-shaped adapter, one of which is a flat end or tailed as described herein, so as to be connected to a nucleic acid molecule, which is also a flat end or tailed with one or more complementary nucleotides in the tail of the adapter. In another exemplary embodiment, the adapter is a bell-shaped adapter, comprising a flat end or tailed end to be connected to a DNA molecule to be analyzed. Other exemplary adapters include T-tailed, C-tailed or hairpin-shaped adapters. For example, a hairpin-shaped adapter can include a complementary double-stranded portion and a loop portion, wherein the double-stranded portion can be attached (e.g., connected) to a double-stranded polynucleotide. A hairpin-shaped sequencing adapter can be attached to the two ends of a polynucleotide fragment to produce a circular molecule, which can be sequenced multiple times. The adapter used in the method of the present disclosure includes one or more known modified nucleosides, such as methylated nucleosides. In the case where two adapters are connected to a sample nucleic acid (one at each end), any one or both of the adapters can include one or more known modified nucleosides. Typically, primer binding sites, sequencing primer binding sites, sample indexes, and / or molecular barcodes (if present) do not contain modified nucleosides known to alter base pairing specificity as a result of the conversion procedure.

[0230] In some embodiments, adapters can be added to DNA or its sub-sample. Adapters can be connected to the DNA at any point in the method herein. In some embodiments, before primers are annealed to DNA to produce capture probes, adapters are connected to the DNA of sample or its sub-sample. In some such embodiments, the DNA to which adapters are connected is amplified before primers are annealed to DNA to produce capture probes. In some embodiments, before DNA contacts with capture probes, adapters are connected to the DNA of sample or its sub-sample. In some embodiments, the DNA connected to the adapters is in the same sample or sub-sample as a template to produce capture probes. In some embodiments, compared with the DNA used as a template to generate capture probes, the DNA connected to the adapters is in different samples or sub-samples (e.g., the second sample or the second sub-sample of the first sample). In some embodiments, adapters are connected to the DNA captured by the capture probe.

[0231] In some embodiments, the primer used to generate the capture probe is not complementary to the adapter, and thus the resulting capture probe does not contain an adapter. Therefore, the DNA to which the adapter is attached can be selectively amplified in the presence of a capture probe that does not contain an adapter. Similarly, the DNA to which the adapter is attached can be separated from the DNA that does not contain an adapter.

[0232] In some embodiments, the disclosed method includes analyzing the DNA in the sample. In such a method, an adapter can be added to the DNA. This can be performed simultaneously with the amplification procedure, for example, by providing an adapter in the 5' part of the primer before or after the amplification step (in the case of using PCR, this can be referred to as library preparation PCR or LP-PCR). In some embodiments, the adapter is added by other methods such as connection. In some such methods, the first adapter is added to the 3' end of the nucleic acid by connection, and the connection may include connection with a single-stranded DNA. In some embodiments, before any partition or capture step, the first adapter is added to the nucleic acid by connection, and the connection may include connection to a single-stranded DNA (for example, connected to its 3' end). In some embodiments, the capture probe can be separated after partitioning and connection. For example, a hypomethylated partition can be connected to an adapter, and a part of the hypomethylated partition connected can be used to generate a capture probe for rearrangement. The adapter can be used as a priming site for the synthesis of the second chain, for example, using a universal primer and DNA polymerase. The second adapter can then be connected to at least the 3' end of the second chain of the now double-stranded molecule. In some embodiments, the first adaptor comprises an affinity tag, such as biotin, and the nucleic acid attached to the first adaptor is bound to a solid support (e.g., a bead), which may comprise 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 preparing sequencing libraries compatible with single-stranded nucleic acids are available, such as those from Swift Biosciences. Methyl-Seq DNA Library Kit. In some embodiments, after adaptor ligation, the nucleic acid is amplified.

[0233] In some embodiments, the adapters comprise a sufficient number of different tags so that the number of tag combinations results in a high probability, such as 95%, 99%, or 99.9%, that two nucleic acids with the same start and end points receive different tag combinations. Adapters with the same or different tags may comprise the same or different primer binding sites, but preferably the adapters comprise the same primer binding sites.

[0234] In some embodiments, after attachment of the adaptor, the nucleic acid is subjected to amplification. Amplification can use, for example, a universal primer that recognizes a primer binding site in the adaptor.

[0235] In some embodiments, after attaching the adapter, the DNA or a subsample or portion of the DNA is partitioned, including contacting the DNA with a reagent that preferentially binds to a nucleic acid carrying an epigenetic modification. The nucleic acid is partitioned into at least two partition subsamples, the difference being the degree of binding of the modified nucleic acid to the agent. For example, if the agent has an affinity for the modified nucleic acid, the modified nucleic acid that is overrepresented (compared to the median representation in the population) is preferentially bound to the agent, while the modified nucleic acid that is not fully represented does not bind the agent or is more easily eluted from the agent. The nucleic acid can then be amplified by a primer that binds to a primer binding site in the adapter. Alternatively, the partition can be performed before the adapter is attached, in which case the adapter can include a differential tag that includes a component that identifies the molecule as appearing in which partition.

[0236] In some embodiments, the nucleic acid is ligated at both ends to Y-shaped adaptors comprising primer binding sites and tags. The molecule is amplified.

[0237] Molecular labeling

[0238] In some embodiments, the DNA molecules of a sample can be tagged with a sample index and / or a molecular barcode (commonly referred to as a "tag").

[0239] A tag can be a molecule, such as a nucleic acid, that contains information indicating the characteristics of the molecule associated with the tag. For example, a DNA molecule can carry a sample tag or sample index (which distinguishes molecules in one sample from molecules in different samples), a partition tag (which distinguishes molecules in one partition from molecules in different partitions), and / or a molecular tag / molecular barcode (which distinguishes different molecules from each other (in both the case of unique tagging and non-unique tagging)).

[0240] The labeling strategy can be divided into a unique labeling strategy and a non-unique labeling strategy. In unique labeling, all or substantially all molecules in the sample carry different labels, so that the reads can be assigned to the original molecules based on the separate label information. The labels used in such methods are sometimes referred to as "unique labels". In non-unique labeling, different molecules in the same sample can carry the same label, so that other information besides the label information is used to assign sequence reads to the original molecules. Such information may include start and end coordinates, coordinates to which the molecules are mapped, separate start or end coordinates, etc. The labels used in such methods are sometimes referred to as "non-unique labels". Therefore, it is not necessary to uniquely label each molecule in the sample. It is sufficient to uniquely label the molecules in the sample that fall into identifiable categories. Therefore, molecules in different identifiable families can carry the same label without losing information about the identity of the labeled molecules.

[0241] In certain embodiments, the label may include a barcode or a combination of more barcodes. As used herein, the term "barcode" refers to a nucleic acid molecule having a specific nucleotide sequence, or to the nucleotide sequence itself, depending on the context. A barcode may have, for example, between 10 and 100 nucleotides. Depending on the needs of a particular purpose, a collection of barcodes may have a degenerate sequence or may have a sequence with a specific Hamming distance. Thus, for example, a molecular barcode may include a combination of one barcode or two barcodes (each barcode being attached to different ends of a molecule). Additionally or alternatively, for different partitions and / or samples, different molecular barcode groups, molecular tag groups, or molecular index groups may be used so that the barcodes are used as molecular tags by their individual sequences and are also used to identify their corresponding partitions and / or samples based on the groups of which they are members.

[0242] Label can be used to mark single polynucleotide population partition, so that label (or more than one label) is associated with specific partition.Alternatively, label can be used in the embodiment of the present disclosure that does not adopt partition step.In some embodiments, single label can be used to mark specific partition.In some embodiments, more than one different label can be used to mark specific partition.In the embodiment that adopts more than one different label to mark specific partition, the label group for marking a partition can be easily distinguished from the label group for marking other partitions.In some embodiments, label can have other functions, such as label can be used to index sample source or be used as unique molecular identifier (it can be used to improve the quality of sequencing data by distinguishing sequencing error from mutation, such as Kinde et al., Proc Nat'zl AcadSci USA 108:9530-9535 (2011), Kou et al., PLoS ONE, 11:e0146638 (2016)) or be used as non-unique molecular identifier, such as described in U.S. Patent No. 9,598,731. Similarly, in some embodiments, the tag can have additional functions, for example, the tag can be used to index the source of the sample or used as a non-unique molecular identifier (which can be used to improve the quality of sequencing data by distinguishing sequencing errors from mutations).

[0243] The label can be incorporated into the adapter or otherwise connected to the adapter by methods such as chemical synthesis, connection (e.g., as described above, such as by blunt end connection or sticky end connection) or overlap extension polymerase chain reaction (PCR). Such an adapter is ultimately connected to the sample DNA molecule. In other embodiments, one or more rounds of amplification cycles (e.g., PCR amplification) can be applied to introduce the sample index into the nucleic acid molecule using conventional nucleic acid amplification methods. Amplification can be carried out in one or more reaction mixtures (e.g., more than one microwell in the array). Molecular barcodes and / or sample indexes can be introduced simultaneously or in any order. In some embodiments, molecular barcodes and / or sample indexes are introduced before and / or after any conversion procedure. In the case of introducing molecular barcodes and / or sample indexes by an amplification process, the conversion step will occur before introducing molecular barcodes and / or sample indexes. In some embodiments, molecular barcodes and / or sample indexes are introduced before and / or after performing a sequence capture step (if any). In some embodiments, only molecular barcodes are introduced before probe capture, and sample indexes are introduced after performing a sequence capture step. In some embodiments, both the molecular barcode and the sample index are introduced before the probe-based capture step (if any) is performed. In some embodiments, the sample index is introduced after the sequence capture step (if any) is performed. In some embodiments, the sample index is incorporated by overlap extension polymerase chain reaction (PCR).

[0244] In some embodiments, the label can be located at one end or both ends of the sample DNA molecule. In some embodiments, the label is an oligonucleotide of a predetermined or random or semi-random sequence. In some embodiments, the length of the label can be less than about 500, 200, 100, 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides. Typically, the length of the label is about 5 to 20 or 6 to 15 nucleotides. The label can be randomly or non-randomly connected to the sample DNA molecule.

[0245] In some embodiments, each sample or partition (discussed below) is uniquely labeled with a sample index or a combination of sample indexes. In some embodiments, each nucleic acid molecule of a sample or subsample is uniquely labeled with a molecular barcode or a combination of molecular barcodes. In other embodiments, more than one barcode may be used so that the molecular barcodes do not have to be unique relative to each other in the more than one barcode (e.g., non-unique molecular barcodes). In these embodiments, molecular barcodes are typically attached (e.g., by connecting as part of an adapter) to individual molecules so that a combination of molecular barcodes and sequences to which they can be attached produces a unique sequence that can be tracked individually. Detection of a non-unique molecular barcode in combination with endogenous sequence information (e.g., corresponding to the start (start) and / or end (stop) genomic loci / positions of the original DNA molecule sequence in the sample, corresponding to the start and end genomic positions of the original DNA molecule sequence in the sample, the start (start) and / or end (stop) genomic loci / positions of sequence reads mapped to a reference sequence, the start and end genomic positions of sequence reads mapped to a reference sequence, a subsequence of the sequence reads at one or both ends, the length of the sequence reads, and / or the length of the original DNA molecule in the sample) generally allows a unique identity to be assigned to a specific molecule. In some embodiments, the start 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 reads aligned to the reference sequence. In some embodiments, the end region includes the last 1, last 2, last 5, last 10, last 15, last 20, last 25, last 30, or at least the last 30 base positions of the 3' end of the sequencing reads aligned to the reference sequence. The length or number of base pairs of the individual sequence reads is also optionally used to assign a unique identity to a given molecule. As described herein, fragments from a single strand of nucleic acid to which a unique identity has been assigned can thereby allow subsequent identification of fragments from a parental strand and / or a complementary strand.

[0246] In certain embodiments of adding non-unique tags, the number of different tags used can be enough to make all DNA molecules of a particular group with different tags 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, for example, randomly attached to the two ends of a molecule, the combination of barcodes can constitute tags together. With regard to this number, it is a function of the number of molecules that fall into the determination. For example, a category can be all molecules mapped to the same start-stop position on a reference genome. A category can be across a specific genetic locus, for example, all molecules mapped to a specific base or a specific region (e.g., up to 100 bases or genes or gene exons).

[0247] In certain embodiments, the number of different tags used to uniquely identify the number of molecules in a class (z) can be between any of 2*z, 3*z, 4*z, 5*z, 6*z, 7*z, 8*z, ​​9*z, 10*z, 11*z, 12*z, 13*z, 14*z, 15*z, 16*z, 17*z, 18*z, 19*z, 20*z, or 100*z (e.g., a lower limit) and any of 100,000*z, 10,000*z, 1000*z, or 100*z (e.g., an upper limit). In some embodiments, molecular barcodes are introduced with a set of identifiers (e.g., a combination of unique or non-unique molecular barcodes) and an expected ratio of molecules in a sample. One example form uses 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 attached to both ends of the target molecule. Alternatively, about 25 to about 1,000,000 different molecular barcode sequences can be used. For example, 20-50×20-50 molecular barcode sequences can be used (i.e., one of 20-50 different molecular barcode sequences can be attached to each end of the target molecule). This number of identifiers is generally sufficient to allow different molecules with the same starting and ending points to have a high probability of receiving different identifier combinations (e.g., at least 94%, 99.5%, 99.99% or 99.999%). In some embodiments, about 80%, about 90%, about 95%, or about 99% of the molecules have the same combination of molecular barcodes. For example, in a sample of about 5 ng to 30 ng of cell-free DNA, about 3000 molecules are expected to map to a particular nucleotide coordinate, and about 3 to 10 molecules with any starting coordinate share the same ending coordinate. Thus, about 50 to about 50,000 different tags (e.g., between about 6 and 220 barcode combinations) can be sufficient to uniquely tag all such molecules. To uniquely tag all 3000 molecules mapped across one nucleotide coordinate, about 1 million to about 20 million different tags would be required.

[0248] In some embodiments, the assignment of unique or non-unique molecular barcodes in the reaction is performed using methods and systems described in, for example, U.S. Patent Application Nos. 20010053519, 20030152490, and 20110160078, and U.S. Patent Nos. 6,582,908, 7,537,898, 9,598,731, and 9,902,992, each of which is incorporated herein by reference in its entirety. Alternatively, in some embodiments, different nucleic acid molecules of a sample can be identified using only endogenous sequence information (e.g., start and / or end position, subsequences at one or both ends of the sequence, and / or length). Tags can be randomly or non-randomly attached to sample nucleic acids.

[0249] In some embodiments, the tagged nucleic acids are sequenced after loading into a microwell plate. The microwell plate can have 96, 384, or 1536 microwells. In some cases, they are introduced at an expected ratio of unique tags to microwells. For example, unique tags can be loaded so that more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags are loaded per genome sample. In some cases, unique tags can 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 sample genome is less than or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 50,000,000, or 1,000,000,000 unique tags per genomic sample.

[0250] In some embodiments, one format uses 20-50 different tags (e.g., barcodes) attached to both ends of the target nucleic acid. For example, 35 different tags (e.g., barcodes) are attached to both ends of the target molecule, resulting in 35×35 arrangements, which is equivalent to 1225 arrangements for 35 tags. Such a number of tags is sufficient so that different molecules with the same starting and ending points have a high probability of receiving different tag combinations (e.g., at least 94%, 99.5%, 99.99%, 99.999%). Other barcode combinations include any number between 10 and 500, for example, about 15×15, about 35×35, about 75×75, about 100×100, about 250×250, about 500×500.

[0251] In some cases, the unique tag can be an oligonucleotide of a predetermined sequence or a random sequence or a semi-random sequence. In other cases, more than one barcode can be used so that the barcodes do not have to be unique relative to each other in the more than one barcode. In this example, the barcode can be connected to a single molecule so that the combination of the barcode and the sequence that can be connected thereto produces a unique sequence that can be traced back individually. As described herein, the detection of non-unique barcodes combined with the sequence data at the beginning (start) and end (termination) parts of the sequence reads can allow a unique identity to be assigned to a specific molecule. The length of a single sequence read or the number of base pairs can also be used to assign a unique identity to such a molecule. As described herein, fragments from a single strand of nucleic acid to which a unique identity has been assigned can thereby allow subsequent identification of fragments from the parental chain.

[0252] In some embodiments, the method includes adding one or more internal control DNA and forward and reverse primers for amplifying the internal control DNA. The internal control DNA can be added using primers annealed upstream and downstream of the rearrangement breakpoint before amplification. The forward and reverse primers for amplifying the internal control DNA can be included or added simultaneously with the primers annealed upstream and downstream of the rearrangement breakpoint. The internal control DNA can include or be composed of a sequence that does not appear in the subject's genome or a sequence that does not appear in the genome (e.g., the human genome) of a species of which the subject is a member. The forward and / or reverse primers for amplifying the internal control DNA can include a sequence that is not complementary to any sequence in the subject's genome (e.g., the human genome). The internal control DNA can be used to ensure that the amplification process is carried out as designed. Therefore, the method can include detecting (e.g., sequencing) molecules amplified from one or more internal control DNAs and / or captured by one or more internal control DNAs. The method can include comparing the amount of the internal control DNA (e.g., the number of molecules or reads corresponding to the internal control DNA sequence detected) with 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 value can be established, for example, based on historical data or by testing the method on DNA samples 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 correctly, thereby reducing the possibility of false negatives.

[0253] Modification-Sensitive Sequencing

[0254] The methods disclosed herein use modification-sensitive sequencing to detect the modification state of at least one type of dNTP containing modified bases used in the end repair reaction. "Modification-sensitive sequencing" refers to any sequencing workflow that can distinguish at least two modification states of nucleotide bases. These two states can be: (i) whether the base is modified (e.g., 5mC and / or 5hmC relative to unmethylated cytosine); or (ii) the type of modification exhibited by the base (e.g., 5mC to 5hmC). Modification-sensitive sequencing does not necessarily require that a particular type of modification be identified as present or absent at a particular position, but only requires determining whether one or more modification types (e.g., 5mC and 5hmC) are present. For example, in some embodiments, modification-sensitive sequencing includes sequencing comprising a bisulfite conversion step, which can distinguish 5mC and 5hmC from unmethylated C, but it cannot distinguish 5mC from 5hmC.

[0255] Of course, the type of modification-sensitive sequencing used will depend on the type of modified base used in the end repair, such that the type of modification-sensitive sequencing will be able to detect at least the presence or absence of at least the modified base. Table 1 summarizes exemplary forms of modification-sensitive sequencing and the types of modified bases that can be detected by these methods. These are described in more detail below.

[0256] Table 1 - Exemplary modification-sensitive sequencing methods

[0257] Sequencing methods Detectable base modifications Bisulfite conversion 5mC and 5hmC can be distinguished from C Oxidative bisulfite conversion 5mC can be distinguished from 5hmC and C Tet-assisted bisulfite (TAB) conversion 5hmC can be distinguished from 5mC and C APOBEC-coupled epigenetic (ACE) transformation 5hmC can be distinguished from 5mC and C Enzymatic methyl sequencing (EM-seq) 5mC and 5hmC can be distinguished from C Single Enzyme 5-Methylcytosine Sequencing (SEM-seq) 5mC and 5hmC can be distinguished from C Tet-assisted pyridine borane sequencing (TAPS) 5mC and 5hmC can be distinguished from C Nanopore-based sequencing 4mC, 5mC, 5hmC, 6mA, BrdU, FldU, IdU and EdU. Single-molecule real-time sequencing (SMRT) 4mC, 5mC, 5hmC, 6mA and 8oxoG.

[0258] As described below, there are multiple methods for detecting and / or identifying the nucleosides of modification, and these methods rely on the conversion program that changes the base pairing specificity of nucleosides based on the modification state of nucleosides.These changes of base pairing specificity can then be detected by sequencing, thereby inferring the modification state of nucleosides.As described herein, conversion program and sequencing itself constitute a form of modification perception sequencing together.

[0259] In some cases, the conversion program used in the method of the present disclosure is to change the base pairing specificity of the nucleoside (such as methylated cytosine) of modification, but does not change the base pairing specificity of the corresponding unmodified nucleoside (such as cytosine) or does not change the base pairing specificity of any unmodified nucleoside (such as cytosine, adenosine, guanosine and thymidine (or uracil)). The advantages of the method for not converting the base pairing specificity of unmodified nucleoside include the loss of reduced sequence complexity, higher sequencing efficiency and reduced comparison loss. In addition, in some cases, methods such as TAPS may be better than methods such as bisulfite sequencing and EM-seq, because they are less destructive (especially important for low-yield samples such as cfDNA or FFPE samples) and do not require denaturation, which means that non-conversion errors are more likely to be random in theory. In the method where denaturation is required to be converted, the denaturation failure of DNA molecules will result in the non-conversion of all bases in DNA molecules. Due to the biological changes in methylation are mainly consistent with the local area of ​​interest, these non-random (local) non-conversion events can be shown as false negatives (non-methylated areas). Random non-conversion methods can maximally affect low percentages of bases within a region, and thus can maximize the specificity of methylation change detection (reduce false positives) by setting a threshold on the percentage of methylated / unmethylated bases within a region. Therefore, in some cases, a conversion procedure that does not involve denaturation is preferred.

[0260] In other cases, the conversion procedure used in the methods of the present disclosure is a conversion procedure that changes the base pairing specificity of unmodified nucleosides (e.g., cytosine) but does not change the base pairing specificity of corresponding modified nucleosides (e.g., methylated cytosine, such as 5hmC and / or 5mC). Such methods include, for example, bisulfite sequencing.

[0261] Skilled artisans can select appropriate methods based on their needs, including which nucleoside modifications to detect and / or identify and which type of modified base to use in the end repair reaction.

[0262] In some embodiments, the conversion procedure converts modified nucleosides. In some embodiments, the conversion procedure for converting modified nucleosides includes Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane, ammonia borane or pyridine borane. In the Tet-assisted pic-borane conversion using a substituted borane reducing agent for conversion, 5mC and 5hmC are converted to 5caC using TET proteins without affecting unmodified C. Then, by treating with 2-methylpyridine borane (pic-borane) or other substituted borane reducing agents (such as pyridine borane, tert-butylamine borane or ammonia borane), 5caC and 5fC (if present) are converted to dihydrouracil (DHU), also without affecting unmodified C. See, e.g., Liu et al., Nature Biotechnology 2019; 37: 424-429 (e.g., in Supplementary Figure 1 and Supplementary Note 7). DHU is read as T in sequencing. Sequencing of the converted DNA identifies positions that are read as cytosine as unmodified C positions. At the same time, positions that are read as T are identified as T, 5mC, 5fC, 5caC, or 5hmC. Therefore, performing TAP conversion helps identify positions containing unmodified C using the obtained sequence reads.

[0263] Thus, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP comprises 5mC or 5hmC, and the regions synthesized during the end repair reaction can be identified as those regions comprising 5mC or 5hmC at non-CpG positions (via being judged as T at positions that are C in the reference). The procedure includes Tet-assisted pyridine borane sequencing (TAPS) as described in further detail in: Liu et al. 2019, supra. In this method, Tet enzymes are used to gradually oxidize 5mC and 5hmC to 5fC or 5caC, and then pyridine borane deaminates 5fC and 5caC to DHU and amplifies to T.

[0264] Alternatively, protection of 5hmC (e.g., using βGT) can be combined with Tet-assisted conversion with a substituted borane reducing agent, such as described above. In this method (TAPS-β), 5hmC can be protected from conversion, for example by glucosylation using β-glucosyltransferase (βGT), forming 5-glucosylhydroxymethylcytosine (5ghmC). This is described in Yu et al., Cell 2012; 149: 1368-80. 5mC is then converted to 5caC with TET proteins such as mTet1, but not C or 5ghmC. 5caC is then converted to DHU by treatment with pic-borane or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane or ammonia borane, without affecting unmodified C or 5ghmC. Sequencing of the converted DNA identifies the position read as cytosine as a 5mC position or an unmodified C position. At the same time, positions that read as T are identified as T, 5fC, 5caC, or 5hmC. Therefore, performing TAPSβ conversion on a sample as described herein helps to distinguish positions containing unmodified C or 5hmC from positions containing 5mC using the sequence reads obtained. Therefore, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP contains 5mC, and the regions synthesized during the end repair reaction can be identified as those regions that contain 5mC at non-CpG positions (via positions that are C in the reference being judged to be T). For an example description of this type of conversion, see, e.g., Liu et al., Nature Biotechnology 2019; 37: 424-429.

[0265] In some embodiments, the conversion procedure converts the modified nucleoside. In some embodiments, the conversion procedure for converting the modified nucleoside includes chemically assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane, pyridine borane pyridine or ammonia borane. In chemically assisted conversion of substituted borane reducing agents, an oxidant such as potassium perruthenate (KRuO 4) (also applicable to ox-BS conversion) is used to specifically oxidize 5hmC to 5fC. Treatment with pic-borane or another substituted borane reducing agent such as borane pyridine, tert-butylamine borane or ammonia borane converts 5fC and 5caC to DHU, but does not affect 5mC or unmodified C. Sequencing the converted DNA identifies positions that read as cytosine as 5mC or unmodified C positions. At the same time, positions that read as T are identified as T, 5fC, 5caC or 5hmC. Therefore, performing this type of conversion as described herein helps, on the one hand, distinguish positions containing unmodified C or 5mC from positions containing 5hmC using the obtained sequence reads. Therefore, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP contains 5hmC, and the regions synthesized during the end repair reaction can be identified as those regions containing 5hmC at non-CpG positions (via being judged as T at positions that are C in the reference). For an example description of this type of transformation, see, e.g., Liu et al., Nature Biotechnology 2019; 37:424-429.

[0266] Exemplary conversion procedures for changing the base pairing specificity of modified cytosine have been described. However, the method described herein can use any modified nucleoside and suitable conversion procedures (i.e., single base epigenetic conversion assay) in principle, which changes the base pairing specificity of the modified nucleoside, thereby allowing the modified base to be distinguished from the corresponding unmodified nucleoside and / or other types of modifications when sequencing. For example, any conversion procedure that allows any one of N6-methyladenine (6mA), N6-hydroxymethyladenine (6hmA) or N6-formyladenine (6fA) to be distinguished from unmodified adenosine can be used.

[0267] In some embodiments, the conversion procedure converts unmodified nucleosides. In some embodiments, the conversion procedure for converting unmodified nucleosides includes bisulfite conversion. Unmodified cytosine and some modified cytosine nucleotides (such as 5-formyl cytosine (5fC) or 5-carboxyl cytosine (5caC)) are converted into uracil with bisulfite treatment, while other modified cytosines (such as 5mC and 5hmC) are not converted. Therefore, when using bisulfite conversion, the converted nucleobase is inferred to include one or more of unmodified cytosine, 5fC, 5caC or other cytosine forms affected by bisulfite. Unconverted nucleobase is inferred to include one or more of 5mC and 5hmC. The sequencing of the DNA treated with bisulfite will be identified as the position of cytosine as 5mC position or 5hmC position. Meanwhile, the position of T is identified as T or bisulfite susceptible form of C, such as unmodified cytosine, 5fC or 5caC. Therefore, performing bisulfite conversion as described herein helps identify positions containing 5mC or 5hmC. Therefore, in these embodiments, the end repair reaction can be performed with dNTPs, wherein at least one type of dNTP contains 5mC and / or 5hmC, and the regions synthesized during the end repair reaction can be identified as those regions containing 5mC or 5hmC (via being judged as C at these positions) at non-CpG positions. For an exemplary description of bisulfite conversion, see, e.g., Moss et al., Nat Commun. 2018; 9: 5068.

[0268] In some embodiments, the program for converting unmodified nucleosides includes oxidative bisulfite (Ox-BS) conversion. The program first converts 5hmC into 5fC susceptible to bisulfite, and then carries out bisulfite conversion. Therefore, when using oxidative bisulfite conversion, the converted nucleobase is inferred to include one or more of unmodified cytosine, 5fC, 5caC, 5hmC or other cytosine forms affected by bisulfite. Unconverted nucleobase is inferred to include 5mC. The sequencing of the DNA converted by Ox-BS will be identified as the position of cytosine read as 5mC position. Meanwhile, the position read as T is identified as T or bisulfite susceptible form C, such as unmodified cytosine, 5fC or 5caC. Therefore, in these embodiments, the end repair reaction can be carried out with dNTP, wherein at least one type of dNTP includes 5mC, and the region synthesized during the end repair reaction can be identified as those regions including 5mC (via being judged as C at these positions) at non-CpG positions. Therefore, performing an Ox-BS conversion helps identify positions containing mC. For an example description of oxidative bisulfite conversion, see, e.g., Booth et al., Science 2012; 336: 934-937.

[0269] In some embodiments, the procedure for converting unmodified nucleosides includes Tet-assisted bisulfite (TAB) conversion. In TAB conversion, 5hmC is protected from conversion, and 5mC is oxidized before bisulfite treatment, so the position initially occupied by 5mC is converted to U, and the position initially occupied by 5hmC 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 5hmC (forming 5-glucosylhydroxymethylcytosine (5ghmC)), then TET proteins such as mTet1 can be used to convert 5mC into 5caC, and then bisulfite treatment can be used to convert C and 5caC into U, while 5ghmC remains unaffected. Therefore, when using TAB conversion, the converted nucleobase is inferred to include one or more of unmodified cytosine, 5fC, 5caC, 5mC or other cytosine forms affected by bisulfite. Unconverted nucleobases are inferred to contain 5hmC. The sequencing of TAB-converted DNA identifies the position read as cytosine as a 5hmC position. At the same time, the position read as T is identified as a bisulfite-sensitive form of T or C, such as unmodified cytosine, 5mC, 5fC or 5caC. Therefore, as described herein, TAB conversion of the first subsample helps to identify the position containing 5hmC. Therefore, in these embodiments, the end repair reaction can be carried out with dNTPs, wherein at least one type of dNTP includes 5hmC, and the region synthesized during the end repair reaction can be identified as those regions that include 5hmC (via being judged as C at these positions) at non-CpG positions.

[0270] In some embodiments, the conversion procedure of converting unmodified nucleosides includes APOBEC coupling epigenetic (ACE) conversion. In ACE conversion, AID / APOBEC family DNA deaminase such as APOBEC3A (A3A) is used to deaminate unmodified cytosine and 5mC, but not 5hmC, 5fC or 5caC. The sequencing of the DNA converted by ACE will identify the position read as cytosine as 5hmC position, 5fC position or 5caC position. At the same time, the position read as T is identified as T, unmodified C or 5mC. Therefore, ACE conversion as described herein helps to distinguish the position containing 5hmC from the position containing 5mC or unmodified C using the sequence read obtained from the first subsample. Therefore, in these embodiments, the end repair reaction can be carried out with dNTP, wherein at least one type of dNTP includes 5hmC, and the region synthesized during the end repair reaction can be identified as those regions containing 5hmC (via being judged as C at these positions) in non-CpG positions. For an example description of ACE conversion, see, e.g., Schutsky et al., Nature Biotechnology 2018; 36: 1083-1090.

[0271] Modification-sensitive sequencing also includes sequencing methods that do not rely on a conversion step, in which the base pairing specificity of a base is altered depending on its modification state. For example, single-molecule technologies such as nanopore-based sequencing and single-molecule real-time sequencing can be used to directly detect modified bases.

[0272] For example, some sequencing reactions involve the use of enzymes to control the passage of nucleic acids through nanopores, and in this case, the reaction data may include the kinetics and other behaviors of the enzymes and the fluctuations of the current through the nanopore. For example, ratchet proteins, helicases, or motor proteins may be used to push or pull nucleic acid molecules through holes in biological or synthetic membranes. The kinetics of these proteins may vary according to the sequence environment of the nucleic acids they act on. For example, they may slow down or pause at modified bases, and this behavior captured as part of the reaction data indicates the presence of modified bases, even if the modified bases are not within the sensing portion of the nanopore. An example of a nanopore sequencing system is commercialized by Oxford Nanopore Technologies (ONT). (See, for example, Weirather et al., F1000 Research, 6: 10 0, 2017). ONT sequencing directly sequences natural single-stranded DNA (ssDNA) molecules by measuring the characteristic current changes when bases are passed through nanopores by molecular motor proteins. ONT sequencing uses a hairpin library structure similar to that of PacBio circular DNA templates: DNA templates and their complements are bound by hairpin adapters. Thus, the DNA template passes through the nanopore, followed by the hairpin, and finally the complement. By removing the adapter, the original read can be split into two "1D" reads ("template" and "complement"). The common sequence of the two "1D" reads is a "2D" read with higher accuracy.

[0273] Nanopore sequencing can be used to detect base modifications including 5mC, 5hmC, 6mA, BrdU, FdU, IdU and EdU (see, for example, Gouil & Keniry Essays in Biochemistry (2019) 63 639-648; Kutyavin, Biochemistry (2008), 47, 51, 13666-1367; Müller et al., Nature Methods (2019), Vol. 16, pp. 429-436; Hennion et al., Genome Biology (2020), Vol. 21, Article No. 125). Therefore, in some embodiments, modification-sensitive sequencing includes nanopore sequencing. In such embodiments, end repair can be performed using dNTPs containing 4mC, 5mC, 5hmC, 6mA, BrdU, FdU, IdU and / or EdU.

[0274] Another modification-sensitive single-molecule sequencing technology is the single-molecule real-time sequencing (SMRT) commercialized by Pacific Biosciences. SMRT sequencing relies on synthetic sequencing, in which the sequence of the circular DNA template is determined by continuous fluorescent pulses, and each fluorescent pulse is produced by adding a labeled nucleotide by the polymerase fixed to the bottom of the hole. Base modifications do not affect the base call order, but they affect the kinetics of the polymerase. By considering the inter-pulse duration (IPD), base modifications can be inferred from the comparison of the modified template with a computer model or an unmodified template. Therefore, such a method can use the pulse width of the signal from the sequencing base, the inter-pulse duration (IPD) of the base and the identity of the base, so as to detect the modification in the base or adjacent base. (See, for example, Weirather et al., F1000 Research, 6: 10 0, 2017).

[0275] Single-molecule real-time sequencing can be used to detect base modifications such as 4mC, 5mC, 5hmC, 6mA, and 8oxoG (Gouil & Keniry Essays in Biochemistry (2019) 63 639-648). Therefore, in some embodiments, modification-sensitive sequencing includes single-molecule real-time sequencing. In such an embodiment, end repair can be performed using dNTPs containing 4mC, 5mC, 5hmC, 6mA, and / or 8oxoG.

[0276] Partition

[0277] In some cases, a heterogeneous nucleic acid sample is partitioned into two or more partitions (subsamples). In some embodiments, each partition is differentially labeled. The labeled partitions can then be pooled together for collective sample preparation and / or sequencing. The partition-labeling-pooling step can occur more than once, with each round of partitioning occurring based on different features and labeled using differential labels that are distinct from other partitions and partitioning means.

[0278] The example of the feature that can be used for partition includes sequence length, methylation level, nucleosome binding, sequence mismatch, immunoprecipitation and / or protein combined with DNA. The partition obtained can include one or more of the following nucleic acid forms: single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), shorter DNA fragments and longer DNA fragments. In some embodiments, partitions based on cytosine modification (e.g., cytosine methylation) or methylation are generally performed, and optionally combined with at least one other partitioning step, which step can be based on any of the aforementioned features or forms of DNA. In some embodiments, the heterogeneous nucleic acid population is partitioned into nucleic acids with one or more base modifications and without the one or more base modifications. The example of base modification is described elsewhere herein. Alternatively or additionally, the heterogeneous nucleic acid population can be partitioned into nucleic acid molecules related to nucleosomes and nucleic acid molecules without nucleosomes. Alternatively or additionally, the heterogeneous nucleic acid population can be partitioned into single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Alternatively or additionally, a heterogeneous nucleic acid population can be partitioned based on nucleic acid length (e.g., molecules up to 160 bp and molecules with a length greater than 160 bp).

[0279] In some cases, different programs are applied to different partitions to determine the different characteristics of the initial sample. According to the method of the disclosure described herein, the DNA of at least one partition is subjected to end repair and modification sensitivity sequencing procedures. In some embodiments, at least one partition is not subjected to end repair and modification sensitivity sequencing procedures according to the method of the disclosure described herein. In the case where the modification sensitivity sequencing procedure includes a conversion procedure, the corresponding sequences from the converted and unconverted partitions can be compared to identify the mononucleotides that have undergone conversion, and therefore identify the corresponding modified nucleosides in the initial sample.

[0280] In one embodiment, labeling the partitions includes labeling the molecules in each partition with a partition label. After the partitions are reassembled (e.g., to reduce the number of sequencing runs required and avoid unnecessary costs) and the molecules are sequenced, the partition label identifies the source partition. In another embodiment, different partitions are labeled with different sets of molecular labels (e.g., comprising a pair of barcodes). In this way, each molecular barcode indicates the source partition and can be used to distinguish molecules within the partition. For example, the first set of 35 barcodes can be used to label the molecules in the first partition, and the second set of 35 barcodes can be used to label the molecules in the second partition.

[0281] In some embodiments, after partitioning and labeling with partition labels, the molecules can be pooled for sequencing in a single run. In some embodiments, sample labels are added to the molecules in a step, such as after adding partition labels and pooling. Sample labels can help pool material generated from more than one sample for sequencing in a single sequencing run.

[0282] Optionally, in some embodiments, partition labels can be associated with samples and partitions. As a simple example, a first label can indicate a first partition of a first sample; a second label can indicate a second partition of the first sample; a third label can indicate a first partition of a second sample; and a fourth label can indicate a second partition of the second sample.

[0283] Although a tag can be attached to a molecule that has been partitioned based on one or more features, the final tagged molecule in the library may no longer have that feature. For example, although a single-stranded DNA molecule may be partitioned and tagged, the final tagged molecule in the library may be double-stranded. Similarly, although DNA can undergo partitioning based on different methylation levels, the tagged molecules derived from these molecules in the final library may be unmethylated. Therefore, the tags attached to molecules in the library generally indicate the characteristics of the "parent molecule" from which the final tagged molecule is derived, and not necessarily the characteristics of the tagged molecule itself.

[0284] As an example, barcodes 1, 2, 3, 4, etc. are used to tag and label the molecules in the first partition; barcodes A, B, C, D, etc. are used to tag and label the molecules in the second partition; and barcodes a, b, c, d, etc. are used to tag and label the molecules in the third partition. The differentially tagged partitions can be pooled and then sequenced. The differentially tagged partitions can be sequenced individually or together simultaneously, for example, in the same flow cell of an Illumina sequencer.

[0285] After sequencing, analysis can be performed on the reads at the level of each partition and at the level of the entire DNA population. Tags are used to sort the reads from different partitions. Analysis can include using sequence information, genome coordinate length, coverage and / or copy number to determine genetic and epigenetic variations (one or more of methylation, chromatin structure, etc.) with computer analysis. In some embodiments, higher coverage can be associated with higher nucleosome occupancy in a genomic region, while lower coverage can be associated with lower nucleosome occupancy or nucleosome loss region (NDR).

[0286] Method disclosed herein includes analyzing the DNA in the sample. In some embodiments described herein, the disclosed method includes partitioning the DNA. In such a method, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more features of DNA. The method can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, the first subsample or aliquot of the sample is subjected to a step for preparing a capture probe as described elsewhere herein, and the second subsample or aliquot of the sample is subjected to partitioning. In some embodiments, the sample or subsample or its aliquot is subjected to partitioning and differential labeling, followed by a capture step, using a capture probe for rearrangement of the sequence and optionally for, for example, another capture probe for sequence variable target region and / or epigenetic target region.

[0287] Methylation spectrum analysis can include determining the methylation pattern throughout the different regions of the genome. For example, after molecular partitioning and sequencing based on the degree of methylation (e.g., the relative number of methylated core bases of each molecule), the sequence of the molecules in the different partitions can be mapped to the reference genome. This can show regions in the genome that are more methylated or less methylated than other regions. In this way, in contrast to single molecules, genomic regions can be different in their degree of methylation.

[0288] The nucleic acid molecules in the sample can be partitioned, for example, by enriching in a rare nucleic acid molecule that is more common in a partition of the sample to increase rare signals. For example, genetic variations present in hypermethylated DNA but less (or not) present in hypomethylated DNA can be more easily detected by partitioning the sample into hypermethylated and hypomethylated nucleic acid molecules. By analyzing more than one partition of the sample, multidimensional analysis of a single molecule can be performed, and therefore higher sensitivity can be achieved. Partitions can include physically partitioning nucleic acid molecules into partitions or subsamples based on the presence or absence of one or more methylated core bases. Samples can be partitioned into partitions or subsamples based on the features indicating differential gene expression or disease states. Samples can be partitioned based on the features or feature combinations that provide signal differences between normal states and diseased states during nucleic acid analysis, such as cell-free DNA (cfDNA), non-cfDNA, tumor DNA, circulating tumor DNA (ctDNA) and cell-free nucleic acid (cfNA).

[0289] In some embodiments, hypermethylated and / or hypomethylated variable epigenetic target regions are analyzed to determine whether they display differential methylation signatures 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.

[0290] In some cases, the heterogeneous DNA in the sample is partitioned into two or more partitions (e.g., at least 3, 4, 5, 6, or 7 partitions). In some embodiments, each partition is differentially labeled. The labeled partitions can then be pooled together for collective sample preparation and / or sequencing. The partition-labeling-pooling step can occur more than once, with each round of partitioning occurring based on a different feature (examples provided herein) and labeled using a differential label that is distinct from other partitions and partitioning means. In other cases, the differentially labeled partitions are sequenced separately.

[0291] The reagent for partitioning the nucleic acid population within the sample can be an affinity reagent, such as an antibody with the desired specificity, a natural binding partner or a variation thereof (Bock et al., Nat Biotech 28: 1106-1114 (2010); Song et al., Nat Biotech 29: 68-72 (2011)), or, for example, an artificial peptide selected by phage display that has specificity for a given target. In some embodiments, the agent for partitioning is an agent that recognizes a modified nucleobase. In some embodiments, the modified nucleobase recognized by the agent is a modified cytosine, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, the modified nucleobase recognized by the agent is the product of a program that differently affects the first nucleobase in the DNA of the sample and the second nucleobase in the DNA. In some embodiments, the modified nucleobase can be a "converted nucleobase", meaning that its base pairing specificity is changed by a program. For example, some procedures convert unmethylated or unmodified cytosine into dihydrouracil, or more generally, at least one modified or unmodified form of cytosine undergoes deamination to produce uracil (considered to be a modified nucleobase in the context of DNA) or a further modified form of uracil. Examples of partitioning agents include antibodies, such as antibodies that recognize modified nucleobases, which can be modified cytosines, such as methylcytosine (e.g., 5-methylcytosine). In some embodiments, partitioning agents are antibodies that recognize modified cytosines such as 5-carboxycytosine (5caC) other than 5-methylcytosine. Alternative partitioning agents include methyl binding domains (MBDs) and methyl binding proteins (MBPs) as described herein, including proteins such as MeCP2.

[0292] Additional non-limiting examples of partitioning agents are histone binding proteins, which can separate nucleic acids bound to histones from free or unbound nucleic acids.Examples of histone binding proteins that can be used in the methods disclosed herein include RBBP4, RbAp48, and SANT domain peptides.

[0293] In some embodiments, partitioning can include binary partitioning and partitioning based on degree of modification / level.For example, methylated fragments can be partitioned by methylated DNA immunoprecipitation (MeDIP), or all methylated fragments and unmethylated fragments can be partitioned using methyl binding domain proteins (e.g., MethylMinder methylated DNA enrichment kit (ThermoFisher Scientific)). Subsequently, other partitioning can include eluting fragments with different methylation levels by adjusting the salt concentration of a solution containing a methyl binding domain and a binding fragment.As salt concentration increases, fragments with greater methylation levels are eluted.

[0294] Analyzing DNA can include detecting or quantifying DNA of interest. Analyzing DNA can include detecting genetic variation and / or epigenetic features (e.g., DNA methylation and / or DNA fragmentation).

[0295] In some embodiments, methylation levels can be determined using partitioning, modification-sensitive conversions such as bisulfite conversion, direct detection during sequencing, methylation-sensitive restriction endonuclease digestion, methylation-dependent restriction endonuclease digestion, or any other suitable method. For example, different forms of DNA (e.g., hypermethylated and hypomethylated DNA) can be physically partitioned based on one or more features of DNA. For example, methylated DNA binding proteins (e.g., MBD, such as MBD2, MBD4, or MeCP2) or antibodies specific to 5-methylcytosine (e.g., in MeDIP) can be used to partition DNA. This method can be used to determine, for example, whether certain sequences are hypermethylated or hypomethylated. In some embodiments, DNA fragmentation patterns can be determined based on endpoints and / or center points of DNA molecules (such as cfDNA molecules).

[0296] In some cases, the final partition is enriched with nucleic acids with varying degrees of modification (overrepresentative or underrepresentative modifications). Overrepresentation and underrepresentation can be defined by the number of modifications carried by the nucleic acid relative to the median of the modifications of each chain in the population. For example, if the median of 5-methylcytosine residues in the nucleic acids in the sample is 2, then the modification of nucleic acids containing more than two 5-methylcytosine residues is overrepresentative, while nucleic acids with 1 or 0 5-methylcytosine residues are underrepresentative. The effect of affinity separation is to enrich for nucleic acids with overrepresented modifications in the binding phase and nucleic acids with underrepresented modifications in the non-binding phase (i.e., in solution). The nucleic acids in the binding phase can be eluted before subsequent processing.

[0297] When using MeDIP or During the methylation DNA enrichment kit (ThermoFisher Scientific), sequential elution can be used to separate the methylation partitions of different levels. For example, low methylation partitions (no methylation) can be separated from methylation partitions by contacting the nucleic acid colony with the MBD attached to the magnetic beads from the kit. Pearls are used to separate methylated nucleic acids from unmethylated nucleic acids. Subsequently, one or more elution steps are sequentially carried out to elute the nucleic acids with various methylation levels. For example, the first group of methylated nucleic acids can be eluted at a salt concentration of 160mM or higher, such as at least 150mM, at least 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or 2000mM. After such methylated nucleic acids are eluted, magnetic separation is used again to separate the methylated nucleic acids of higher levels from the nucleic acids with lower methylation levels. The elution and magnetic separation steps can be repeated to produce various partitions, such as a hypomethylated partition (enriched in nucleic acids containing no methylation), a methylated partition (enriched in nucleic acids containing low levels of methylation), and a hypermethylated partition (enriched in nucleic acids containing high levels of methylation).

[0298] In some methods, the nucleic acid bound to the agent for affinity separation is subjected to a washing step based on the partition. The washing step washes away the nucleic acid weakly bound to the affinity agent. Such nucleic acid can be enriched with nucleic acids that are close to the mean or median (i.e., the intermediate value between the nucleic acid bound to the solid phase and the nucleic acid not bound to the solid phase when the sample is initially contacted with the agent) degree of modification.

[0299] Affinity separation results in at least two and sometimes three or more partitions of nucleic acids with different degrees of modification. When the partitions are still separated, the nucleic acids of at least one partition and usually two or three (or more) partitions are connected to nucleic acid tags, which are usually provided as components of adapters, and the nucleic acids in different partitions receive different tags that distinguish members of one partition from members of another partition. The tags connected to the nucleic acid molecules of the same partition can be the same or different from each other. But if different from each other, the tags can have a part of the common coding so that the molecules to which they are attached are identified as belonging to a specific partition.

[0300] For more details on partitioning nucleic acid samples based on features such as methylation, see WO2018 / 119452, which is incorporated herein by reference.

[0301] In some embodiments, nucleic acid molecules can be partitioned into different partitions based on nucleic acid molecules that bind to a specific protein or fragment thereof and nucleic acid molecules that do not bind to the specific protein or fragment thereof.

[0302] Nucleic acid molecules can be partitioned based on DNA-protein binding. Protein-DNA complexes can be partitioned based on specific protein properties. Examples of such properties include various epitopes, modifications (e.g., histone methylation or acetylation) or enzymatic activity. Examples of proteins that can be combined with DNA and used as a classification basis can include but are not limited to protein A and protein G. Any suitable method can be used for partitioning nucleic acid molecules based on protein binding regions. Examples for methods for partitioning nucleic acid molecules based on protein binding regions include but are not limited to SDS-PAGE, chromatin immunoprecipitation (ChIP), heparin chromatography and asymmetric flow field flow classification (AF4).

[0303] In some embodiments, partitioning includes contacting DNA with a methylation-sensitive restriction enzyme (MSRE) and / or a methylation-dependent restriction enzyme (MDRE). After treating the DNA with MSRE or MDRE, the DNA partitioning can be based on size to produce a subsample of hypermethylation (the longest DNA molecule after MSRE treatment and the shortest DNA fragment after MDRE treatment), medium methylation (MSRE or MDRE treatment of medium-length DNA molecules) and hypomethylation (the shortest DNA molecule after MSRE treatment and the longest DNA fragment after MDRE treatment).

[0304] In some embodiments, 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 an intact MBP. In some embodiments, the MBD binds to 5-methylcytosine (5mC), and the MBP comprises the MBD and is referred to interchangeably herein as a methyl binding protein or a methyl binding domain protein. In some embodiments, the MBD is attached to a paramagnetic bead (such as a 5-methylcytosine protein) via a biotin linker. M-280 streptavidin) coupled. Partitioning into fractions with different degrees of methylation can be performed by eluting the fractions with increasing NaCl concentrations.

[0305] In some embodiments, bound DNA is eluted by contacting the antibody or MBD with a protease such as proteinase K. This can be done instead of or in addition to the elution step using NaCl as discussed above.

[0306] Examples of agents that recognize modified nucleobases contemplated herein include, but are not limited to:

[0307] (a) The protein MeCP2 preferentially binds to 5-methyl-cytosine compared to unmodified cytosine.

[0308] (b) RPL26, PRP8, and the DNA mismatch repair protein MHS6 preferentially bind to 5-hydroxymethyl-cytosine compared to unmodified cytosine.

[0309] (c) FOXK1, FOXK2, FOXP1, FOXP4, and FOXI3 that preferentially bind to 5-formyl-cytosine over unmodified cytosine (Iurlaro et al., Genome Biol. 14:R119 (2013)).

[0310] (d) Antibodies specific for one or more methylated or modified nucleobases or their conversion products such as 5mC, 5caC or DHU.

[0311] Typically, elution varies with the number of modifications such as the number of methylation sites per molecule, wherein the molecule with more methylation is eluted under increased salt concentration. In order to elute DNA into different populations based on the degree of methylation, people can use a series of elution buffers that increase the NaCl concentration. The salt concentration can be in the range of about 100nM to about 2500mM NaCl. In an embodiment, the process results in three (3) partitions. The molecule is contacted with a solution of a first salt concentration, and the solution comprises a molecule of an agent containing a modified nucleobase, which can be attached to a capture portion, such as streptavidin. At a first salt concentration, a molecular population will be combined with an agent, and a population will remain unbound. Unbound populations can be separated into "hypomethylation" populations. For example, the first partition of DNA enriched in hypomethylated form is a partition that remains unbound at low salt concentrations (e.g., 100mM or 160mM). The second partition of the enriched moderately methylated DNA is eluted using a medium salt concentration (e.g., a concentration between 100mM and 2000mM). This is also separated from the sample. The third partition, enriched for the highly methylated form of DNA, is eluted using a high salt concentration (eg, at least about 2000 mM).

[0312] In some embodiments, monoclonal antibodies raised against 5-methylcytidine (5mC) are used to purify methylated DNA. For example, at 95°C, the DNA is denatured to produce single-stranded DNA fragments. Protein G coupled to a standard or magnetic beads and washes after incubation with an anti-5mC antibody are used to immunoprecipitate DNA bound to the antibody. Such DNA can then be eluted. Partitions can include unprecipitated DNA and one or more partitions eluted from the beads.

[0313] In some embodiments, partitions of DNA are desalted and concentrated in preparation for enzymatic steps of library preparation.

[0314] Sequences containing abnormally high copy numbers may tend to be hypermethylated. Therefore, in some embodiments, the DNA contacted with a capture probe specific to a member of an epigenetic target region group comprises at least a portion of a hypermethylated partition, wherein the epigenetic target region group comprises more than one target region that is both a type-specific differentially methylated region and a copy number variation. DNA from or comprising at least a portion of a hypermethylated partition may or may not be combined with DNA from or comprising at least a portion of one or more other partitions (such as a medium partition or a hypomethylated partition).

[0315] Amplification

[0316] The adapted DNA can be amplified (e.g., by PCR) prior to or as part of modification-sensitive sequencing. For example, in a modification-sensitive sequencing procedure that includes a conversion step, the adapted DNA can be amplified after the conversion step. In a modification-sensitive sequencing procedure involving single-molecule sequencing (such as nanopore-based sequencing or SMRT sequencing), there may be no amplification step.

[0317] Amplification is typically initiated by primers that bind to primer binding sites in adapters flanking the DNA molecule to be amplified. Amplification methods can include cycles of denaturation, annealing, and extension produced by thermal cycling, or can be isothermal cycling as in transcription-mediated amplification. Other amplification methods include ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and replication based on self-sustained sequences.

[0318] In some embodiments, the methods of the present invention use T-tailed and C-tailed adapters for dsDNA ligation. Adding C-tailed adapters can increase the efficiency of ligation because when A-tailing is performed in the presence of dGTP, such as when A-tailing is performed in the same reaction as end repair, the A-tailing reaction can also add G-tails to a small portion of the DNA molecule. The use of T-tailed and C-tailed adapters can result in the amplification of at least 50%, 60%, 70% or 80% of double-stranded nucleic acids. Preferably, the method increases the amount or number of amplified molecules by at least 10%, 15% or 20% relative to a control method performed with a T-tailed adapter alone.

[0319] In some embodiments, the DNA that is adapted is amplified before sequencing. In some cases, amplification can be before one or more capture steps. In some embodiments, the connection step occurs after the conversion step. In some embodiments, connection occurs before amplification or simultaneously with amplification.

[0320] Enrichment, capture, and use of capture probes

[0321] The DNA molecules in the sample can undergo a capture step, wherein molecules with target sequences are captured for subsequent analysis. In some embodiments, the method disclosed herein includes a step of capturing one or more target area groups of DNA such as cfDNA. Any suitable method known in the art can be used to capture. Target capture can include the use of a bait group, which includes an oligonucleotide bait labeled with a capture portion (such as, biotin or other examples mentioned below). The probe can have a sequence selected to tile across a group of regions (such as genes). Under conditions that allow target molecules to hybridize with baits, these bait groups are combined with samples. Then, the captured molecules are separated using a capture portion. For example, a biotin capture portion of streptavidin based on beads. Such a method is further described in, for example, U.S. Patent No. 9,850,523, issued on December 26, 2017, which is incorporated herein by reference.

[0322] Capture moieties include, but are not limited to, biotin, avidin, streptavidin, nucleic acids comprising specific nucleotide sequences, haptens recognized by antibodies, and magnetically attractable particles. Extraction moieties may be members of a binding pair, such as biotin / streptavidin or haptens / antibodies. In some embodiments, the capture moiety attached to the analyte is captured by its binding pair, which is attached to a separable portion, such as magnetically attractable particles or large particles that can be precipitated by centrifugation. Capture moieties may be any type of molecule that allows affinity separation of nucleic acids with capture moieties from nucleic acids lacking capture moieties. Example capture moieties are biotin or oligonucleotides, the biotin allowing affinity separation by binding to streptavidin attached or connectable to a solid phase, the oligonucleotides allowing affinity separation by binding to complementary oligonucleotides attached or connectable to a solid phase.

[0323] A set of targeted enriched regions can be selected so that they do not contain regions known to include base modifications used in the end repair reaction. When end repair is performed with dNTPs containing 5mC or 5hmC, a set of targeted enriched regions can be selected so that they do not contain CpH dinucleotides known to be naturally methylated in a subject (e.g., a human). Such CpH dinucleotides can be identified by using publicly available resources (e.g., MethBank3.0: a database of DNAmethylomes across a variety of species Nucleic Acids Res 2018). The advantage of this approach is that any detected methylated CpH dinucleotides can be unambiguously attributed to the region synthesized in the end repair.

[0324] In some embodiments, capture includes contacting the DNA to be captured with a target-specific probe group. The target-specific probe group can have any feature of the target-specific probe group described herein, including but not limited to the features in the embodiment set forth above and the parts related to the probe below. Capture can be performed on one or more subsamples prepared in the method process disclosed herein. In some embodiments, DNA is captured from at least the first subsample or the second subsample, for example, at least the first subsample and the second subsample. In some embodiments, differential labeling (e.g., as described herein) is performed on the subsamples, and then collected before experiencing capture.

[0325] The capture step can be performed using conditions suitable for specific nucleic acid hybridization, which conditions generally depend to some extent on the characteristics of the probe, such as length, base composition, etc. In view of the general knowledge of nucleic acid hybridization in the art, those skilled in the art will be familiar with appropriate conditions. In some embodiments, a complex of a target-specific probe and DNA is formed.

[0326] In some embodiments, the methods described herein include capturing more than one target group of cfDNA obtained from a subject. The target area includes an epigenetic target area, which can show differences in methylation levels and / or fragmentation patterns, depending on whether they are derived from tumor cells or from healthy cells. The target area also includes a sequence variable target area, which can show sequence differences, depending on whether they are derived from tumor cells or from healthy cells. The capture step produces a capture group of cfDNA molecules, and in the capture group of cfDNA molecules, the cfDNA molecules corresponding to the sequence variable target area group are captured with a greater capture yield than the cfDNA molecules corresponding to the epigenetic target area group. For further discussion of capture steps, capture yields, and related aspects, see WO2020 / 160414, which is incorporated herein by reference for all purposes.

[0327] In some embodiments, the methods described herein include contacting cfDNA obtained from a subject with a target-specific probe set, wherein the target-specific probe set is configured to capture cfDNA corresponding to a set of sequence variable target regions with a greater capture yield than cfDNA corresponding to a set of epigenetic target regions.

[0328] It is beneficial to capture cfDNA corresponding to a set of sequence variable target regions at a greater capture yield than cfDNA corresponding to a set of epigenetic target regions because a greater sequencing depth may be required to analyze sequence variable target regions with sufficient confidence or accuracy than an epigenetic target region. The amount of data required to determine fragmentation patterns (e.g., testing perturbations of transcription start sites or CTCF binding sites) or fragment abundance (e.g., in hypermethylated and hypomethylated partitions) is typically less than the amount of data required to determine the presence or absence of cancer-associated sequence mutations. Capturing target region sets at different yields can facilitate sequencing target regions to different sequencing depths in the same sequencing run (e.g., using pooled mixtures and / or in the same sequencing pool).

[0329] In various embodiments, the method further comprises sequencing the captured cfDNA to, for example, varying degrees of sequencing depth for a set of epigenetic target regions and a set of sequence variable target regions, consistent with discussion herein.

[0330] In some embodiments, the complex of the target-specific probe and DNA is separated from DNA that is not bound to the target-specific probe. For example, where the target-specific probe is covalently or non-covalently bound to a solid support, a washing or aspiration step can be used to separate the unbound material. Alternatively, where the complex has chromatographic properties different from those of the unbound material (e.g., where the probe comprises a ligand that binds to a chromatographic resin), chromatography can be used.

[0331] As discussed in detail elsewhere herein, target-specific probe groups can include more than one group, such as probes for a sequence variable target group and probes for an epigenetic target group. In some such embodiments, the capture step is performed using probes for sequence variable target groups and probes for epigenetic target groups simultaneously in the same container, for example, probes for sequence variable target groups and epigenetic target groups are in the same composition. This method provides a relatively more efficient workflow. In some embodiments, the concentration of probes for sequence variable target groups is greater than the concentration of probes for epigenetic target groups.

[0332] Optionally, the capture step is performed in the first container with a sequence variable target probe set and in the second container with an epigenetic target probe set, or the contacting step is performed at a first time and in the first container with a sequence variable target probe set and at a second time before or after the first time with an epigenetic target probe set. The method allows the preparation of separate first and second compositions, which contain captured DNA corresponding to the sequence variable target set and captured DNA corresponding to the epigenetic target set. The compositions can be processed separately as desired (e.g., graded based on methylation, as described elsewhere herein) and recombined in appropriate proportions to provide materials for further processing and analysis such as sequencing.

[0333] In some embodiments, a capture group of DNA (e.g., cfDNA) is provided. For the disclosed method, the capture group of DNA can be provided, for example, by performing a capture step before a sequencing step as described herein. The capture group can include DNA corresponding to a sequence variable target group, DNA corresponding to an epigenetic target group, or a combination thereof. In some embodiments, the capture step is performed before or after the conversion step.

[0334] In some embodiments, capture (e.g., capture from a sample or a first subsample) a first target group, which includes at least an epigenetic target region. The epigenetic target region captured from the first subsample may include a hypermethylated variable target region. In some embodiments, a hypermethylated variable target region is a CpG-containing region that is unmethylated or has low methylation (e.g., methylation below average relative to a large amount of cfDNA (bulk cfDNA)) in cfDNA from a healthy subject. In some embodiments, a hypermethylated variable target region is a region that shows lower methylation in healthy cfDNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells can shed more DNA into the bloodstream than healthy cells of the same tissue type. Therefore, the distribution of cfDNA-derived tissues can change when cancerous. Therefore, an increase in the level of hypermethylated variable target regions in the first subsample can be an indicator of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0335] In some embodiments, the second target group is captured from the second subsample, including at least an epigenetic target area. The epigenetic target area may include a low methylation variable target area. In some embodiments, the low methylation variable target area is a CpG-containing area that is methylated in cfDNA from a healthy subject or has high methylation (e.g., methylation higher than the average relative to a large amount of cfDNA). In some embodiments, the low methylation variable target area is a region that shows high methylation in healthy cfDNA than in at least one other tissue type. Without wishing to be bound by any particular theory, cancer cells can shed more DNA into the bloodstream than healthy cells of the same tissue type. Therefore, the distribution of cfDNA source tissues can change when cancerous. Therefore, the increase in the level of low methylation variable target areas in the second subsample can be an indicator of cancer presence (or recurrence, depending on the medical history of the subject).

[0336] In some embodiments, the amount of sequence variable target region DNA captured is greater than the amount of epigenetic target region DNA captured when normalized for differences in the size of the target regions (footprint size).

[0337] Alternatively, a first capture group and a second capture group may be provided that include DNA corresponding to a set of sequence variable target regions and DNA corresponding to a set of epigenetic target regions, respectively. The first capture group and the second capture group may be combined to provide a combined capture group.

[0338] In some embodiments of capture sets (including combined capture sets as discussed above) in which DNA corresponding to a set of sequence variable target regions and a set of epigenetic target regions is included, the DNA corresponding to the set of sequence variable target regions can be present at a greater concentration than the DNA corresponding to the set of epigenetic target regions, e.g., 1.1 to 1.2 times greater concentration, 1.2 to 1.4 times greater concentration, 1.4 to 1.6 times greater concentration, 1.6 to 1.8 times greater concentration, 1.8 to 2. .0 times greater concentration, 2.0 times to 2.2 times greater concentration, 2.2 times to 2.4 times greater concentration, 2.4 times to 2.6 times greater concentration, 2.6 times to 2.8 times greater concentration, 2.8 times to 3.0 times greater concentration, 3.0 times to 3.5 times greater concentration, 3.5 times to 4.0, 4.0 times to 4.5 times greater concentration, 4.5 times to 5.0 times greater concentration, 5.0 times to 5.5 times greater concentration, 5.5 times to 6.0 times greater concentration, 6.0 times to 6.5 times greater concentration, 6.5 times to 7.0 times greater concentration, 7.0 times to 7.5 times greater concentration, 7.5 times to 8.0 times greater concentration, 8.0 times to 8.5 times greater concentration, 8.5 times to 9.0 times greater concentration, 9.0 times to 9.5 times greater concentration, 9.5 times to 10.0 times greater concentration, 10 times to 11 times greater concentration, 11 times to 12 times greater concentration, 12 times to 13 times greater concentration, 13 times to 14 times greater concentration, 14 times to 15 times greater concentration. The concentration difference is calculated by normalization for the target footprint size, as discussed in the definitions section.

[0339] In some embodiments, the captured DNA comprises an intronic region. In some embodiments, the intronic region comprises one or more introns that may distinguish DNA from neoplastic (e.g., tumor or cancer) cells and healthy cells (e.g., non-neoplastic circulating cells). For example, introns comprising rearrangements known to be present in some neoplastic cells but not present in healthy cells can be used to distinguish DNA from neoplastic (e.g., tumor or cancer) cells and from healthy cells. In some embodiments, the rearrangement is a translocation.

[0340] 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 has a footprint in the range of 30bp-1000kb, for example, 30bp-100bp, 100bp-200bp, 200bp-500bp, 500bp-1kb, 1kb-2kb, 2kb-5kb, 5kb-10kb, 10kb-20kb, 20kb-50kb, 50kb-100kb, 100kb-200kb, 200kb-300kb, 300kb-400kb, 400kb-500kb, 500kb-600kb, 600kb-700kb, 700kb-800kb, 800kb-900kb, and 900kb-1000kb.

[0341] Exemplary rearrangements that can be detected using the methods described herein, such as intronic translocations, include but are not limited to translocations in which at least one of the two genes involved in the translocation is a receptor tyrosine kinase. Exemplary translocation products are BCR-ABL fusions, and fusions comprising any one of ALK, FGFR2, FGFR3, NTRK1, RET or ROS1.

[0342] In some embodiments, the captured DNA comprises a target region with a type-specific epigenetic variation. In some embodiments, the epigenetic target region group consists of a target region with a type-specific epigenetic variation. In some embodiments, type-specific epigenetic variation (e.g., differential methylation or type-specific fragmentation pattern) may distinguish DNA from one or more related cells or tissue types from DNA from other cells or tissue types present in a sample or subject.

[0343] In some embodiments, nucleic acids captured or enriched using the methods described herein include captured DNA, such as one or more captured DNA groups. In some embodiments, the captured DNA is included in target regions that are differentially methylated in different immune cell types. In some embodiments, immune cell types include rare or closely related immune cell types, such as activated and immature lymphocytes or myeloid cells at different stages of differentiation.

[0344] In some embodiments, the captured epigenetic target region group captured from the sample or the first subsample includes a high methylation variable target region. In some embodiments, the high methylation variable target region is differentially or exclusively high methylated in one or more related cells or tissue types. In some embodiments, the high methylation variable target region is differentially or exclusively high methylated in one cell type or in one immune cell type or in one immune cell type within a cluster. In some embodiments, the high methylation variable target region is high methylated to a degree that is distinguishably higher or only present in one cell type or one immune cell type or one immune cell type within a cluster. Such a high methylation variable target region may be high methylated in other cells or tissue types, but not to the extent observed in one or more related cells or tissue types. In some embodiments, the high methylation variable target region shows lower methylation in healthy cfDNA than in at least one other tissue type. In some embodiments, the high methylation variable target region shows even higher methylation in the cfDNA of diseased cells from one or more related cells or tissue types. In some embodiments, the target region comprises a hypermethylated region with an abnormally high copy number. In some such embodiments, the target region is hypermethylated in healthy and diseased colon tissues and has an abnormally high copy number in precancerous or cancerous colon tissues. Examples of such target regions are shown in Table 1 below.

[0345] Table 1: Hypermethylated target regions with high copy number abnormalities in colon cancer or precancerous lesions

[0346]

[0347]

[0348] Table 2. Example hypermethylated target regions based on lung cancer research

[0349]

[0350]

[0351] In some embodiments, the epigenetic target region set captured from the sample or subsample includes a hypomethylated variable target region. In some embodiments, the hypomethylated variable target region is hypomethylated only in one or more related cells or tissue types. In some embodiments, the hypomethylated variable target region is exclusively hypomethylated in one cell type or in one immune cell type or in one immune cell type within a cluster. In some embodiments, the hypomethylated variable target region is hypomethylated to the extent that it is present only in one cell type or one immune cell type or one immune cell type within a cluster. Such a hypomethylated variable target region may be hypomethylated in other cells or tissue types, but not to the extent observed in one or more cells 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.

[0352] Without wishing to be bound by any particular theory, in individuals with cancer, proliferating or activated immune cells (and / or dying cancer cells) can shed more DNA into the bloodstream than immune cells in healthy individuals (and / or healthy cells of the same tissue type, respectively). Therefore, the distribution of cell types and / or tissues from which cfDNA is derived can change when carcinogenesis occurs. Therefore, the presence and / or level of cfDNA derived from certain cell or tissue types can be an indicator of disease. Changes in hypermethylation and / or hypomethylation can be an indicator of disease. For example, after the partitioning step, an increase in the level of hypermethylated variable target regions and / or hypomethylated variable target regions in the subsample can be an indicator of the presence (or recurrence, depending on the subject's medical history) of cancer.

[0353] Exemplary hypermethylated variable target regions and hypomethylated variable target regions that can be used to distinguish various cell types (including but not limited to immune cell types) have been identified by analyzing DNA obtained from various cell types via whole-genome bisulfite sequencing, as described in, for example, 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 / s13059-020-02065-5). Whole-genome bisulfite sequencing data are available from the Blueprint consortium and available on the Internet at dcc.blueprint-epigenome.eu.

[0354] In some embodiments, the first captured target area group and the second captured target area group respectively include DNA corresponding to the sequence variable target area group and DNA corresponding to the epigenetic target area group, for example, as described in WO 2020 / 160414. The first capture group and the second capture group can be combined to provide a combined capture group. The sequence variable target area group and the epigenetic target area group can have any of the features described for such a group in WO 2020 / 160414 (incorporated herein by reference in its entirety). In some embodiments, the epigenetic target area group includes a high methylation variable target area group. In some embodiments, the epigenetic target area group includes a low methylation variable target area group. In some embodiments, the epigenetic target area group includes a CTCF binding region. In some embodiments, the epigenetic target area group includes a fragmented variable target area. In some embodiments, the epigenetic target area group includes a transcription start site. In some embodiments, the epigenetic target region group includes regions that can show local amplification in cancer, for example, 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, the epigenetic target region group includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the aforementioned targets.

[0355] In some embodiments, the sequence variable target group includes more than one region known to undergo somatic mutations in cancer. In some aspects, the sequence variable target group targets more than one different gene or genomic region ("panel") selected so that a determined proportion of subjects with cancer exhibit genetic variation or tumor markers in one or more different genes or genomic regions in the group. The group can be selected to limit the region for sequencing to a fixed number of base pairs. The group can be selected to sequence the desired amount of DNA, for example, by adjusting the affinity and / or amount of the probe, as described elsewhere herein. The group can also be selected to achieve the desired sequence read depth. The group can be selected to achieve the desired sequence read depth or sequence read coverage for a certain amount of sequencing base pairs. The group can be selected to achieve theoretical sensitivity, theoretical specificity and / or theoretical accuracy for one or more genetic variations in the detection sample.

[0356] The probe for detecting the group of regions may include a probe for detecting a genomic region of interest (hotspot region). Information about chromatin structure may be considered when designing the probe, and / or the probe may be designed to maximize the possibility that a specific site (e.g., KRAS codons 12 and 13) may be captured, and may be designed to optimize capture based on analyzing cfDNA coverage and fragment size variation and GC sequence composition affected by nucleosome binding patterns. The region used herein may also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0357] The probe for detecting the group of regions may include a probe for detecting a genomic region of interest (hotspot region). Information about chromatin structure may be considered when designing the probe, and / or the probe may be designed to maximize the possibility that a specific site (e.g., KRAS codons 12 and 13) may be captured, and may be designed to optimize capture based on analyzing cfDNA coverage and fragment size variation and GC sequence composition affected by nucleosome binding patterns. The region used herein may also include non-hotspot regions optimized based on nucleosome positions and GC models.

[0358] Some 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 comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, or 70 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 comprises at least a portion of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, or 73 genes in Table 4 of WO 2020 / 160414. Additionally or alternatively, suitable target groups can be obtained from the literature. For example, Gale et al., PLoS One 13: e0194630 (2018), which is incorporated herein by reference, describes a group of 35 cancer-related gene targets that can be used as part or all of a sequence variable target group. These 35 targets are AKT1, ALK, BRAF, CCND1, CDK2A, CTNNB1, EGFR, ERBB2, ESR1, FGFR1, FGFR2, FGFR3, FOXL2, GAT GATA3, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MED 12, MET, MYC, NFE2L2, NRAS, PDGFRA, PIK3CA, PPP2R1A, PTEN, RET, STK11, TP53, and U2AF1.

[0359] In some embodiments, the set of sequence variable target regions comprises target regions from at least 10, 20, 30, or 35 cancer-related genes, such as the cancer-related genes listed above and in WO 2020 / 160414.

[0360] In some embodiments, a collection of capture probes is used in the methods described herein, e.g., comprising capture probes prepared by any method disclosed herein for doing so. In some embodiments, the collection of capture probes also comprises target binding probes specific to a sequence variable target region group and / or target binding probes specific to an epigenetic target region group. In some embodiments, the capture yield of the target binding probes specific to the sequence variable target region group is higher (e.g., at least 2 times higher) than the capture yield of the target binding probes specific to the epigenetic target region group. In some embodiments, the collection of capture probes is configured to have a capture yield specific to a sequence variable target region that is higher (e.g., at least 2 times higher) than its capture yield specific to an epigenetic target region.

[0361] In some embodiments, the capture yield of a target binding probe specific for a set of sequence variable target regions is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than the capture yield of a target binding probe specific for a set of epigenetic target regions. In some embodiments, the capture yield of a target binding probe specific for a set of sequence variable target regions is 1.25-fold to 1.5-fold, 1.5-fold to 1.75-fold, 1.75-fold to 2-fold, 2-fold to 2.25-fold, 2.25-fold to 2.5-fold, 2.5-fold to 2.75-fold, 2.75-fold to 3-fold, 3-fold to 3.5-fold, 3.5-fold to 4-fold, 4-fold to 4.5-fold, 4.5-fold to 5-fold, 5-fold to 5.5-fold, 5.5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, 9-fold to 10-fold, 10-fold to 11-fold, 11-fold to 12-fold, 13-fold to 14-fold, or 14-fold to 15-fold higher than the capture yield of a target binding probe specific for a set of epigenetic target regions.

[0362] In some embodiments, the collection of capture probes is configured to have a capture yield specific for a set of sequence variable target regions that is at least 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than its capture yield for a set of epigenetic target regions. In some embodiments, the collection of capture probes is configured to have a capture yield specific for a set of sequence variable target regions that is 1.25-fold to 1.5-fold, 1.5-fold to 1.75-fold, 1.75-fold to 2-fold, 2-fold to 2.25-fold, 2.25-fold to 2.5-fold, 2.5-fold to 2.75-fold, 2.75-fold to 3-fold, 3-fold to 3.5-fold, 3.5-fold to 4-fold, 4-fold to 4.5-fold, 4.5-fold to 5-fold, 5-fold to 5.5-fold, 5.5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, 9-fold to 10-fold, 10-fold to 11-fold, 11-fold to 12-fold, 13-fold to 14-fold, or 14-fold to 15-fold higher than its capture yield specific for a set of epigenetic target regions.

[0363] The collection of probes can be configured to provide higher capture yields for sets of sequence-variable targets in a variety of ways, including concentration, varying lengths, and / or chemistries (e.g., chemistries that affect affinity), and combinations thereof. Affinity can be modulated by adjusting probe length and / or including nucleotide modifications, as discussed below.

[0364] In some embodiments, capture probes specific for the set of sequence variable target regions are present at a higher concentration than 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 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 2.25-fold, 2.5-fold, 2.75-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold 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 for a set of sequence variable target regions is 1.25-fold to 1.5-fold, 1.5-fold to 1.75-fold, 1.75-fold to 2-fold, 2-fold to 2.25-fold, 2.25-fold to 2.5-fold, 2.5-fold to 2.75-fold, 2.75-fold to 3-fold, 3-fold to 3.5-fold, 3.5-fold to 4-fold, 4-fold to 4.5-fold, 4.5-fold to 5-fold, 5-fold to 5.5-fold, 5.5-fold to 6-fold, 6-fold to 7-fold, 7-fold to 8-fold, 8-fold to 9-fold, 9-fold to 10-fold, 10-fold to 11-fold, 11-fold to 12-fold, 13-fold to 14-fold, or 14-fold to 15-fold greater than the concentration of target binding probes specific for a set of epigenetic target regions. In such embodiments, the concentration may refer to the average mass / volume concentration of the individual probes in each set.

[0365] In some embodiments, a capture probe specific to a sequence variable target region group has a higher affinity for its target than a capture probe specific to an epigenetic target region group. Affinity can be adjusted in any manner known to those skilled in the art, including by using different probe chemistries. For example, certain nucleotide modifications, such as cytosine 5-methylation (in the case of certain sequences), modifications providing heteroatoms at the 2' sugar position, and LNA nucleotides, can increase the stability of double-stranded nucleic acids, which means that oligonucleotides with such modifications have a relatively higher affinity for 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. In addition, longer sequence lengths will generally provide increased affinity. Other nucleotide modifications, such as substitution of the nucleobase hypoxanthine for guanine, reduce affinity by reducing the amount of hydrogen bonding between the oligonucleotide and its complementary sequence. In some embodiments, capture probes specific for a sequence variable target region group have modifications that increase their affinity for their target. In some embodiments, alternatively or additionally, capture probes specific for an epigenetic target region group have modifications that reduce their affinity for their target. In some embodiments, capture probes specific for a sequence variable target region group have a longer average length and / or a higher average melting temperature than capture probes specific for an epigenetic target region group. These embodiments can be combined with each other and / or have concentration differences as discussed above to achieve a desired multiple difference in capture yield, such as any multiple difference described above or a range thereof.

[0366] In some embodiments, the capture probe comprises a capture moiety. The capture moiety can be any capture moiety described herein, such as biotin. In some embodiments, the capture probe is connected to a solid support, for example, covalently or non-covalently, such as by the interaction of the binding pair of the capture moiety. In some embodiments, the solid support is a bead, such as a magnetic bead.

[0367] In some embodiments, capture probes specific for a set of sequence variable target regions and / or capture probes specific for a set of epigenetic target regions are capture probe sets as discussed above, e.g., probes comprising a capture moiety and a sequence selected to tile across a set of regions, such as genes.

[0368] In some embodiments, the capture probes are provided in a single composition. The single composition can be a solution (liquid or frozen). Alternatively, the single composition can be a lyophilisate.

[0369] Alternatively, the capture probes can be provided as more than one composition, for example, including 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 proportions to provide a probe composition having a combination of any of the aforementioned fold differences in concentration and / or capture yield. Alternatively, they can be used in separate capture procedures (e.g., for aliquots of a sample or sequentially for the same sample) to provide a first composition and a second composition comprising a captured epigenetic target region and a captured sequence variable target region, respectively.

[0370] Probes specific for epigenetic targets

[0371] The probes for the epigenetic target region group can include probes specific to one or more types of target regions that can distinguish DNA from neoplastic (e.g., tumor or cancer) cells from healthy cells (e.g., non-neoplastic circulating cells). Exemplary types of such regions are discussed in detail herein (e.g., in the above section on capture groups). The probes for the epigenetic target region group can also include probes for one or more control regions, for example, as described herein.

[0372] In some embodiments, the probes for the epigenetic target region set have a footprint of at least 100 kbp, e.g., at least 200 kbp, at least 300 kbp, or at least 400 kbp. In some embodiments, the epigenetic target region set has a footprint in the range of 100 kbp-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.

[0373] Hypermethylated variable target regions

[0374] In some embodiments, probes for the epigenetic target region group include probes specific to one or more hypermethylated variable target regions. Hypermethylated variable target regions may also be referred to herein as hypermethylated DMRs (differentially methylated regions). Hypermethylated variable target regions may be any hypermethylated variable target regions listed above. For example, probes specific to hypermethylated variable target regions include probes specific to more than one locus 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, probes specific to hypermethylated variable target regions include probes specific to more than one locus 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, probes specific for hypermethylated variable target regions include probes specific for more than one locus listed in Table 1 or Table 2 (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the loci listed in Table 1 or Table 2). In some embodiments, for each locus included as a target region, there can be one or more probes having a hybridization site that binds between the transcription start site and the stop codon (the last stop codon for alternatively spliced ​​genes) of the gene. In some embodiments, one or more probes bind within 300 bp (e.g., within 200 bp or 100 bp) of the listed positions. In some embodiments, the probes have hybridization sites that overlap with the positions listed above. In some embodiments, probes specific to hypermethylated target regions include probes specific to one, two, three, four, or five subsets of hypermethylated target regions, which collectively show hypermethylation in one, two, three, four, or five of breast cancer, colon cancer, kidney cancer, liver cancer, and lung cancer.

[0375] Hypomethylated variable target regions

[0376] In some embodiments, probes for the epigenetic target region group include probes specific to one or more hypomethylated variable target regions. Hypomethylated variable target regions may also be referred to herein as hypomethylated DMRs (differentially methylated regions). Hypomethylated variable target regions may be any of the hypomethylated target regions listed above. For example, probes specific to one or more hypomethylated variable target regions may include probes for the following regions: such as repetitive elements (e.g., LINE1 elements, Alu elements, centromere tandem repeats, centromere peri-tandem repeats, and satellite DNA) and intergenic regions, which are typically methylated in healthy cells and may show reduced methylation in tumor cells.

[0377] In some embodiments, probes specific for hypomethylated variable target regions include probes specific for repetitive elements and / or intergenic regions. In some embodiments, probes specific for repetitive elements include probes specific for one, two, three, four, or five of LINE1 elements, Alu elements, centromeric tandem repeats, pericentromeric tandem repeats, and / or satellite DNA.

[0378] Exemplary probes specific for genomic regions showing 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 or containing nucleotides 8403565-8953708 and / or 151104701-151106035 of human chromosome 1.

[0379] CTCF binding region

[0380] In some embodiments, probes for the epigenetic target group include probes specific to CTCF binding regions. In some embodiments, probes specific to CTCF binding regions include probes specific to 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, for example, such as the CTCF binding regions described above, or CTCFBSDB or one or more of the articles cited above, Cuddapah et al., Martin et al., or Rhee et al. In some embodiments, probes for the epigenetic target group 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 of the upstream and downstream regions of the CTCF binding site.

[0381] Transcription start site

[0382] In some embodiments, probes for the epigenetic target region group include probes specific for transcription start sites. In some embodiments, probes specific for transcription start sites include probes 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, for example, probes specific for transcription start sites such as those listed in DBTSS. In some embodiments, probes for the epigenetic target region group 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.

[0383] Focused Amplification

[0384] As mentioned above, although focal amplifications are somatic mutations, they can be detected by sequencing based on read frequency in a manner similar to methods for detecting certain epigenetic changes such as methylation changes. Therefore, regions in cancer that may show focal amplification can be included in the epigenetic target group, as discussed above. In some embodiments, probes specific to the epigenetic target group include probes specific for focal amplification. In some embodiments, probes specific for focal amplification include probes specific for one or more of AR, BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA, and RAF1. For example, in some embodiments, probes specific for focused amplification 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.

[0385] Control area

[0386] It is useful to include control regions to aid in data validation. In some embodiments, probes specific to a panel of epigenetic target regions include probes specific to a control methylated region that is expected to be methylated in substantially all samples. In some embodiments, probes specific to a panel of epigenetic target regions include probes specific to a positive control hypomethylated region that is expected to be methylated in substantially all samples.

[0387] Probes specific for sequence variable target regions

[0388] Probes for sequence variable target groups can include probes specific to more than one region known to undergo somatic mutations in cancer. Probes can be specific to any sequence variable target group described herein. Example sequence variable target groups are discussed in detail herein (e.g., in the section above on capture groups).

[0389] In some embodiments, the sequence variable target 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 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 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 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 probe set has a footprint of at least 2 Mbp.

[0390] In some embodiments, 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 70 genes in Table 3. In some embodiments, 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 SNVs in Table 3. In some embodiments, 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 fusions in Table 3. In some embodiments, 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, or 3 indels in Table 3. In some embodiments, 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, at least 70, or 73 genes in Table 4. In some embodiments, 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, at least 70, or 73 SNVs in Table 4. In some embodiments, 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 fusions in Table 4. In some embodiments, 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 indels in Table 4. In some embodiments, probes specific for a 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 in Table 5.

[0391] Table 3

[0392]

[0393]

[0394] Table 4

[0395]

[0396] Table 5

[0397]

[0398]

[0399]

[0400] In some embodiments, probes specific for a set of sequence variable target regions include probes specific for targets from at least 10, 20, 30, or 35 cancer-related 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 U2AF1.

[0401] Sequencing

[0402] Typically, the sample nucleic acid flanked by adapters can be sequenced in the case of pre-amplification or without pre-amplification. Sequencing methods include, for example, Sanger sequencing, high-throughput sequencing, pyrophosphate sequencing, synthetic sequencing, single-molecule sequencing, nanopore sequencing, semiconductor sequencing, connection sequencing, hybridization sequencing, digital gene expression (Helicos), next-generation sequencing (NGS), single-molecule synthetic sequencing (SMSS) (Helicos), massively parallel sequencing, cloned single-molecule arrays (Solexa), shotgun sequencing, Ion Torrent, Oxford nanopore, Roche Genia, Maxim-Gilbert sequencing, primer walking (primerwalking) and sequencing using PacBio, SOLiD, Ion Torrent or nanopore platforms. Sequencing reactions can be carried out in various sample processing units, and the sample processing unit may include multiple lanes, multiple channels, multiple holes or other devices that substantially process more than one sample group at the same time. The sample processing unit may also include more than one sample chamber so that more than one operation can be processed simultaneously.

[0403] In some embodiments, the sequence coverage of a genome can be, for example, less than 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 100%. In some embodiments, a sequence reaction can provide a sequence coverage of, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, or 80% of a genome. Sequence coverage can be performed for, for example, 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.

[0404] Multiple sequencing can be used to perform simultaneous sequencing reactions. In some cases, the cell-free nucleic acid can be sequenced with at least, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. In other cases, the cell-free nucleic acid can be sequenced with less than, for example, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 50000, or 100,000 sequencing reactions. The sequencing reactions can be performed sequentially or simultaneously. Subsequent data analysis can be performed on all or a portion of the 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 is 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, 1000-10000, or 1000-20000 reads per locus (base).

[0405] Typically, sequencing of epigenetic target regions (e.g., for analyzing modified nucleotide profiles of DNA) requires a smaller sequencing depth than sequencing of sequence variable target regions (e.g., for analyzing mutations). Therefore, as described herein, a smaller sequencing depth may be sufficient for the methods described herein in some cases.

[0406] a. Differential sequencing depth

[0407] In some embodiments, nucleic acids corresponding to sequence variable target groups are sequenced to a greater sequencing depth than nucleic acids corresponding to epigenetic target groups. In some embodiments, nucleic acids corresponding to hydroxymethylation variable target groups are sequenced to a greater sequencing depth than nucleic acids corresponding to at least one other target group. For example, the depth of sequencing of nucleic acids corresponding to sequence variable and / or hydroxymethylation variable target groups can be at least 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, or 15 times greater than the depth of sequencing of nucleic acids corresponding to epigenetic target groups or nucleic acids corresponding to at least one other target group, or 1.25 to 1.5 times greater. In some embodiments, the sequencing depth is at least 2 times larger. In some embodiments, the sequencing depth is at least 5 times larger. In some embodiments, the sequencing depth is at least 10 times larger. In some embodiments, the sequencing depth is 4 to 10 times larger. In some embodiments, the sequencing depth is 4 to 100 times greater. Each of these embodiments involves sequencing nucleic acids corresponding to a set of sequence variable target regions to a greater degree of sequencing depth than nucleic acids corresponding to a set of epigenetic target regions.

[0408] In some embodiments, captured cfDNA corresponding to a set of sequence variable target regions and captured cfDNA corresponding to a set of epigenetic target regions are sequenced simultaneously, for example, in the same sequencing pool (such as a flow cell of an Illumina sequencer) and / or in the same composition; the same composition can be a pooled composition generated by recombining individually captured groups, or a composition obtained by capturing cfDNA corresponding to a set of sequence variable target regions and captured cfDNA corresponding to a set of epigenetic target regions in the same container.

[0409] In some embodiments, captured cfDNA corresponding to a hydroxymethylation variable target region group and captured cfDNA corresponding to at least one other target region group are sequenced simultaneously, for example, in the same sequencing pool (such as a flow cell of an Illumina sequencer) and / or in the same composition; the same composition can be a pooled composition generated by recombining individually captured groups, or a composition obtained by capturing cfDNA corresponding to a hydroxymethylation variable target region group and captured cfDNA corresponding to at least one other target region group in the same container.

[0410] analyze

[0411] Defined regions of end-repaired DNA synthesized during end repair

[0412] Typically, the method described herein relies on using at least one dNTP comprising a modified base in the end repair reaction, and using modification sensitivity sequencing that can detect the modified base. This allows identification of the region synthesized in the end repair in the sequencing data. This is important because these synthetic regions can cause artificial data in the typical sequencing reactions that do not control these synthetic regions. For example, when performing end repair with unmodified dNTP before methylation sensitivity sequencing (such as bisulfite sequencing), end repair can cause filling of 5' overhangs, nick translation and filling gaps with dCTP comprising unmodified cytosine. These unmodified cytosines may not reflect the original methylation state (that is, the methylation state before forming overhangs, nicks and gaps) at these positions in the original DNA molecule, and therefore end repair may cause artificial methylation information. Method disclosed herein avoids such artificial information by identifying the sequencing data corresponding to these synthetic regions. Then it can be, for example, filtered such regions so that they are not used to classify the methylation state of DNA molecules.

[0413] Regions synthesized during end repair can be classified in a variety of ways, and the exact method will depend on the identity of the modified bases used in the end repair reaction and the modification-sensitive sequencing method used. In addition, the user can determine the exact end points of regions that are classified as synthesized during end repair. A basic step in identifying regions synthesized during an end repair reaction is to identify the presence of base modifications in at least one type of dNTP used in the end repair reaction.

[0414] In some embodiments, end repair is performed with dNTPs containing 5mC or 5hmC. Both 5mC and 5hmC are naturally occurring base modifications, so when identifying these modified bases, sequencing data can be derived from: (i) modified bases present in the original DNA molecule; or (ii) modified bases introduced in the end repair reaction. However, when 5mC and 5hmC occur in a non-CpG sequence environment, they can be classified as being introduced in the end repair reaction. Although CpH (i.e., CpA, CpT, CpC) methylation has been described in humans, it is believed that it accounts for 0.02% of total methylcytosine in differentiated somatic cells (Jang et al., Genes (Basel). 2017Jun; 8(6): 148). Therefore, methylated cytosines in the CpH sequence environment can be reliably attributed to regions synthesized during end repair. This is especially true when the disclosed method includes enriching a sequence group, wherein the sequence group does not include a region known to contain a methylated CpH site. Alternatively, the classification of whether a methylated CpH is part of a synthesized region can be determined by considering: (i) the specific position of the CpH site in the reference sequence; and / or (ii) the methylation status of the surrounding CpH sites. For example, if a CpH site is known to be methylated in nature (e.g., by comparison with reference data), the methylation detected at the CpH site can be ignored when defining regions synthesized during end repair. Similarly, the methylation detected at such a CpH site can be determined as the true methylation state in the DNA sample. If a CpH site known to be methylated in nature is detected as methylated in the sequencing data, but is contained in a string of other methylated CpH sites, some of which are not found to be methylated in nature (e.g., by comparison with reference data), the region can still be classified as being synthesized during end repair.

[0415] In embodiments where end repair is performed with dNTPs containing 5mC and / or 5hmC, the region in the one or more regions synthesized during the end repair in the end-repaired DNA is defined as: (i) a sequence between two non-methylated cytosines spanning a methylated non-CpG cytosine; and / or (ii) a sequence between a non-methylated cytosine and the end of a sequence read, wherein no additional non-methylated cytosine exists between the non-methylated cytosine and the end of the sequence read. Alternatively, the one or more regions synthesized during the end repair in the end-repaired DNA may be defined as: (i) a sequence from the first methylated non-CpG cytosine to the last methylated non-CpG cytosine in one or more consecutive methylated non-CpG cytosines; and / or (ii) a sequence from a methylated cytosine (5mC or 5hmC) in a non-CpG context to the end of a sequence read, wherein no non-methylated cytosine exists between the methylated cytosine in a non-CpG context and the end of the sequence read. "The end of a sequence read" refers to the portion of the sequence read that corresponds to the end-repaired DNA molecule and does not include, for example, adapter sequences.

[0416] In some embodiments, end repair is performed with a dNTP comprising a base modification, and the base modification is not naturally occurring in the subject of the DNA sample source, or only exists at a very low frequency. For example, 4mC does not occur in mammals (such as humans), and 6mA only occurs at a very low frequency (Xiao et al., Molecular Cell Vol. 71, No. 2, July 19, 2018, pp. 306-318. e7). In these embodiments, the region of the end repair DNA synthesized during the end repair can be simply classified as any region in which the modified base is detected. Although this method may cause the naturally occurring low-frequency base modification to be mistakenly classified as the result of the end repair, this will be rare, and may simply cause the corresponding sequence data to not be used for further analysis. This preferably uses the sequence data from the region synthesized during the end repair, and these data may include artificial data that may cause the erroneous inference about the corresponding DNA sample and the subject.

[0417] Thus, in some embodiments in which the modified base is not 5mC or 5hmC, the region in the one or more regions is defined as: (i) a sequence between two non-modified bases spanning a modified base, wherein the two non-modified bases have the same identity as a base present in at least one type of dNTP comprising the modified base; and / or (ii) a sequence between the non-modified base and an end of a sequence read, wherein there are no additional non-modified bases between the non-modified base and the end of the sequence read, wherein the additional non-modified bases have the same identity as a modified base present in at least one type of dNTP comprising the modified base. Optionally, one or more regions synthesized during end repair in the end-repaired DNA can be defined as: (i) a sequence from a first modified base to a last modified base in one or more consecutive modified bases, wherein the bases have the same identity as bases present in at least one type of dNTP comprising the modified base; and / or (ii) a sequence from a modified base to the end of a sequence read, wherein there are no non-modified bases between the modified base and the end of the sequence read, wherein the modified base and the non-modified base have the same identity as at least one type of dNTP comprising the modified base.

[0418] Once identified, regions synthesized during the end repair in DNA that is classified as end-repaired can be filtered out from the sequence data so that they are not used for further analysis, such as variation determination or for determining the modification state of bases in the original DNA molecule (i.e., before the end repair). Therefore, in some embodiments, the methods disclosed herein also include analyzing at least some of the sequence data corresponding to regions that are not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations in the DNA sample. The disclosed methods for identifying regions synthesized during end repair are superior to prior art methods that use uninformed "end shearing" because these prior art methods potentially remove regions that are not synthesized in the end repair reaction and therefore represent the original DNA molecule.

[0419] Example Applications

[0420] The methods disclosed herein allow for the detection of regions synthesized during end repair in end-repaired DNA. This information has utility in a wide range of contexts, including determining the methylation state of DNA in a DNA sample (i.e., before end repair) and detecting mutations in DNA.

[0421] The method proposed herein can be used as a part of any method for accurately determining the mutation of DNA in any DNA sample and / or the nucleoside spectrum of the accurate modification of DNA in any DNA sample. This is because the method disclosed herein allows identification of the region synthesized during the end repair in the DNA molecule corresponding to the end repair, and therefore may not represent the sequencing data of the original DNA molecule. The identification in these regions avoids relying on such potentially artificial data to carry out subsequent analysis, such as mutation determination and / or subsequent methylation analysis.

[0422] For example, the classification of whether there is or is not a variation in a DNA molecule depends on whether there is double-stranded supporting evidence for the variation. Double-stranded support refers to the presence of sequencing data derived from two DNA chains that support the existence of a variation. However, in the synthesized region, double-stranded supporting evidence can be artificially introduced by using a complementary strand as a template to synthesize the end repair and / or A-tailing reaction of the region. This may occur when there is a mismatch at an equivalent position in the original DNA molecule, and therefore before end repair and / or A-tailing, the variation is not present in the two chains of the original DNA molecule. Therefore, the method disclosed herein can be used to identify the region of the synthesis and filter the variation in those regions, otherwise these variations will be mistakenly classified as having double-stranded supporting evidence. When the modification sensitivity sequencing method used does not require the conversion of unmethylated cytosine and is therefore compatible with high-sensitivity variation determination, it is particularly advantageous to use the disclosed method in variation determination. Therefore, in some embodiments, the method disclosed herein is used to detect SNV, wherein modification sensitivity sequencing is based on nanopore sequencing, single molecule real-time (SMRT) sequencing, or Tet-assisted pyridine borane sequencing (TAPS).

[0423] An important exemplary application of the methods of the present disclosure is the use of the resulting sequencing data in the diagnosis and prognosis of cancer or other genetic diseases or conditions.

[0424] Thus, in some embodiments, the methods described herein include identifying or predicting the presence or absence of DNA 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 described herein.

[0425] The present method can be used to diagnose a condition in a subject, particularly the presence or absence of cancer, to characterize the condition (e.g., to stage cancer or determine the heterogeneity of cancer), to monitor the response of the condition to treatment, and to achieve a prognosis of the risk of the development of the condition or the subsequent course of the condition. The present disclosure can also be used to determine the efficacy of a particular treatment option. If the treatment is successful, a successful treatment regimen may increase the amount of rare mutations detected in the subject's blood because more cancers may die and shed DNA. In other instances, this may not happen. In another instance, perhaps certain treatment options may be associated with the genetic spectrum of the cancer over time. This correlation can be used to select therapy. In some embodiments, the target areas are analyzed to determine whether they show multiple methylation characteristics of tumor cells or cells that do not typically contribute significantly to cfDNA, and / or the target areas are analyzed to determine whether they show a single methylation characteristic of tumor cells or cells that do not typically contribute significantly to cfDNA.

[0426] In some embodiments, the method of the present invention is used to screen for cancer, such as metastasis, or is used in a method for screening for cancer, such as in a method for detecting the presence or absence of metastasis. For example, the sample can be a sample from a subject previously diagnosed with or not diagnosed with cancer. In some embodiments, such as before and / or after the subject is diagnosed with cancer, one or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples are collected from a subject as described herein. In some embodiments, the subject may suffer from or may not suffer from cancer. In some embodiments, the subject may suffer from or may not suffer from early stage cancer. In some embodiments, the subject has one or more cancer risk factors, such as tobacco use (e.g., smoking), overweight or obesity, with high body mass index (BMI), advanced age, malnutrition, high alcohol consumption or family history of cancer.

[0427] In some embodiments, the subject has used tobacco, for example, at least 1 year, 5 years, 10 years or 15 years. In some embodiments, the subject has a high BMI, for example, 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 years old, 45 years old, 50 years old, 55 years old, 60 years old, 65 years old, 70 years old, 75 years old or 80 years old. In some embodiments, the subject suffers from malnutrition, for example, with high consumption of one or more of red meat and / or processed meat, trans fats, saturated fats and refined sugars, and / or low consumption of fruits and vegetables, complex carbohydrates and / or unsaturated fats. High consumption and low consumption can be defined, for example, as exceeding or falling short of the recommendations in the Dietary Guidelines for Americans 2020-2025, respectively, which can be obtained at dietaryguidelines.gov / sites / default / files / 2021-03 / Dietary_Guidelines_for_Americans-2020-2025.pdf. In some embodiments, the subject has high alcohol consumption, for example, an average of at least three, four, or five drinks per day (wherein the beverage is about one ounce or 30 ml of 80-proof liquor or equivalent). In some embodiments, the subject has a family history of cancer, for example, at least one, two, or three blood relatives have been 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 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 a full sibling).

[0428] Additionally, if a cancer is observed to be in remission following treatment, the present method can be used to monitor for residual disease or recurrence of disease.

[0429] Typically, the disease under consideration is a type of cancer, such as any of the cancers mentioned herein. The types and numbers of cancers that may be detected may include blood cancer, brain cancer, lung cancer, skin cancer, nose cancer, throat cancer, liver cancer, bone cancer, lymphoma, pancreatic cancer, skin cancer, intestinal cancer, rectal cancer, thyroid cancer, bladder cancer, kidney 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 nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular 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 myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), liver cancer, hepatoepithelial carcinoma (liver carcinoma), hepatoma, 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 cancer, oral squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary tumor, acinar cell carcinoma, prostate cancer, prostate adenocarcinoma, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric carcinoma, gastrointestinal stromal tumor (GIST), uterine cancer or uterine sarcoma.

[0430] The type and / or stage of cancer can be detected based on genetic variations including mutations, rare mutations, insertions / deletions, copy number variations, transversions, translocations, inversions, deletions, aneuploidy, partial aneuploidy, polyploidy, chromosomal instability, chromosomal structural changes, gene fusions, chromosome fusions, gene truncations, gene amplifications, gene duplications, chromosomal damage, DNA damage, abnormal changes in nucleic acid chemical modifications, abnormal changes in epigenetic patterns such as 5mC and 5mC spectra. Therefore, in some cases, the methods of the present invention can be used in combination with methods for detecting other genetic / epigenetic variations (e.g., methods used in methods for detecting or characterizing cancer or other methods described herein).

[0431] In some embodiments, the methods described herein include identifying the presence of a target region and / or DNA produced by a tumor (or neoplastic cell or cancer cell) or by a precancerous cell. In some embodiments, the methods described herein include determining the level of a target region and / or identifying the presence of DNA produced by a tumor (or neoplastic cell or cancer cell) or by a precancerous cell. In some embodiments, determining the level of a target region includes determining an increased level or decreased level of a target region, wherein the increased or decreased level of a target region is determined by comparing the level of the target region to a threshold level / value.

[0432] Genetic and / or epigenetic data can also be used to characterize specific forms of cancer. Cancer is usually heterogeneous in both composition and staging. Genetic and / or epigenetic profile data can allow characterization of a specific subtype of cancer, which may be important in the diagnosis or treatment of the specific subtype. This information can also provide clues about the prognosis of a specific type of cancer to a subject or practitioner, and allow the subject or practitioner to adjust treatment options based on the progression of the disease. Some cancers can progress and become more aggressive and genetically unstable. Other cancers can remain benign, inactive, or dormant. The systems and methods of the present disclosure can be used to determine disease progression.

[0433] In addition, the method of the present disclosure can be used to characterize the heterogeneity of the abnormal condition of the subject.Such a method may include, for example, generating a genetic and / or epigenetic spectrum derived from the cfDNA of the subject, wherein the genetic and / or epigenetic spectrum includes more than one data generated by copy number variation and rare mutation analysis.In some embodiments, the abnormal condition is cancer, such as cancer as described herein.In some embodiments, the abnormal condition can be a condition that causes a heterogeneous genome population.In the example of cancer, it is known that some tumors include tumor cells in different stages of cancer.In other examples, heterogeneity may include multiple lesions of the disease, such as one or more lesions (such as one or more tumor lesions) are the result of the transfer spread from the primary site of cancer.The tissue of the source can be used to identify organs affected by cancer, including primary cancer and / or metastatic tumors.

[0434] The method of the present invention can also be used to quantify the level of different cell types, such as immune cell types, including rare immune cell types, such as activated lymphocytes and myeloid cells in a specific differentiation stage. Such quantification can be based on the number of molecules corresponding to a given cell type in a sample. The sequence information obtained in the method of the present invention can include sequence reads of nucleic acids generated by a nucleic acid sequencer. In some embodiments, the nucleic acid sequencer performs pyrophosphate sequencing, single molecule sequencing, nanopore sequencing, semiconductor sequencing, synthetic sequencing, 5-letter sequencing, 6-letter sequencing, connection sequencing or hybrid sequencing to the nucleic acid to generate sequencing reads. In some embodiments, the method also includes grouping sequence reads into sequence read families, each family including sequence reads generated from nucleic acids in the sample. In some embodiments, the method includes determining that a subject from which a sample is obtained suffers from cancer or precancerous lesions or has the possibility of metastasis associated with a change in the ratio of the type of immune cells.

[0435] The method can be used to generate or analyze a fingerprint or data set that is a sum of genetic and / or epigenetic information from different cells in a heterogeneous disease. The data set may include separate or combined copy number variation, epigenetic variation, and mutation analysis.

[0436] The present method can be used for diagnosis, prognosis, monitoring or observation of cancer or other diseases. In some embodiments, the methods herein do not involve diagnosis, prognosis or monitoring of a fetus, and therefore do not involve non-invasive prenatal testing. In other embodiments, these methods can be used in pregnant subjects to diagnose, prognose, monitor or observe cancer or other diseases in unborn subjects, whose DNA and other polynucleotides can co-circulate with maternal molecules.

[0437] Non-limiting examples of other genetically based diseases, disorders, or conditions that are optionally assessed using the methods and systems disclosed herein include achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Charcot-Marie-Tooth disease (CMT), Cry-a-Cat syndrome, Crohn's disease, cystic fibrosis, Dercum's disease, Down syndrome, Duane syndrome, Duchenne muscular dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington 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 disease, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, Wilson disease, etc.

[0438] In some embodiments, the method can provide a measure of the extent of DNA damage by quantifying the region synthesized during end repair. The methods disclosed herein can also be used to quantify the level of DNA damage present in an original DNA sample. This is because the level of end repair will depend in part on the amount of DNA damage (e.g., nicks, cuts, and overhangs) present in the DNA, as it is this damage that can serve as a priming site for synthesis during end repair (see Figure 1-6 and corresponding description).

[0439] In some embodiments, the method also includes calculating a synthesis index, which is a quantitative measure of the region synthesized in the end repair. The synthesis index can be at a molecular level and / or at a sample level. The synthesis index can be the ratio of the sequencing data corresponding to the region synthesized. In some embodiments, the method also includes comparing the synthesis index with one or more reference values ​​to classify the DNA sample. Classification can be whether the DNA sample is derived from a subject suffering from or not suffering from cancer. The reference value can be derived from one or more control DNA samples known to have a specific property, such as derived from a subject known to suffer from cancer (e.g., a specific type of cancer). Reference values ​​can be obtained by performing a method for obtaining a synthesis index (i.e., using the same end repair, connection and sequencing method) on the control sample.

[0440] In some embodiments, the methods described herein include detecting the presence or absence of DNA derived from or originating from tumor cells using a sequence information set obtained as described herein at a preselected time point after a previous cancer treatment in a subject previously diagnosed with cancer. The method may also include determining a cancer recurrence score indicating the presence or absence of DNA derived from or originating from tumor cells of the subject.

[0441] In the case where a cancer recurrence score is determined, it can be further used to determine a cancer recurrence state. For example, when the cancer recurrence score is above a predetermined threshold, the cancer recurrence state may be at risk of cancer recurrence. For example, when the cancer recurrence score is below a predetermined threshold, the cancer recurrence state may be at a low or lower risk of cancer recurrence. In a specific embodiment, a cancer recurrence score equal to a predetermined threshold can result in a cancer recurrence state that is at risk of cancer recurrence or at a low or lower risk of cancer recurrence.

[0442] In some embodiments, the cancer recurrence score is compared to a predetermined cancer recurrence threshold, and when the cancer recurrence score is above the cancer recurrence threshold, the subject is classified as a candidate for subsequent cancer treatment, or when the cancer recurrence score is below the cancer recurrence threshold, the test subject is classified as a non-candidate for treatment. In specific embodiments, a cancer recurrence score equal to the cancer recurrence threshold can result in a classification as a candidate for subsequent cancer treatment or a non-candidate for treatment.

[0443] The methods discussed above may also include any compatible features or features described elsewhere herein, including in the section on methods of determining a subject's risk of cancer recurrence and / or classifying a subject as a candidate for subsequent cancer treatment. Methods of determining a subject's risk of cancer recurrence and / or classifying a subject as a candidate for subsequent cancer treatment

[0444] In some embodiments, the methods provided herein are or include methods for determining the risk of cancer recurrence in a subject. In some embodiments, the methods provided herein are or include methods for detecting the presence or absence of metastasis in a subject. In some embodiments, the methods provided herein are or include methods for classifying a subject as a candidate for subsequent cancer treatment.

[0445] Any such method may include collecting a sample (such as DNA, such as DNA derived from or derived from tumor cells) from a subject diagnosed with cancer at one or more preselected time points after one or more prior cancer treatments have been performed on the subject. The subject may be any subject described herein. The sample may comprise chromatin, cfDNA or other cellular material. The sample, such as a DNA sample, may be a tissue sample.

[0446] Any of such methods may include capturing more than one target region set from DNA from a subject, wherein the more than one target region set includes a sequence variable target region set and an epigenetic target region set, thereby generating a captured set of DNA molecules. The capture step may be performed according to any embodiment described elsewhere herein.

[0447] In any of such methods, prior cancer treatment may include surgery, administration of a therapeutic composition, and / or chemotherapy.

[0448] Any of such methods may include sequencing the captured DNA molecules, thereby generating a sequence information set.The captured DNA molecules of the sequence variable target region set may be sequenced to a greater sequencing depth than the captured DNA molecules of the epigenetic target region set.

[0449] Any of such methods may include using the sequence information set to detect the presence or absence of DNA derived from or derived from tumor cells at a preselected time point. Detection of the presence or absence of DNA derived from or derived from tumor cells may be performed according to any of its embodiments described elsewhere herein.

[0450] The method for determining the risk of cancer recurrence in a subject may include determining a cancer recurrence score, which indicates the presence or absence or amount of DNA (such as genomic regions and target regions of interest) derived from or derived from a tumor cell of a subject. The cancer recurrence score may be further used to determine a cancer recurrence state. For example, when the cancer recurrence score is higher than a predetermined threshold, the cancer recurrence state may be at a risk of cancer recurrence. For example, when the cancer recurrence score is lower than a predetermined threshold, the cancer recurrence state may be at a low or lower risk of cancer recurrence. In a specific embodiment, a cancer recurrence score equal to a predetermined threshold can be obtained to be at a risk of cancer recurrence or at a low or lower risk of cancer recurrence.

[0451] Methods for detecting the presence or absence of metastasis in a subject may include comparing the presence or level of tissue-specific cellular material with the presence or level of tissue-specific cellular material obtained from the subject at a different time, a reference level of tissue-specific cellular material, or a comparison cellular material. The methods herein may include an additional step of determining whether metastasis is present.

[0452] The method of classifying a subject as a candidate for a subsequent cancer treatment may include comparing the cancer recurrence score of the test subject to a predetermined cancer recurrence threshold, thereby classifying the subject as a candidate for a subsequent cancer treatment when the cancer recurrence score is above the cancer recurrence threshold, or classifying the subject as a non-candidate for 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 can result in a classification as a candidate for a subsequent cancer treatment or a non-candidate for treatment. In some embodiments, the subsequent cancer treatment includes chemotherapy or administration of a therapeutic composition.

[0453] Any of such methods can include determining a disease-free survival (DFS) period for the subject based on the cancer recurrence score; for example, the DFS period can be 1 year, 2 years, 3 years, 4 years, 5 years, or 10 years.

[0454] In some embodiments, obtaining a sequence variable target region sequence and determining a cancer recurrence score may include determining at least a first subscore indicating the amount of SNVs, indels, CNVs, and / or fusions present in the sequence variable target region sequence.

[0455] In some embodiments, the number of mutations selected from 1, 2, 3, 4, or 5 in the sequence variable target region is sufficient for the first subscore to result in the cancer recurrence score being classified as cancer recurrence positive. In some embodiments, the number of mutations is selected from 1, 2, or 3.

[0456] In some embodiments, obtaining an epigenetic target region sequence and determining a cancer recurrence score includes determining a second subscore indicating the amount of molecules (obtained from the epigenetic target region sequence) that represent an epigenetic state that is different from DNA found in a corresponding sample from a healthy subject (e.g., cfDNA found in a blood sample from a healthy subject, or DNA found in a tissue sample from a healthy subject, where the tissue sample is the same type of tissue as the tissue obtained from the subject). These abnormal molecules (i.e., molecules with an epigenetic state that is different from DNA found in a corresponding sample from a healthy subject) can be consistent with epigenetic changes associated with cancer (such as associated metastasis), for example, methylation of a hypermethylated variable target region and / or perturbed fragmentation of a fragmented variable target region, where "perturbed" means different from DNA found in a corresponding sample from a healthy subject.

[0457] In some embodiments, a ratio of molecules corresponding to the hypermethylated variable target region group and / or the fragmented variable target region group that is greater than or equal to a value in the range of 0.001%-10% indicating hypermethylation in the hypermethylated variable target region group and / or aberrant fragmentation in the fragmented variable target region group is sufficient for the subscore to be classified as positive for cancer recurrence. The range may be 0.001%-1%, 0.005%-1%, 0.01%-5%, 0.01%-2%, or 0.01%-1%.

[0458] In some embodiments, any of such methods may include determining a tumor DNA score based on the fraction of molecules in a sequence information group indicating one or more features indicating a source from a tumor cell. This can be used for molecules corresponding to some or all of the target regions, for example, including one or more of a hypermethylated variable target region, a hypomethylated variable target region, and a fragmented variable target region (hypermethylation of a hypermethylated variable target region and / or abnormal fragmentation of a fragmented variable target region can be considered to indicate a source from a tumor cell). This can be used for molecules corresponding to sequence variable target regions, for example, molecules containing changes consistent with cancer (such as SNVs, insertions / deletions, CNVs, and / or fusions). The tumor DNA score can be determined based on a combination of molecules corresponding to epigenetic target regions and molecules corresponding to sequence variable target regions.

[0459] The determination of a cancer recurrence score can be based at least in part on a tumor DNA score greater than 10 -11 to 1 or 10 -10 In some embodiments, a tumor DNA fraction greater than or equal to 10 is sufficient for the cancer recurrence score to be classified as positive for cancer recurrence. -10 Up to 10 -9 , 10 -9 Up to 10 -8 , 10 -8 Up to 10 -7 , 10 -7 Up to 10 -6 , 10 -6 Up to 10 -5 , 10 -5 Up to 10 -4 , 10 -4 Up to 10 -3 , 10 -3 Up to 10 -2 or 10 -2 Up to 10 -1 A tumor DNA fraction within a threshold range of 10 to 20 is sufficient for the cancer recurrence score to be classified as cancer recurrence positive. In some embodiments, a tumor DNA fraction greater than at least 10 -7The tumor DNA score of the threshold value of the tumor DNA score is sufficient to classify the cancer recurrence score as positive for cancer recurrence. The tumor DNA score can be determined to be greater than the threshold value based on the cumulative probability, such as the threshold value corresponding to any of the aforementioned embodiments. For example, if the cumulative probability that the tumor score is greater than the threshold value in any of the aforementioned 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, the sample is considered positive. In some embodiments, the probability threshold is at least 0.95, such as 0.99.

[0460] In some embodiments, the sequence information group includes sequence variable target region sequences and epigenetic target region sequences, and determining the cancer recurrence score includes determining a sub-score indicating the amount of SNVs, insertions / deletions, CNVs and / or fusions present in the sequence variable target region sequences and a sub-score indicating the amount of abnormal molecules in the epigenetic target region sequences, and combining the sub-scores to provide a cancer recurrence score. In the case of combined sub-scores, they can be combined in the following ways: thresholds are applied independently to each sub-score (e.g., greater than a predetermined number of mutations in the sequence variable target region (e.g., >1), and greater than a predetermined score of abnormal molecules in the epigenetic target region (i.e., molecules with epigenetic states different from those found in corresponding samples from healthy subjects; e.g., tumors), or training a machine learning classifier to determine a state based on more than one positive training sample and a negative training sample.

[0461] In some embodiments, a value for the combined score in 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.

[0462] In any embodiment in which the cancer recurrence score is classified as cancer recurrence positive, the cancer recurrence status of the subject may be at risk for cancer recurrence and / or the subject may be classified as a candidate for subsequent cancer treatment.

[0463] In some embodiments, the cancer is any of the cancer types described elsewhere herein, e.g., colorectal cancer.

[0464] Methods for monitoring cancer in a subject over time: collecting samples at two or more time points

[0465] In some embodiments, the methods of the invention can be used to monitor one or more aspects of a condition in a subject over time, such as the subject's response to receiving treatment for the condition (such as a chemotherapeutic or immunotherapeutic agent), the severity of the condition in the subject (such as the stage of cancer), recurrence of a condition (such as cancer), and / or the subject's risk of developing a condition (such as cancer) and / or to monitor the subject's health as part of a preventive health monitoring program (such as determining if and / or when the subject requires further diagnostic screening). In some embodiments, monitoring comprises analyzing at least two samples collected from the subject at at least two different time points as described herein.

[0466] Methods according to the present disclosure can be used to predict the response of a subject to a specific treatment option, such as over a period of time. As described elsewhere herein, successful treatment options can increase the amount of cancer-related DNA sequences detected in the subject's blood, such as if the treatment is successful, because more cancers may die and shed DNA. In such instances, certain treatment options may be associated with the genetic profile of the cancer over time. This correlation can be used to select therapy.

[0467] As disclosed herein, in some embodiments, the amount of each of more than one cell type (such as an immune cell type) is determined based on sequencing and analysis (such as determination of epigenetic and / or genomic features) of DNA isolated from at least one cell-containing sample (such as a tissue sample or blood sample, e.g., a whole blood sample, a buffy coat sample, a white blood cell apheresis sample, or a PBMC sample) from a subject. In some embodiments, differences in the levels and / or presence of specific genetic and / or epigenetic features in DNA isolated from a blood sample from a subject can be used to quantify cell types, such as immune cell types, within a sample. Therefore, comparisons of genetic and / or epigenetic features disclosed in DNA isolated from blood samples collected from a subject at two or more time points can be used to monitor changes in the amount of cell types in a subject under different conditions (such as before and after treatment) or over time (e.g., as part of a preventive health monitoring program).

[0468] The disclosed method may include evaluating (such as quantifying) and / or explaining the cell types (such as immune cell types) present in one or more samples (such as tissue samples or blood samples, for example, whole blood samples, buffy coat samples, leukocyte single sample samples or PBMC samples) collected from a subject at one or more time points compared to a selected baseline value or reference standard (or a selected set of baseline values ​​or reference standards). A baseline value or reference standard may be the amount of cell types measured in one or more samples collected from a subject at one or more time points, such as before receiving treatment, before diagnosing a condition (such as cancer) or as part of a preventive health monitoring program (such as the average amount or amount range of cell types present in at least two samples). A baseline value or reference standard may be the amount of cell types measured in one or more samples collected from one or more subjects without conditions (such as healthy subjects without cancer), one or more subjects with a good response to treatment, or one or more subjects who have not received treatment at one or more time points (such as the average amount or amount range of cell types present in at least two samples). In certain embodiments, the baseline value or reference standard used is a standard or spectrum derived from a single reference subject. In other embodiments, the baseline value or reference standard used is a standard or spectrum derived from the average data derived from multiple reference subjects. In various embodiments, the reference standard can be a single value, mean, average, numerical mean or range of numerical mean values, numerical patterns or graphical patterns created by cell type quantity data derived from a single reference subject or from multiple reference subjects. The selection of a specific baseline value or reference standard, or the selection of one or more reference subjects, depends on, for example, the use to which the method described herein will be applied by a research scientist or clinician (such as a physician).

[0469] In some embodiments, one or more samples (such as tissue samples or blood samples, such as whole blood samples, buffy coat samples, leukocyte single sample samples or PBMC samples) can be collected from a subject at two or more time points to assess the change of cell types (such as the change of the amount of cell types) between two or more time points. In some embodiments, the sample collected at the first time point is a tissue sample or a blood sample, and the sample collected at a subsequent time point (such as a second time point) is a blood sample. In some embodiments, the sample collected at the first time point is a tissue sample, and the sample collected at a subsequent time point (such as a second time point) is a blood sample. By monitoring cell types and identifying the difference between the cell types in the samples collected from a subject at two or more time points, the present method can be used for example to determine the presence or absence of a condition (such as cancer), the subject's response to treatment, one or more features of a condition (such as a cancer stage) in a subject, the recurrence of a condition (such as cancer) and / or the risk of a subject developing a condition (such as cancer). Thus, in some embodiments, methods are provided in which the amount of cell types present in at least one sample (such as at least one tissue sample and / or at least one sample, e.g., a whole blood sample, a buffy coat sample, a single sample of white blood cells, or a PBMC sample) collected from a subject at one or more time points (such as before receiving treatment) is compared with the amount of cell types present in at least one sample collected from the subject at one or more different time points (such as after receiving treatment). The disclosed methods can allow patient-specific monitoring, such that, for example, differences in cell type quantification between samples collected from a subject at different time points can indicate changes (such as the presence or absence of a condition, response to treatment, prognosis, etc.) that are significant relative to the subject but may still fall within the normal range of a general healthy population.

[0470] As disclosed herein, methods for monitoring one or more aspects of a subject's condition over time are provided, such as, but not limited to, a subject's response to receiving treatment for a condition (such as a response to a chemotherapeutic or immunotherapeutic agent). In certain embodiments, one or more samples are collected from a subject at least 1-10, at least 1-5, at least 2-5, or 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 15, or at least 20 time points before the subject receives treatment. In certain embodiments, one or more samples are collected from a subject at least 1-10, at least 1-5, at least 2-5, or 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 15, or at least 20 time points after the subject receives treatment. Samples can continue to be collected from a subject during and / or after treatment to monitor the subject's response to treatment.

[0471] In some embodiments, samples are not collected from subjects before diagnosing a condition (such as cancer) or before receiving treatment. In such embodiments, where the course or stage of a condition (such as cancer) in a subject is monitored over time, cell types are compared between samples collected at least 2-10, at least 2-5, at least 3-6, or at least 2, such as 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 15, or at least 20 time points collected after the subject has been diagnosed and / or after the subject has received treatment. Samples can continue to be collected from subjects to monitor the subject's response to treatment during and / or after treatment.

[0472] In some embodiments of the disclosed method, at least once a year, such as about 1-12 times or about 2-6 times a year, such as about 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times or 12 times, one or more samples (such as one or more tissues, whole blood, buffy coat, leukocyte apheresis or PBMC samples) are collected from the subject. In other embodiments, less than once a year, such as about every 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 months, one or more samples are collected from the subject. In some embodiments, about once every 1-5 years or about once every 1-2 years, such as about every 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years or 5 years, one or more samples are collected from the subject.

[0473] In other embodiments of the disclosed method, at least once a week, such as every 1-4 days, 1-2 days or 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, one or more samples (such as one or more tissue samples or blood samples, for example, or one or more buffy coat samples, whole blood samples, leukocyte single sampling samples or PBMC samples) are collected from the subject. In certain embodiments, at least once a month, such as monthly 1-15 times, 1-10 times, 2-5 times or 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times, one or more samples are collected from the subject. In other embodiments, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or every 12 months, one or more samples are collected from the subject. In some embodiments, one or more samples are collected from a subject at least once a day, such as once, twice, three times, four times, five times, or six times a day. The selection of one or more sample collection time points (e.g., the frequency of sample collection) or the number of samples to be collected at each time point depends on the use to which the methods described herein will be applied by, for example, a research scientist or a clinician (such as a physician).

[0474] Treatment and related administration

[0475] In certain embodiments, methods disclosed herein relate to identifying and administering customized therapy, such as customized therapy for a patient. In some embodiments, a patient or subject suffers from a given disease, disorder or condition, such as any cancer or other condition described elsewhere herein. Substantially any cancer treatment (e.g., surgical treatment, radiotherapy, chemotherapy, immunotherapy, etc.) may be included as part of these methods. In certain embodiments, the treatment administered to a subject includes at least one chemotherapeutic agent. In some embodiments, the chemotherapeutic drugs may include alkylating agents (such as, but not limited to, chlorambucil, cyclophosphamide, cisplatin, and carboplatin), nitrosoureas (such as, but not limited to, carmustine and lomustine), antimetabolites (such as, but not limited to, fluorouracil, methotrexate, and fludarabine), plant alkaloids and natural products (such as, but not limited to, vincristine, paclitaxel, and topotecan), antitumor antibiotics (such as, but not limited to, bleomycin, doxorubicin, and mitoxantrone), hormonal agents (such as, but not limited to, prednisone, dexamethasone, tamoxifen, and leuprolide), and biological response modifiers (such as, but not limited to, Herceptin and Avastin, Erbitux, and rituximab). In some embodiments, the chemotherapy administered to the subject may include FOLFOX or FOLFIRI. In certain embodiments, the subject may be administered a treatment comprising at least one PARP inhibitor. In certain embodiments, PARP inhibitors may include OLAPARIB, TALAZOPARIB, RUCAPARIB, NIRAPARIB (trade name ZEJULA), etc. Typically, treatment includes at least one immunotherapy (or immunotherapeutic agent). Immunotherapy generally refers to a method of enhancing the immune response to a given cancer type. In certain embodiments, immunotherapy refers to a method of enhancing the T cell response to a tumor or cancer.

[0476] In some embodiments, immunotherapy or immunotherapeutic agents target immune checkpoint molecules. Certain tumors are able to evade the immune system by co-opting immune checkpoint pathways. Therefore, targeting immune checkpoints has become an effective method to combat the ability of tumors to evade the immune system and activate anti-tumor immunity against certain cancers. Pardoll, Nature Reviews Cancer, 2012, 12: 252-264.

[0477] In certain embodiments, immune checkpoint molecules are inhibitory molecules that reduce the signals involved in the response of T cells to antigens. For example, CTLA4 is expressed on T cells and plays 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. In addition, PD-1 ligands (PD-L1 or PD-L2) are usually upregulated on the surface of many different tumors, resulting in downregulation of anti-tumor immune responses in tumor microenvironments. In certain embodiments, inhibitory immune checkpoint molecules are CTLA4 or PD-1. In other embodiments, inhibitory immune checkpoint molecules are ligands of PD-1, such as PD-L1 or PD-L2. In other embodiments, inhibitory immune checkpoint molecules are ligands of 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).

[0478] Antagonists targeting these immune checkpoint molecules can be used to enhance antigen-specific T cell responses against certain cancers. Therefore, in certain embodiments, immunotherapy or immunotherapeutic agents are antagonists of inhibitory immune checkpoint molecules. 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 the 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 monocl...

Claims

1. A method, comprising: (a) subjecting a DNA sample to end repair to produce end-repaired DNA, wherein the end repair is performed with deoxynucleotide triphosphates (dNTPs), wherein at least one type of dNTP comprises a modified base; (b) performing a ligation reaction to connect the adaptor to the end-repaired DNA to generate an adapted DNA, wherein the ligation reaction also seals the nick present in the end-repaired DNA; (c) subjecting the adapted DNA to modification-sensitive sequencing to obtain sequencing data derived from the DNA sample, wherein the modification-sensitive sequencing is capable of identifying base modifications in the at least one type of dNTP; and (d) analyzing the sequence data obtained in step (c) to identify one or more regions in the end-repaired DNA synthesized during the end-repair by base modifications present in the at least one type of dNTP used in step (a). 2 . The method according to claim 1 , wherein the end repair is performed using a DNA polymerase that does not have 5′-3′ exonuclease activity and / or is not a strand-displacing DNA polymerase.

3. The method according to claim 2, wherein the DNA polymerase is T4 DNA polymerase, T7 DNA polymerase or Klenow fragment. 4 . The method according to claim 1 , wherein the end repair is performed using a DNA polymerase having 5′-3′ exonuclease activity and / or a strand-displacing DNA polymerase.

5. The method according to any one of claims 1 to 4, wherein the at least one type of dNTP containing a modified base includes a dNTP containing 4-methylcytosine (4mC), a dNTP containing 5-methylcytosine (5mC), a dNTP containing 5-hydroxymethylcytosine (5hmC), a dNTP containing N6-methyladenosine (6mA), a dNTP containing bromodeoxyuridine (BrdU) and / or a dNTP containing 8-oxoguanine (8oxoG).

6. The method according to any one of claims 1 to 5, further comprising performing an A-tailing reaction between steps (a) and (b).

7. The method of claim 6, wherein the end repair and the A-tailing reaction are performed in the same reaction mixture, optionally wherein the end repair and the A-tailing reaction are performed in a single tube and / or optionally wherein the end repair and the A-tailing reaction are performed without an intermediate clean-up step.

8. The method of claim 7, wherein the A-tailing is performed using a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, optionally wherein the DNA polymerase is HemoKlen Taq.

9. The method of claim 7, wherein the A-tailing is performed using a thermostable DNA polymerase.

10. The method of claim 7, wherein the A-tailing is performed using Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, Large Fragment or Tth DNA polymerase.

11. The method of claim 6, wherein the end repair and the A-tailing reaction are performed as separate reactions, wherein a reaction cleanup step is performed after the end repair and before the A-tailing reaction.

12. The method of claim 11, wherein the reaction cleaning step removes unincorporated dNTPs.

13. The method according to claim 11 or 12, wherein the A-tailing is performed using a DNA polymerase without 3'-5' exonuclease activity, optionally wherein the DNA polymerase is a Klenow fragment lacking 3'-5' exonuclease activity.

14. The method according to claim 7 or claim 11, wherein the A-tailing is performed using a DNA polymerase having 5'-3' exonuclease activity and / or being a strand-displacing DNA polymerase.

15. The method according to any one of claims 6-14, wherein the A-tailing reaction is performed at a temperature higher than the end repair, optionally wherein the end repair is performed at about 15°C-35°C and / or the A-tailing is performed at a temperature greater than about 60°C, and optionally wherein the temperature greater than 60°C is about 60°C-75°C.

16. The method according to any one of claims 1 to 5, wherein the ligation reaction is a blunt-end ligation reaction.

17. The method according to any one of claims 6 to 15, wherein the ligation reaction is a sticky end ligation reaction.

18. The method according to any one of claims 1-17, wherein the modification-sensitive sequencing comprises a conversion procedure that changes the base pairing specificity of the base or does not change the base pairing specificity of the base according to the modification status of the base.

19. The method of claim 18, wherein the modified base is a methylated cytosine, and wherein the conversion procedure converts the methylated cytosine, for example wherein the conversion procedure is a Tet-assisted conversion with a substituted borane reducing agent, optionally wherein the substituted borane reducing agent is 2-methylpyridine borane, pyridine borane, tert-butylamine borane or ammonia borane.

20. The method of claim 18, wherein the modified base is a methylated cytosine, and wherein the conversion procedure converts unmethylated cytosine, for example, wherein the conversion procedure is bisulfite conversion, oxidative bisulfite (Ox-BS) conversion, Tet-assisted bisulfite (TAB) conversion, APOBEC-coupled epigenetic (ACE) conversion, enzymatic methyl sequencing (EM-seq) or single enzyme 5-methylcytosine sequencing (SEM-seq) method.

21. The method of any one of claims 1-17, wherein the modification-sensitive sequencing comprises nanopore-based sequencing or single-molecule real-time (SMRT) sequencing.

22. The method of claim 21, wherein the modification-sensitive sequencing comprises nanopore-based sequencing, and wherein the at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA, a dNTP comprising BrdU, a dUTP, a dNTP comprising FldU, a dNTP comprising IdU, and / or a dNTP comprising EdU.

23. The method of claim 21, wherein the modification-sensitive sequencing comprises single-molecule real-time (SMRT) sequencing, and wherein the at least one type of dNTP comprising a modified base comprises a dNTP comprising 4mC, a dNTP comprising 5mC, a dNTP comprising 5hmC, a dNTP comprising 6mA and / or a dNTP comprising 8oxoG.

24. The method according to any one of claims 1-23, wherein the modified base is 5mC.

25. The method of any one of claims 1-23, wherein the modified base is 5hmC.

26. The method of any one of claims 1-25, wherein analyzing the sequence data obtained in step (c) comprises identifying regions containing the modified bases in a non-CpG sequence context and classifying these regions as regions synthesized during the end repair.

27. The method of any one of claims 1-23, wherein the modified base is not 5mC or 5hmC, and a region in the one or more regions is defined as: (i) a sequence between two non-modified bases spanning a modified base, wherein the two non-modified bases have the same identity as the modified base present in the at least one type of dNTP; and / or (ii) a sequence between a non-modified base and an end of a sequence read, wherein there are no additional non-modified bases between the non-modified base and the end of the sequence read, wherein the additional non-modified bases have the same identity as the modified base present in the at least one type of dNTP.

28. The method of any one of claims 1-23, wherein the modified base is a methylated cytosine, such as 5mC or 5hmC, and the region in the one or more regions is defined as: (i) a sequence between two non-methylated cytosines spanning a methylated non-CpG cytosine; and / or (ii) a sequence between an unmethylated cytosine and an end of a sequence read, wherein no additional unmethylated cytosines are present between said unmethylated cytosine and said end of said sequence read.

29. The method according to any one of claims 1 to 28, further comprising: (i) filtering out sequence data from the one or more regions identified as being synthesized during the end-repair so that these sequence data are not used for subsequent analysis; or (ii) marking the sequence data from the one or more regions identified as being synthesized during the end-repair as potentially containing artificial sequence data that may not be representative of the DNA sample.

30. The method of any one of claims 1-29, further comprising analyzing at least some of the sequence data corresponding to regions not identified as being synthesized during the end repair to detect the presence or absence of base modifications or mutations in the DNA sample.

31. A method according to any one of claims 1-30, wherein the method is used to detect the methylation state of cytosine in the DNA sample, and wherein analyzing the sequence data includes filtering out the one or more regions in the end-repaired DNA that are identified as being synthesized during the end repair, so that the one or more regions are not used to determine the methylation state of cytosine in the DNA sample.

32. The method of any one of claims 1-31, wherein the method is used to detect single nucleotide variants (SNVs) in the DNA sample, and wherein analyzing the sequence data comprises classifying all base calls within the one or more regions as having no double-stranded supporting evidence.

33. The method of any one of claims 1-32, wherein the method further comprises quantifying DNA damage in the DNA sample by identifying one or more regions in the end-repaired DNA that are synthesized during the end-repair.

34. The method of claim 33, wherein the level of DNA damage is used to predict whether a portion of the DNA in the DNA sample originates from a cancer cell.

35. The method of any one of claims 1-34, wherein the DNA sample comprises cell-free DNA (cfDNA), and the method further comprises analyzing the sequence data obtained in step (c) to determine the level of artifacts measured in the cfDNA.

36. The method of claim 35, further comprising comparing the measured level of artifact to one or more reference levels.

37. The method of claim 35 or 36, further comprising (i) using the measured level of artifacts and / or a comparison of the measured level of artifacts with one or more reference levels to predict whether a portion of the cfDNA is derived from cancer cells, or (ii) using the measured level of artifacts and / or a comparison of the measured level of artifacts with one or more reference levels to determine the likelihood that a portion of the cfDNA is derived from cancer cells.

38. The method of any one of claims 1-34, wherein the DNA sample comprises cell-free DNA or DNA from a formalin-fixed paraffin-embedded sample.

39. The method according to the preceding claim, wherein the DNA sample comprises cell-free DNA.

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

41. The method according to any one of the preceding claims, further comprising at least one DNA amplification step, optionally wherein the DNA amplification step is performed after step (b) and before step (c).

42. The method according to the preceding claim, wherein the DNA amplification step comprises PCR.

43. The method of any of the preceding claims, wherein the adaptor comprises a molecular barcode.

44. The method of any of the preceding claims, further comprising enriching more than one target region of the DNA prior to step (c).

45. The method of claim 44, wherein the more than one target regions comprise epigenetic target regions.

46. ​​The method of claim 45, wherein the epigenetic target region comprises a hypermethylated variable target region.

47. The method of claim 45 or 46, wherein the epigenetic target region comprises a hypomethylated variable target region.

48. The method of any one of claims 44-47, wherein the more than one target region comprises a sequence variable target region.

49. A kit comprising: a) a first reagent for end repair to produce end-repaired DNA, wherein the first reagent comprises at least one type of dNTP, the dNTP comprising a modified base; b) a second reagent for ligating an adaptor to the end-repaired DNA to generate adapted DNA, wherein the second reagent also seals the nick present in the end-repaired DNA; c) reagents for modification-sensitive sequencing capable of identifying base modifications in said at least one type of dNTP, and / or a DNA polymerase for incorporating said first reagent into DNA during end repair; and d) Library adapters with different molecular barcodes.

50. The kit of claim 49, further comprising more than one oligonucleotide probe and / or primer for sequencing.

51. A kit according to claim 49 or 50, wherein the first reagent comprises at least one type of dNTP containing a modified base selected from the following: dNTPs containing 4-methylcytosine (4mC), dNTPs containing 5-methylcytosine (5mC), dNTPs containing 5-hydroxymethylcytosine (5hmC), dNTPs containing N6-methyladenosine (6mA), dNTPs containing bromodeoxyuridine (BrdU), dNTPs containing 8-oxoguanine (8oxoG), dUTP, dNTPs containing fluorodeoxyuridine (FldU), dNTPs containing iododeoxyuridine (IdU), and / or dNTPs containing ethynyldeoxyuridine (EdU).

52. The kit according to any one of claims 49-51, wherein the DNA polymerase does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase.

53. The kit according to any one of claims 49-52, wherein the DNA polymerase is T4 DNA polymerase, T7 DNA polymerase or Klenow fragment.

54. The kit according to claim 49, wherein the DNA polymerase has 5'-3' exonuclease activity and / or is a strand-displacing DNA polymerase.

55. The kit according to any one of claims 49-54, further comprising reagents for performing an A-tailing reaction.

56. The kit according to the preceding claim, further comprising reagents for a clean-up step after the A-tailing reaction, optionally wherein the reagents for the clean-up step are used to remove unincorporated dNTPs.

57. The kit according to claim 55 or 56, wherein the reagent for performing the A-tailing reaction comprises a DNA polymerase that does not have 5'-3' exonuclease activity and / or is not a strand-displacing DNA polymerase, optionally wherein the reagent for performing the A-tailing reaction is HemoKlen Taq.

58. The kit according to any one of claims 55-57, wherein the reagents for performing the A-tailing reaction include Taq DNA polymerase, Tfl DNA polymerase, Bst DNA polymerase, Large Fragment or Tth DNA polymerase.

59. The kit of any one of claims 55-58, wherein the reagents for performing an A-tailing reaction comprise a thermostable DNA polymerase.

60. The kit of any one of claims 55-56, wherein the reagent for performing an A-tailing reaction comprises a DNA polymerase without 3'-5' exonuclease activity, optionally wherein the reagent for performing an A-tailing reaction is a Klenow fragment lacking 3'-5' exonuclease activity.

61. The kit according to any one of claims 55-56, wherein the reagent for performing an A-tailing reaction comprises a DNA polymerase having 5'-3' exonuclease activity and / or being a strand-displacing DNA polymerase.

62. The kit according to any one of claims 49 to 61, wherein: a) the more than one oligonucleotide probe selectively hybridizes to at least 5, 6, 7, 8, 9, 10, 20, 30, 40 or all genes selected from the group consisting of ALK, APC, BRAF, CDKN2A, EGFR, ERBB2, FBXW7, KRAS, MYC, NOTCH1, NRAS, PIK3CA, PTEN, RBI, TP53, MET, AR, ABL1, AKT1, ATM, CDH1, CSFIR, CTNNB1, ERBB4, EZH2, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, MLH1, MPL, NPM1, PDGFRA, PROC, P TPN11, RET, SMAD4, SMARCB1, SMO, SRC, STK11, VHL, TERT, CCND1, CDK4, CDKN2B, RAF1, BRCA1, CCND2, CDK6, NF1, TP53, ARID 1A, BRCA2, CCNE1, ESR1, RIT1, GATA3, MAP2K1, RHEB, ROS1, ARAF, MAP2K2, NFE2L2, RHOA and NTRKl; b) the library adaptors do not comprise flow cell sequences or sequences that allow formation of hairpin loops for sequencing; c) the library adaptors are blunt-ended and Y-shaped; and / or d) The length of the library adaptor is less than or equal to 40 nucleic acid bases.

63. The kit of any one of claims 49-62, further comprising instructions for performing the method of any one of claims 1-48.

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