Uracil reduction by polymerase

Abstract

Described herein are methods of removing false positive uracil resulting from the deamination of unmethylated cytosine in assays that use engineered cytosine deaminases to deaminate methylated cytosine, using enzymes that differentiate uracil residues, such as enzymes that differentiate uracil residues. The present invention relates to a method for the removal of false positive uracil residues from cytidine deaminase-mediated methylation sequencing assays, such as, for example, uracil-intolerant polymerases, uracil DNA glycosylase (UDG) and / or USERTM (uracil-specific excising reagents) enzymes, to remove false positive uracil residues from cytidine deaminase-mediated methylation sequencing assays.

Description

[0001] Continuing application data

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 437,413, filed on January 6, 2023, which is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically to the United States Patent and Trademark Office as an XML file named "531_002461WO01_ST26", 46 kilobytes in size and created on December 8, 2023. The information contained in the sequence listing is incorporated herein by reference. Technical Field

[0005] Embodiments of the present disclosure relate to preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in assays that use cytosine deaminase to selectively deaminate methylated cytosine. In particular, embodiments of the methods, compositions, and kits provided herein utilize uracil-intolerant polymerases and / or treatment with uracil DNA glycosylase (UDG) to reduce the likelihood of detecting such false positive conversions in the final sequencing library. Background Art

[0006] Modified DNA cytosines, including 5-methylcytosine (5mC), are intensively studied epigenetic modifications that play fundamental roles in human development and disease. Its genome-wide distribution differs between tissue types and between healthy and disease states. In recent years, 5mC has also become increasingly important as a clinical diagnostic tool. For example, its distribution in cell-free DNA (cfDNA) obtained from liquid biopsies can be used for tissue-specific prediction of early-stage cancer. Therefore, the development of methods to map 5mC with single-base resolution with minimal loss of sample DNA quantity, quality, and complexity has received close attention.

[0007] 5mC bases treated with cytosine deaminase generate thymine bases, thereby providing a signal for evaluating the sequence-specific methylation state of cytosine during sequencing. APOBEC3A is a cytidine deaminase that recognizes single-stranded DNA and catalyzes the deamination of cytosine (C) to uracil (U), 5-methylcytosine (5mC) to thymine (T), and 5-hydroxymethylcytosine to 5-hydroxymethyluracil. Protein engineering of APOBEC3A has produced mutant APOBEC proteins that are selective for the deamination of 5mC, with reduced activity for the deamination of C, but the residual activity for the deamination of C still exists. This undesirable deamination of unmethylated cytosine leads to false positive detection of 5mC (and 5hmC), in which uracil bases are interpreted as thymine bases in the assay. Summary of the invention

[0008] In one aspect, the present disclosure describes a method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay that uses a cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: providing a sample comprising DNA library fragments, wherein the cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5' and 3' library adapters; subjecting the sample comprising the DNA library fragments to at least one round of second strand synthesis by contacting the sample with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA library fragments that do not contain uracil. In some aspects, the method further comprises: performing polymerase chain reaction (PCR) amplification on the sample of double-stranded DNA library fragments not containing uracil using a uracil-resistant polymerase or a uracil-intolerant polymerase. In some aspects, the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi.

[0009] In one aspect, the present disclosure describes a method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay that uses a cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: providing a sample comprising library DNA fragments, wherein the cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5'-end and 3'-end library adapters; and The sample is contacted with uracil DNA glycosylase (UDG) and an endonuclease to produce a DNA library fragment cut at a uracil residue; and the sample of the DNA library fragment cut at the uracil residue is amplified by polymerase chain reaction (PCR) by contacting the DNA library fragments contained in the uracil residue with a polymerase, dNTPs and primers complementary to the 5' end and 3' end library adapters under conditions for providing second strand synthesis, thereby producing a double-stranded DNA library fragment without uracil. In some aspects, the polymerase comprises a polymerase that is not resistant to uracil. In some aspects, the polymerase that is not resistant to uracil comprises KAPA HiFi, Ultra IIQ5 or PhusionHiFi.

[0010] In one aspect, the present disclosure describes a method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay that uses cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: providing a sample comprising original DNA library fragments, wherein the cytosine deaminase deaminates methylated cytosine, wherein the original DNA library fragments comprise 5' and 3' library adapters; and combining the sample comprising the original DNA library fragments with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters in a second step for providing a second DNA library fragment. The method comprises contacting the sample of the second strand of the single-stranded synthesis under the conditions of chain synthesis to perform second strand synthesis on the sample, thereby generating a DNA library fragment containing no uracil residues in the synthesized second strand; contacting the sample of the DNA library fragment containing no uracil residues in the synthesized second strand with an endonuclease to digest the original DNA library fragment, thereby generating a single-stranded synthesized second strand; and contacting the sample of the second strand of the single-stranded synthesis with a polymerase, dNTPs and primers complementary to the 5' end and 3' end library adapters under conditions for providing second strand synthesis to perform polymerase chain reaction (PCR) amplification on the sample of the second strand of the single-stranded synthesis, thereby generating a double-stranded DNA library fragment containing no uracil. In some aspects, the polymerase intolerant to uracil comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi. In some aspects, PCR amplification of the sample of the second strand of the single-stranded synthesis comprises contacting the sample of the second strand of the single-stranded synthesis with a uracil-resistant polymerase.

[0011] In one aspect, the present disclosure describes a method for removing DNA fragments containing uracil residues from a sample, the method comprising: providing a sample containing DNA fragments, wherein the DNA fragments contain 5' and 3' library adapters; and contacting the sample containing DNA library fragments with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis to perform polymerase chain reaction (PCR) amplification on the sample, thereby producing double-stranded DNA fragments that do not contain uracil. In some aspects, the method further comprises: performing polymerase chain reaction (PCR) amplification on the sample of double-stranded DNA library fragments that do not contain uracil using a uracil-resistant polymerase or a uracil-intolerant polymerase. In some aspects, the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5, or PhusionHiFi.

[0012] In one aspect, the present disclosure describes a method for removing DNA fragments containing uracil residues from a sample, the method comprising: providing a sample containing DNA fragments, wherein the DNA fragments contain 5' and 3' library adapters; and contacting the sample containing DNA fragments with uracil DNA glycosylase (UDG) and an endonuclease to produce DNA fragments cut at uracil residues; and contacting the DNA fragments cut at uracil residues with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis to amplify the sample containing DNA fragments cut at uracil residues by polymerase chain reaction (PCR), thereby producing double-stranded DNA fragments without uracil. In some aspects, the polymerase comprises a polymerase that is not resistant to uracil. In some aspects, the polymerase comprises a polymerase that is not resistant to uracil. In some aspects, the polymerase that is not resistant to uracil comprises KAPA HiFi, Ultra IIQ5, or Phusion HiFi.

[0013] In one aspect, the present disclosure describes a method for removing DNA fragments containing uracil residues from a sample, the method comprising: providing a sample containing original DNA fragments, wherein the original DNA fragments contain 5' and 3' library adapters; subjecting the sample containing the original DNA fragments to second strand synthesis by contacting the sample containing the original DNA fragments with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby producing DNA fragments that do not contain uracil residues in the synthesized second strands; contacting the sample containing the DNA fragments that do not contain uracil residues in the synthesized second strands with an endonuclease to digest the original DNA fragments, thereby producing single-stranded synthesized second strands; and subjecting the sample containing the single-stranded synthesized second strands to polymerase chain reaction (PCR) amplification by contacting the sample containing the single-stranded synthesized second strands with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby producing double-stranded DNA fragments that do not contain uracil. In some aspects, the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi. In some aspects, PCR amplifying the sample comprising the single-stranded synthesized second strand comprises contacting the sample comprising the single-stranded synthesized second strand with a uracil-resistant polymerase.

[0014] In some aspects of the methods disclosed herein, the DNA library fragments comprise single-stranded DNA library fragments. In some aspects, the DNA library fragments comprise double-stranded DNA library fragments.

[0015] In some aspects of the methods disclosed herein, the cytosine deaminase comprises an altered cytosine deaminase.

[0016] In some aspects, the cytosine deaminase of change is a member of following subfamily: AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, APOBEC3G subfamily, APOBEC3G subfamily, APOBEC3H subfamily or APOBEC4 subfamily, or their change. In some aspects, the cytosine deaminase of change comprises the APOBEC3A of change.

[0017] In some aspects, the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in a wild-type APOBEC3A protein and / or an amino acid substitution mutation at a position functionally equivalent to Tyr132 in a wild-type APOBEC3A protein. In some aspects, the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 and Tyr132 in a wild-type APOBEC3A protein.

[0018] In some aspects, the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine, or threonine.

[0019] In some aspects, the substitution mutation at a position functionally equivalent to Tyrl30 comprises a mutation to Ala, Val, or Trp.

[0020] In some aspects, the substitution mutation at a position functionally equivalent to Tyrl32 comprises a mutation to His, Arg, GIn, or Lys.

[0021] In some aspects, the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in a wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp.

[0022] In some aspects, the (Tyr / Phe)130 is Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:12.

[0023] In some aspects, the altered cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the rate at which cytosine (C) is converted to uracil (U) by deamination. In some aspects, the rate is at least 100 times greater.

[0024] In some aspects, the altered cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the rate at which 5-hydroxymethylcytosine (5hmC) is converted to 5-hydroxymethyluracil (5hmU) by deamination. In some aspects, the conversion of 5hmC to 5hmU by deamination is undetectable.

[0025] In some aspects, the altered cytidine deaminase comprises a ZDD motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1).

[0026] In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX24SW(S / T)PCX[2-4]CX6FX8LX5R(L / I)YX[8-11]LX2LX

[10] M (SEQ ID NO:2), wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is a Tyr (Y) amino acid of the ZDD motif.

[0027] In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises X[16-26]-GRXXTXLCYXV-X15-GXXXN-X12-HAEXXF-X14-YXXTWXXSWSPC-X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X[2-6]

[0028] (SEQ ID NO:3).

[0029] In some aspects, the altered cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0030] In some aspects of the methods disclosed herein, the DNA library fragments are about 100 bp to about 300 bp in length.

[0031] In some aspects of the methods disclosed herein, the method further comprises: sequencing the double-stranded DNA library fragments that do not contain uracil.

[0032] In some aspects of the methods disclosed herein, the method further comprises: treating the double-stranded DNA library fragments that do not contain uracil to generate a sequencing library. In some aspects, the method also comprises: sequencing the sequencing library.

[0033] On the one hand, the present disclosure describes a test kit, the test kit includes a cytosine deaminase and a polymerase that is not resistant to uracil. In some aspects, the polymerase that is not resistant to uracil includes KAPA HiFi, Ultra IIQ5 or Phusion HiFi. In some aspects, the cytosine deaminase includes the cytosine deaminase of change. In some aspects, the cytosine deaminase of change is a member of the following subfamily: AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, APOBEC3G subfamily, APOBEC3G subfamily, APOBEC3H subfamily or APOBEC4 subfamily, or their changes. In some aspects, the cytosine deaminase of change includes the APOBEC3A of change. In some aspects, the changed cytidine deaminase is included in an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe) 130 in the wild-type APOBEC3A protein and / or an amino acid substitution mutation at a position functionally equivalent to Tyr132 in the wild-type APOBEC3A protein. In some aspects, the changed cytidine deaminase is included in an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe) 130 and Tyr132 in the wild-type APOBEC3A protein. In some aspects, the substitution mutation at a position functionally equivalent to Tyr130 includes a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine or threonine. In some aspects, the substitution mutation at a position functionally equivalent to Tyr130 includes a mutation to Ala, Val or Trp. In some aspects, the substitution mutation at the position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln or Lys. In some aspects, the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp. In some aspects, the (Tyr / Phe)130 is Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:12. In some aspects, the altered cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination, and its conversion rate is greater than the rate at which cytosine (C) is converted to uracil (U) by deamination. In some aspects, the rate is at least 100 times greater.In some aspects, the cytidine deaminase of this change converts cytosine (C) into uracil (U) by deamination and converts 5-methylcytosine (5mC) into thymidine (T) by deamination, and its conversion rate is greater than the rate at which 5-hydroxymethylcytosine (5hmC) is converted into 5-hydroxymethyluracil (5hmU) by deamination. In some aspects, it is not possible to detect that 5hmC is converted into 5hmU by deamination. In some aspects, the cytidine deaminase of this change comprises the ZDD motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1). In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX24SW(S / T)PCX[2-4]CX6FX8LX5R(L / I)YX[8-11]LX2LX

[10] M (SEQ ID NO:2), wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is a Tyr (Y) amino acid of the ZDD motif. In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises X[16-26]-GRXXTXLCYXV-X15-GXXXN-X12-HAEXXF-X14-YXXTWXXSWSPC-X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X[2-6](SEQ ID NO:3). In some aspects, the altered cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0034] In one aspect, the present disclosure describes a kit comprising cytosine deaminase, uracil DNA glycosylase (UDG) and AP endonuclease. In some aspects, the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi. In some aspects, the AP endonuclease comprises endonuclease IV and / or AP endonuclease I. In some aspects, the cytosine deaminase comprises a changed cytosine deaminase. In some aspects, the changed cytosine deaminase is a member of the following subfamily: AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, APOBEC3G subfamily, APOBEC3G subfamily, APOBEC3H subfamily or APOBEC4 subfamily, or their changes. In some aspects, the changed cytosine deaminase comprises a changed APOBEC3A. In some aspects, the changed cytidine deaminase is included in an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe) 130 in the wild-type APOBEC3A protein and / or an amino acid substitution mutation at a position functionally equivalent to Tyr132 in the wild-type APOBEC3A protein. In some aspects, the changed cytidine deaminase is included in an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe) 130 and Tyr132 in the wild-type APOBEC3A protein. In some aspects, the substitution mutation at a position functionally equivalent to Tyr130 includes a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine or threonine. In some aspects, the substitution mutation at a position functionally equivalent to Tyr130 includes a mutation to Ala, Val or Trp. In some aspects, the substitution mutation at the position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln or Lys. In some aspects, the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp. In some aspects, the (Tyr / Phe)130 is Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:12. In some aspects, the altered cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination, and its conversion rate is greater than the rate at which cytosine (C) is converted to uracil (U) by deamination. In some aspects, the rate is at least 100 times greater.In some aspects, the cytidine deaminase of this change converts cytosine (C) into uracil (U) by deamination and converts 5-methylcytosine (5mC) into thymidine (T) by deamination, and its conversion rate is greater than the rate at which 5-hydroxymethylcytosine (5hmC) is converted into 5-hydroxymethyluracil (5hmU) by deamination. In some aspects, it is not possible to detect that 5hmC is converted into 5hmU by deamination. In some aspects, the cytidine deaminase of this change comprises the ZDD motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1). In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX24SW(S / T)PCX[2-4]CX6FX8LX5R(L / I)YX[8-11]LX2LX

[10] M (SEQ ID NO:2), wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is a Tyr (Y) amino acid of the ZDD motif. In some aspects, the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises X[16-26]-GRXXTXLCYXV-X15-GXXXN-X12-HAEXXF-X14-YXXTWXXSWSPC-X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X[2-6](SEQ ID NO:3). In some aspects, the altered cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0035] Unless otherwise indicated, the terms used herein should be understood to have their ordinary meanings in the relevant art. Several terms used herein and their meanings are listed below.

[0036] As used herein, the term "nucleic acid" is intended to be consistent with its use in the art, and includes naturally occurring nucleic acids or functional analogs thereof. Particularly useful functional analogs can hybridize with nucleic acids in a sequence-specific manner or can be used as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids generally have a backbone comprising a phosphodiester bond. Similar structures may have alternative backbone bonds, including any of a variety of backbone bonds known in the art. Naturally occurring nucleic acids generally have deoxyribose (e.g., present in deoxyribonucleic acid (DNA)) or ribose (e.g., present in ribonucleic acid (RNA)). Nucleic acids may include any of a variety of analogs of these sugar moieties known in the art. Nucleic acids may include natural or non-natural bases. In this regard, natural deoxyribonucleic acids may have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and ribonucleic acids may have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine. Useful non-natural bases that may be included in nucleic acids are known in the art. Unless expressly stated otherwise, the terms "template" and "target" when used in reference to a nucleic acid are intended as semantic identifiers of the nucleic acid in the context of the methods or compositions described herein and do not necessarily limit the structure or function of the nucleic acid.

[0037] As used herein, the term "target nucleic acid" is intended as a semantic identifier for a nucleic acid in the context of the methods or compositions or kits described herein, and does not necessarily limit the structure or function of the nucleic acid, unless otherwise expressly stated. Unless otherwise indicated, reference to a nucleic acid such as a target nucleic acid includes both single-stranded and double-stranded nucleic acids, and both DNA and RNA.

[0038] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. These terms should be understood to include analogs of DNA, RNA, cDNA, or antibody-oligonucleotide conjugates made from nucleotide analogs as equivalents, and are applicable to single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, i.e., complementary DNA or copy DNA produced by an RNA template, for example, by the action of a reverse transcriptase.

[0039] As used herein, the term "primer" and its derivatives generally refer to any polynucleotide that can hybridize with a target sequence of interest. Typically, a primer is used as a substrate to which nucleotides can be polymerized by a polymerase; however, in some embodiments, a primer can be incorporated into a synthetic nucleic acid chain and provide a site to which another primer can hybridize to initiate the synthesis of a new chain complementary to the synthetic nucleic acid molecule. A primer may include any combination of nucleotides or their analogs. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide. The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may include ribonucleotides, deoxyribonucleotides, their analogs, or their mixtures. These terms should be understood to include analogs of DNA or RNA made from nucleotide analogs as equivalents, and are applicable to single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, i.e., complementary DNA or copy DNA produced by an RNA template, for example, by the action of a reverse transcriptase. The term refers only to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), and triple-, double-, and single-stranded ribonucleic acid ("RNA").

[0040] As used herein, the term "flow cell" refers to a chamber comprising a solid surface through which one or more fluid reagents can flow. Examples of flow cells and related fluid systems and detection platforms that can be readily used in the methods of the present disclosure are described in, for example, Bentley et al., 2008, Nature 456:53-59; WO 04 / 018497; US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US2008 / 0108082. Exemplary flow cells and substrates for making flow cells that can be used in the methods and compositions as described herein include, but are not limited to, flow cells and substrates commercially available from Illumina, Inc. (San Diego, CA).

[0041] As used herein, the term "amplicon" when used to refer to nucleic acid, means a product of replicating the nucleic acid, wherein the product has a nucleotide sequence identical or complementary to at least a portion of the nucleotide sequence of the nucleic acid. Amplicon can be produced by any amplification method in a variety of amplification methods using nucleic acid or its amplicon as a template, and the amplification method includes, for example, PCR, rolling circle amplification (RCA), connection extension or ligase chain reaction. Amplicon can be a nucleic acid molecule with a single copy (for example, PCR product) of a specific nucleotide sequence or multiple copies of the nucleotide sequence (for example, the tandem product of RCA). The first amplicon of a target nucleic acid is generally a complementary copy. Subsequent amplicon is a copy formed by the target nucleic acid or by the first amplicon after generating the first amplicon. Subsequent amplicon can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.

[0042] As defined herein, "multiplex amplification" refers to the selective and non-random amplification of two or more target sequences in a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed so that some or all of the target sequences are amplified in a single reaction vessel. The "multiplicity" or "multiplicity" of a given multiplex amplification generally refers to the number of different target-specific sequences amplified during the single multiplex amplification. In some embodiments, the multiplicity may be about 12-fold, 24-fold, 48-fold, 96-fold, 192-fold, 384-fold, 768-fold, 1536-fold, 3072-fold, 6144-fold or higher. The amplified target sequence can also be detected by several different methods (e.g., gel electrophoresis, followed by densitometry, quantification with a bioanalyzer or quantitative PCR, hybridization with a labeled probe; incorporation of a biotinylated primer, followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates into the amplified target sequence).

[0043] As used herein, the term "amplification site" refers to a site in or on an array where one or more amplicons can be generated. An amplification site can also be configured to contain, retain or connect at least one amplicon generated at the site.

[0044] As used herein, the term "array" refers to a group of sites that can be distinguished from each other according to relative positions. Different molecules located at different sites of the array can be distinguished from each other according to the position of the site in the array. A single site of an array may contain one or more specific types of molecules. For example, a site may contain a single target nucleic acid molecule with a specific sequence, or a site may contain several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of an array may be different features located on the same substrate. Exemplary features include, but are not limited to, droplets, holes in a substrate, beads (or other particles) in or on a substrate, protrusions of a substrate, ridges on a substrate, or channels in a substrate. The sites of an array may be separate substrates each with different molecules. Different molecules attached to a separate substrate may be identified according to the position of the substrate on a surface associated with the substrate, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which separate substrates are located on the surface include, but are not limited to, those arrays with beads in the holes.

[0045] As used herein, the term "clonal population" refers to a nucleic acid population that is homologous relative to a particular nucleotide sequence. Homologous sequences are typically at least 10 nucleotides long, but can even be longer, including, for example, at least 50, 100, 250, 500 or 1000 nucleotides long. The clonal population can be derived from a single target nucleic acid or template nucleic acid. Typically, all nucleic acids in the clonal population will have the same nucleotide sequence. It should be understood that, without departing from clonality, a small amount of mutation (e.g., due to amplification artifacts) may occur in the clonal population.

[0046] The term "sensitivity" as used herein is equal to the number of true positives divided by the sum of true positives and false negatives.

[0047] The term "specificity" as used herein is equal to the number of true negatives divided by the sum of true negatives and false positives.

[0048] As used herein, "providing" in the context of a sample of protein, DNA or RNA, or a composition means preparing a sample of protein, DNA or RNA, or a composition, purchasing a sample of protein, DNA or RNA, or a composition, or obtaining a sample of protein, DNA or RNA, or a composition.

[0049] The term "isolated" as used herein refers to material that has been removed from its original environment (eg, the natural environment in the case of natural occurrence) and thus altered "by the hand of man" from its natural state.

[0050] As used herein, the terms "each," when used in reference to a collection of reference items, are intended to identify an individual item in the collection, but not necessarily every item in the collection unless the context clearly indicates otherwise.

[0051] As used in this specification and the appended claims, the term "or" is generally employed in a sense that includes "and / or" unless the context clearly dictates otherwise. The term "and / or" means one or all of the listed elements, or a combination of any two or more of the listed elements. The use of "and / or" in some cases does not imply that the use of "or" in other cases may not mean "and / or".

[0052] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably to mean one or more than one.

[0053] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. In addition, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present disclosure.

[0054] As used herein, “have / has / having,” “include / includes / including / comprise / comprises / comprising,” and the like are used in their open inclusive sense, and generally mean “include, but not limited to / includes, but not limited to or including, but not limited to.”

[0055] It should be understood that anywhere an embodiment is described herein with the language "having," "including," "comprising," etc., other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided. The term "consisting of" is meant to include and be limited to whatever follows the phrase "consisting of." That is, "consisting of" indicates that the listed elements are required or mandatory, and that other elements may not be present. The term "consisting essentially of" indicates that any elements listed after the phrase are included, and other elements in addition to those listed elements may be included, provided that those elements do not interfere with or contribute to the activities or actions specified in the disclosure of the listed elements.

[0056] "Suitable" conditions or "appropriate" conditions for an event to occur are conditions that do not prevent such an event from occurring. Thus, these conditions allow, enhance, promote and / or facilitate the event.

[0057] References throughout this specification to "one embodiment," "embodiment," "certain embodiments," or "some embodiments" and the like mean that a particular feature, configuration, composition, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present disclosure. Therefore, multiple appearances of such phrases throughout this specification do not necessarily refer to the same embodiment of the present disclosure. Furthermore, in one or more embodiments, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner.

[0058] Throughout the present disclosure, various aspects of the present disclosure can be presented in range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Therefore, the description of the range should be considered to have all possible sub-ranges disclosed specifically and a single numerical value within the range. For example, the description of a range such as 1 to 6 should be considered to have a specific disclosed sub-range (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.), and a single number within the range, such as 1, 2, 2.7, 3, 4, 4.5, 5, 5.3 and 6. Regardless of the breadth of the scope, this applies.

[0059] In the description herein, specific embodiments may be described in isolation for clarity.Unless otherwise explicitly stated that features of a specific embodiment are incompatible with features of another embodiment, certain embodiments may include a combination of compatible features described herein in conjunction with one or more embodiments.

[0060] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. Also, any combination of two or more steps may be performed simultaneously, as appropriate.

[0061] Unless otherwise indicated, all numbers used in the specification and claims expressing the amount of components, molecular weight, etc., are to be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the specification and claims are approximate values, which may vary depending on the desired properties sought to be obtained by the present invention. At least, and not attempting to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying customary rounding techniques.

[0062] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0063] In several places throughout this application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the cited lists serve only as representative groups and should not be construed as exclusive lists. It should be understood that specific embodiments, materials, amounts, and procedures should be broadly interpreted according to the scope and spirit of the invention as described herein.

[0064] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading unless so specified.

[0065] The above summary of the invention provided above of the present disclosure is not intended to describe each disclosed embodiment or each implementation of the present disclosure. The following description more specifically illustrates exemplary embodiments. In several places throughout the application, guidance is provided by lists of examples, which can be used in various combinations. In each case, the cited lists are used only as representative groups and should not be interpreted as exclusive lists. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A and Figure 1B : Overview of the enzymatic method described herein for preventing detection of false positive uracil residues. Figure 1A is a schematic diagram illustrating how library reads that do not contain false-positive uracil residues are amplified preferentially over library reads that contain false-positive uracil residues when a U-intolerant polymerase is employed. Figure 1B is a schematic diagram illustrating an alternative approach in which a uracil excision enzyme such as USER or UDG is used to remove uracil residues from library fragments, thereby preventing the incorporation of uracil residues into the final library reads.

[0067] Figure 2 : A binomial model predicting the incidence of false positive uracil residues. The proportion of library fragments containing at least one uracil is plotted at different fragment lengths using different off-target C>U conversion rates.

[0068] Figure 3 : Workflow diagram illustrating how false positive uracil bases can be removed by enzymatic deamination and uracil excision prior to library preparation.

[0069] Figure 4 : Observation of global CG methylation levels using an alternative uracil discrimination workflow. Global methylation levels of different gene sets when assayed using uracil-tolerant (+) polymerase, uracil-intolerant (-) polymerase, or both USER treatment and uracil (-) polymerase.

[0070] Figure 5: Library complexity of libraries processed using different uracil discrimination strategies. Uracil (+) and uracil (-) refer to uracil-tolerant polymerase and uracil-intolerant polymerase, respectively.

[0071] Figure 6 : Lambda global methylation values ​​for alternative polymerases in the methylation assay. The lambda control genome is completely unmethylated and the methylation signal indicates a false positive.

[0072] FIG. 7A to FIG. 7D : Sequencing metrics comparing the performance of Q5 and Q5U. Fig. 7A Shown is the false positive rate observed in the lambda genome in this experiment. Figure 7B Duplicate readings are presented. Figure 7C Average autosomal coverage over the human genome is presented. Fig.7D The median mean deviation (MAD) of coverage is presented.

[0073] Figure 8 : Correlation between regional methylation levels of CpG islands and EM-Seq data.

[0074] Fig. 9 : Visualization of DMRs identified by EMSeq and APOBEC assays using different polymerases.

[0075] Fig. 10A and Fig. 10B : The workflow diagram illustrates a method that involves a single extension using a uracil-intolerant polymerase to create copies of the original library fragment, followed by the use of a targeted endonuclease to cut or destroy the original library fragment, and then PCR using either a uracil-tolerant polymerase or a uracil-intolerant polymerase. Fig. 10A Utilize Fpg / OGG or Endo V. Fig. 10B Use either lambda exonuclease or T7 exonuclease. For lambda exonuclease, the original library 5' adapter should be labeled with a 5' phosphate to facilitate degradation. For T7 exonuclease, primers for U-intolerant polymerases should contain phosphorothioate bonds to confer stability DETAILED DESCRIPTION

[0076] The methods described herein utilize enzymes that discriminate against uracil residues, such as uracil-intolerant polymerases, uracil DNA glycosylase (UDG), and / or USER TM(Uracil-specific excision reagent) enzymes that remove false positive uracil residues from cytidine deaminase-mediated methylation sequencing assays. Cytidine deaminase catalyzes the deamination of cytosine bases. For example, the cytidine deaminase class APOBEC3A can deaminate cytosine to uracil and deaminate methylcytosine to thymine (Schutsky et al., 2017, Nucleic Acids Research; 45(13): 7655–65). Protein engineering of APOBEC3A has produced mutant APOBEC proteins that are selective for the deamination of 5mC, have reduced activity for the deamination of C, but still have residual activity for the deamination of C. These false positive conversions of APOBEC enzymes result in conversion to uracil (U), while true positive conversions result in conversion of 5mC to T. Examples of such engineered APOBEC3A cytidine deaminases include, for example, any of those described in International Patent Application No. PCT / US2023 / 017846, filed on April 7, 2023 (“Altered Cytidine Deaminases and Methods of Use”), the entire contents of which are hereby incorporated by reference.

[0077] Treatment with uracil-intolerant polymerase

[0078] In some embodiments of the methods described herein, the problem of false positive conversions of cytosine to uracil in cytosine deaminase-mediated methylation detection assays is addressed by utilizing a uracil-intolerant polymerase to reduce the likelihood of detecting such false positive conversions in the final sequencing library. Figure 1A A schematic diagram illustrating the uracil reduction by polymerase (RUBY) method is shown in . Briefly, using the RUBY method described herein, after treating a DNA fragment preparation from an input sample with a cytosine deaminase to deaminate 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) residues and possibly including one or more off-target conversions of cytosine to uracil, the DNA fragment preparation is subjected to at least one round of extension using a uracil-intolerant polymerase to selectively extend reads lacking false positive C>U deamination events ( Figure 1A ).

[0079] In some embodiments, the preparation of DNA fragments is subjected to an initial round of extension using a uracil-intolerant polymerase. The preparation of DNA fragments may then be subjected to additional rounds of extension using a uracil-intolerant polymerase or a uracil-tolerant polymerase. In some embodiments, after an initial round of extension using a uracil-intolerant polymerase, the preparation of DNA fragments may be subjected to polymerase chain reaction (PCR) amplification using a uracil-intolerant polymerase or a uracil-tolerant polymerase.

[0080] In some embodiments, the fragmented dsDNA sample can first be subjected to library preparation conditions based on adapter ligation to facilitate downstream next generation sequencing (NGS). Such dsDNA samples can include, for example, cfDNA, FFPE, or high quality gDNA samples. After the adapter is connected, the DNA sample can be treated with an engineered APOBEC enzyme to promote selective mC>T deamination. Importantly, using the RUBY method described herein, a single C>U deamination event within a library fragment will be detrimental to the amplification of the entire fragment.

[0081] The probability that a fragment contains a C>U deamination event depends on the rate of off-target cytosine deamination using a given APOBEC enzyme and reaction conditions. Using a binomial model, the probability that a random DNA fragment of a given length (in nucleotides) contains a single C>U event was calculated using a range of off-target deamination rates ( Figure 2 ). Based on this model, low levels of false positive deaminations can be tolerated while still retaining much of the complexity of the library. Additionally, fragment length can be used to improve performance by minimizing the probability that a fragment contains a FP event. Loss of library fragments can be particularly severe in regions with high GC content due to the higher density of C residues that may be incorrectly converted to uracil. Therefore, minimizing the baseline C>U deamination rate will provide the best performance of this strategy.

[0082] In some embodiments of the methods described herein, in addition to extension using a uracil-intolerant polymerase, uracil DNA glycosylase (UDG), endonuclease and / or USER TM The preparation of DNA fragments is treated with enzymes to further increase the stringency of uracil discrimination / removal. TM Treatment may be performed before or after extension with a uracil-intolerant polymerase.

[0083] To adjust the level of discrimination of uracil and the uniformity of coverage in the assay, any of a number of strategies can be employed. For example, optimization of the deaminase construct and the deamination reaction conditions can be used to minimize the amount of off-target C>U conversion. In addition, different uracil-intolerant polymerases with different levels of discrimination and different amplification biases can be used. Uracil-intolerant polymerases can be used in combination with uracil-tolerant polymerases to adjust the level of discrimination of uracil residues and optimize uniformity of coverage.

[0084] DNA polymerase

[0085] It is known that B-family polymerases commonly used for PCR amplification exhibit a "uracil look-ahead" effect that causes the polymerase to stall at uracil residues (Greagg et al., 1999, PNAS; 96(16):9045–50). Specifically, archaeal B-family polymerases including, but not limited to, from the organisms Pyrococcus furiosus (Pfu), Thermococcus kodakarensis (KOD), Thermococcus litoralis (Tli / Vent), Pyrococcus woesei (Pwo), and Thermococcus fumicolans (Tfu) contain this function. A skilled artisan can readily identify members of each of the subfamilies by using publicly available databases, such as the Protein Database available from the National Center for Biotechnology Information. Structural and sequence motifs responsible for this uracil look-ahead function have been reported. See, e.g., published U.S. Application No. 20060057682A1; Wardle et al., 2008, Nucleic Acids Research; 36(3):705–11; Kropp et al., 2021, ChemBioChem; 22(21):3060–66; and Fogg et al., 2002, Nat Struct Biol; 9:922–92.

[0086] Due to the uracil intolerance of these enzymes, commonly used existing methylation next generation sequencing (NGS) assays typically employ uracil-resistant (U-resistant) polymerases to accommodate the amplification of templates containing uracil residues. Examples of U-resistant polymerases include commercially available, but not limited to, exonuclease-deficient Taq polymerases, KapaU polymerases (Roche), Q5U polymerases (New England Biolabs), and Phusion U polymerases (Fisher Scientific). For U-intolerant polymerases, if uracil is detected in DNA, DNA synthesis will stop. Examples of commercially available U-intolerant polymerases include, but are not limited to, Kapa HiFi polymerases (Roche), Ultra II Q5 polymerases (NEB), and Phusion HiFi polymerases (Fisher Scientific).

[0087] Any of the many schemes that can be used to synthesize the complementary second DNA chain is compatible with the method described herein. A mixture of DNA polymerase, all four deoxyribonucleoside 5'-triphosphates (dNTPs) and appropriate primers are provided for synthesizing the second complementary chain. These four dNTPs include adenine (dATP), cytosine (dCTP), guanine (dGTP) and thymine (dTTP). Primers include but are not limited to primers complementary to the 3' end library adapters and random oligonucleotides of about 18 to 22 bases in length.

[0088] Degradation at uracil residues

[0089] In some embodiments, an alternative strategy for reducing false positive uracil residue detections utilizes enzymes that specifically cleave library fragments at uracil residues, such as, for example, uracil DNA glycosylase (UDG), endonucleases (such as endonuclease IV or AP endonuclease I), and / or USER TM enzyme mixture, thereby preventing the incorporation of uracil residues into the final library reads. Figure 1B This approach is shown in .

[0090] Some embodiments of the methods described herein remove false positive uracil residues prior to sequencing by first subjecting a preparation of DNA fragments to enzymatic deamination prior to library preparation. Figure 3 ). First, the input DNA can be minimally fragmented to produce fragments less than about 1 kb to facilitate proper enzymatic deamination, as long fragments may not be good substrates for the reaction. The fragmented DNA is then denatured to obtain a preparation of single-stranded DNA (ssDNA) fragments, which is then enzymatically deaminated using a cytidine deaminase, including an engineered cytidine deaminase. The ssDNA fragments are then treated with uracil DNA glycosylase (UDG / UNG) and an endonuclease such as endonuclease IV or AP endonuclease I.

[0091] Alternatively, you can use USER TM ssDNA fragments are treated with (uracil-specific excision reagent) enzyme, which is a mixture of Escherichia coli uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII. TM The enzyme combines these two enzymatic activities to create a single nucleotide gap at the position of a uracil residue. UDG catalyzes the excision of the uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact, and the lyase activity of endonuclease VIII cleaves the phosphodiester backbone on the 3' and 5' sides of the abasic site, releasing the abasic deoxyribose. TMExamples of enzymes include thermolabile USER II enzyme (NEB #M5508) and thermostable USER III enzyme (NEB #M5509).

[0092] Use UDG and endonuclease or USER TM Treatment results in the removal of uracil residues and cleavage of the phosphodiester backbone, thereby generating single-stranded fragments from which uracil residues have been removed, which can be used as substrates for single-stranded library preparation methods. Single-stranded library preparation methods are well known in the art and include ligation-based methods (Troll et al., 2019, BMC Genomics; 20(1): 1–14; and Raine et al., 2017, Nucleic Acids Research; 45(6): e36) and commercial kits ((xGen Methyl Sequencing DNA Library Preparation Kit and Adaptase, Integrated DNA Technologies).

[0093] Degradation of the original library strand

[0094] In some embodiments of the methods described herein, a single extension is performed using a U-intolerant polymerase to a preparation of DNA library fragments from an input sample that has been treated with cytosine deaminase to deaminize 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) residues and may include one or more off-target conversions of cytosine to uracil to create a copy of the original library fragment. Since the U-intolerant polymerase is stalled at any uracil residue present in the original library fragment, a complete copy of the original library fragment with complete adapters 5' and 3' adapters can only be obtained from the original fragment without uracil residues. The original library fragment is then cut or destroyed using a targeted endonuclease. The remaining copies of the original library fragment are then PCR amplified. U-resistant enzymes, U-intolerant enzymes, or other enzymes can be selected for PCR amplification. Fig. 10A and Fig. 10B The method is exemplified and involves a single extension using a uracil-intolerant polymerase to create copies of the original library fragments, followed by the use of a targeted endonuclease to cleave or destroy the original library fragments, followed by PCR amplification.

[0095] Various methods can be used to cut or destroy the original library fragments. For example, in some embodiments, inosine or 8-oxoguanine residues can be incorporated into the original ligation adapter. After the second chain is generated with a U-intolerant polymerase, the presence of 8-oxoguanine or inosine residues enables selective cutting of adapter sequences from the original library fragments. Formamidopyrimidine-DNA glycosylase (FPG) or oxoguanine glycosylase (OGG) can be used to cut adapter sequences containing 8-oxoguanine residues (see, e.g., Murphy and George, 2005, Biochem Biophys Res Commun; 329 (3): 869-872; and Murphy and Guo, 2010, Biochem Biophys Res Commun; 392 (3): 335-339) and endonuclease V can be used to cut adapter sequences containing inosine residues (see, e.g., Cao, 2013, Cell Mol Life Sci; 70 (17): 3145-56). In some embodiments, a 5' phosphate can be incorporated into the original library fragment, thereby marking it for degradation by lambda exonuclease. Also in some embodiments, primers containing phosphorothioate bonds can be used to generate a second strand using a U-intolerant polymerase, thereby conferring protection to that strand. Treatment with T7 exonuclease will preferentially degrade the original library fragment.

[0096] Uracil deglycosylation

[0097] Using the methods described herein, a preparation of double-stranded DNA (dsDNA) fragments or single-stranded DNA (ssDNA) fragments can be contacted with uracil-DNA-glycosylase. Uracil-DNA-glycosylase (UDG) (also known as uracil-N-glycosylase (UNG)) is a highly conserved repair enzyme that catalyzes the excision of uracil from single-stranded and double-stranded DNA containing uracil, but is inactive against RNA. It is a monomeric protein with relatively stable physicochemical properties, a small molecular weight of 25KDa, and is widely present in various prokaryotic and eukaryotic organisms. See, e.g. et al., 2019, Scientific Reports;9:17822; Schormann et al., 2014, Protein Sci;23:1667-1685; Zharkov et al., 2010, Mutation Research 685,11–20; Stivers et al., 2001, Arch Biochem Biophys;396,1–9; Parikh et al., 2000, Proc Natl Acad Sci USA;97:5083; Pearl, 2000, Mutat Res 460,165–181; Lindahl, 1982, Annu Rev Biochem;51:61–87; and Lindahl et al., 1977, J Biol Chem;252:3286-3294.

[0098] UDG excises uracil from DNA by hydrolyzing the N-glycosidic bond between the uracil base and the sugar-phosphate backbone in single-stranded and double-stranded DNA (Bellamy et al., 2007, Nucleic Acids Res; 35: 1478–1487; Slupphaug et al., 1996, Nature 384, 87–92; Stivers et al., 1999, Biochemistry; 38: 952–963; and Parikh et al., 2000, Mutat Res; 460: 183–199), resulting in the formation of an abasic site (AP site) with hemiacetal formation.

[0099] Various UDG enzymes are commercially available, including, for example, Escherichia coli uracil-DNA glycosylase (UDG) (New England Biolabs, catalog number M0280S, see the World Wide Web website neb.com / products / m0280-uracil-dna-glycosylase-udg#Product%20Information) and heat-labile uracil DNA glycosylase (UDG / UNG) isolated from psychrophilic marine bacteria (Yeasen Biotechnology (Shanghai) Co., Ltd., catalog number 10707ES, see the World Wide Web website yeasenbiotech.com / solutiondetail / 79?gclid=EAIaIQobChMI_Oie4unY-gIV3xCtBh0hRwGHEAAYASAAEgKsx_D_BwE). In some embodiments, UDG is commercially available.

[0100] Reaction conditions suitable for UDG-mediated excision of uracil from DNA include, but are not limited to, the concentration of single-stranded or double-stranded DNA substrates, pH, reaction temperature, reaction time, and the concentration of UDG enzyme. It is expected that UDG can work in substantially any buffer. Examples of useful buffers include, but are not limited to, 1X UDG reaction buffer (New England Biolabs, catalog number B0280S, see the World Wide Web website neb.com / products / m0280-uracil-dna-glycosylase-udg#Product%20Information), which is 20mM Tris-HCl, 1mM DTT, 1mM EDTA (pH 8 at 25°C). Uracil-DNA glycosylase is active over a wide pH range, with optimal activity at pH 8.0, does not require divalent cations, and is inhibited by high ionic strength (>200 μM). Uracil-DNA glycosylase is active at a temperature of 25°C to 37°C, and in some embodiments, the reaction can continue at a temperature of 25°C to 37°C. In some embodiments, the reaction can be continued at 37°C. In some embodiments, the reaction can be continued for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or any range thereof. In some embodiments, the reaction can include about 0.001 U / μl to about 1 U / μl UDG enzyme, where one unit is defined as the amount of enzyme that catalyzes the release of 60 pmol of uracil from double-stranded uracil-containing DNA per minute. The reaction is performed by catalyzing the release of 60 pmol of uracil from double-stranded uracil-containing DNA in a 37°C incubation period of 30 minutes. 4 -10 5 cpm / μg) in 50 μl of reaction [ 3 H]-uracil to measure activity (see the World Wide Web website neb.com / products / m0280-uracil-dna-glycosylase-udg#Product%20Information). In some embodiments, the reaction may include about 0.05U / μl UDG. In some embodiments, the reaction may include nucleic acid at a concentration of about 1ng to about 1ug of the input nucleic acid. In some embodiments, the reaction may include nucleic acid at a concentration of about 10pM to about 200nM. In some embodiments, the reaction may include nucleic acid at a concentration of about 200pM to about 20nM.

[0101] Using the methods described herein, target nucleic acids (also referred to herein as "DNA fragments" or "preparation of DNA fragments from an input sample") can be essentially any nucleic acid of known or unknown sequence.

[0102] The target nucleic acid is usually derived from the primary nucleic acid present in the sample (such as a biological sample). The primary nucleic acid can originate from DNA or RNA. The DNA primary nucleic acid can be derived from the double-stranded DNA (dsDNA) form (e.g., genomic DNA, genomic DNA fragments, cell-free DNA, etc.) of the sample, or can be derived from the single-stranded form of the sample. The RNA primary nucleic acid can be mRNA or non-coding RNA, e.g., microRNA or small interfering RNA. The prepared product of the DNA fragment from the input sample can be single-stranded or double-stranded DNA.

[0103] Primary nucleic acid molecules can represent the entire genetic complement of an organism, for example, genomic DNA molecules comprising both intron sequences and exon sequences, and non-coding regulatory sequences such as promoter sequences and enhancer sequences. Primary nucleic acid molecules can represent the entire genetic complement of a specific cell of an organism, for example, from tumor cells, genomic DNA molecules comprising both intron sequences and exon sequences, and non-coding regulatory sequences such as promoter sequences and enhancer sequences. In one embodiment, a specific subset of genomic DNA can be used, for example, a specific chromosome, a DNA associated with an open chromatin, a DNA associated with a closed chromatin, or a region of one or more specific sequences such as a specific gene (for example, targeted sequencing). In one or more embodiments, primary nucleic acid molecules can represent a specific subset of DNA, for example, a DNA with a specific sequence annealed to a primer (such as a primer for a target sequence or target enrichment). In one embodiment, a specific subset of DNA can be used, for example, cell-free DNA, and the DNA can include the DNA of a subject, including DNA from normal cells, DNA from diseased cells (such as tumor cells) and / or DNA from fetal cells.

[0104] Primary nucleic acid molecules can represent the entire transcriptome of cells of an organism, e.g., mRNA molecules. Primary nucleic acid molecules can represent the entire transcriptome of a specific cell of an organism, e.g., from a tumor cell or, e.g., a cell of a tissue. In one embodiment, primary nucleic acid molecules can represent a specific subset of mRNA, e.g., mRNA with a specific sequence annealing to a primer, such as a primer for a target sequence or target enrichment.

[0105] Samples (such as biological samples) may include nucleic acid molecules obtained from biopsy tissue, tumors, scrapings, swabs, blood, mucus, urine, plasma, semen, hair, laser capture microdissection, surgical excision and other clinical or laboratory samples. In some embodiments, the sample may be an epidemiological sample, an agricultural sample, a forensic sample or a pathogenic sample. In some embodiments, the sample may include cultured cells. In some embodiments, the sample may include nucleic acid molecules obtained from animals (such as humans or mammalian sources). In another embodiment, the sample may include nucleic acid molecules obtained from non-mammalian sources (such as plants, bacteria, viruses or fungi). In some embodiments, the source of nucleic acid molecules may be archived or extinct samples or species.

[0106] Other non-limiting examples of biological sample sources may include whole organisms and samples obtained from subjects or patients. Biological samples can be obtained from any biological fluid or tissue, and can be in various forms (including liquid fluids and tissues, solid tissues) and preservation forms (such as drying, freezing and fixing forms). Samples can be any biological tissue, cell or fluid. Such samples include but are not limited to sputum, blood, serum, plasma, blood cells (e.g., leukocytes), ascites, urine, saliva, tears, sputum, vaginal fluid (secretions), rinsing fluids obtained during medical procedures (e.g., pelvic rinsing fluids or other rinsing fluids obtained during biopsy, endoscopy or surgery), tissues, nipple extracts, core or fine needle biopsy tissue samples, body fluids containing cells, peritoneal fluid and pleural effusions or cells therefrom, and free floating nucleic acids such as cell-free circulating DNA. Biological samples may also include tissue sections, such as frozen or fixed sections adopted for histological purposes, or microdissected cells or their extracellular parts. In some embodiments, the sample may be a blood sample, such as a whole blood sample. In another example, the sample is an unprocessed dried blood spot (DBS) sample. In yet another example, the sample is a formalin fixed paraffin embedded (FFPE) sample. In yet another example, the sample is a saliva sample. In yet another example, the sample is a dried saliva spot (DSS) sample.

[0107] Exemplary biological samples from which target nucleic acids can be derived include, for example, those from eukaryotic organisms, e.g., mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans, or non-human primates; plants such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophilamelanogaster, mosquitoes, fruit flies, bees, or spiders; fish such as zebrafish; reptiles; amphibians such as frogs or African toads (Xenopus laevis); Dictyostelium discoideum; fungi such as Pneumocystis carinii. carinii), Takifugu rubripes, yeast, Saccharamoyces cerevisiae, or Schizosaccharomyces pombe; or protozoa, such as Plasmodium falciparum. The target nucleic acid may also be derived from a prokaryotic organism, such as a bacterium, Escherichia coli, Staphylococcus, or Mycoplasma pneumoniae; an archaea; a virus, such as hepatitis C virus or human immunodeficiency virus; or a viroid. The target nucleic acid may be derived from a homogenous culture or population of organisms as described herein, or alternatively from a collection of several different organisms (e.g., in a community or ecosystem).

[0108] In some embodiments, the biological sample includes a tissue processed to obtain the desired primary nucleic acid. In some embodiments, cells are used to obtain the desired primary nucleic acid. In some embodiments, cell nuclei are used to obtain the desired primary nucleic acid. The method may also include dissociating cells and / or separating the cell nuclei from cells. A method for separating cells and cell nuclei from tissues (WO 2019 / 236599) may be used.

[0109] In some embodiments, nucleic acids present in tissues, cells or isolated nuclei can be processed according to the desired reads. For example, nucleic acids can be fixed during processing, and effective fixation methods can be used (WO 2019 / 236599). Fixation can be used to preserve samples or keep analytes in contact with samples, cells or nuclei. The fixation method preserves and stabilizes the morphology and structure of tissues, cells and nuclei, inactivates proteolytic enzymes, strengthens samples, cells and nuclei, so they can withstand further processing and staining, and prevent pollution. Examples of methods that can be effectively fixed include but are not limited to whole genome sequencing of isolated nuclei and chromosome conformation capture methods (such as Hi-C). Common fixation methods include perfusion, immersion, freezing and drying (Srinivasan et al., Am J Pathol. December 2002; 161 (6): 1961–1971. doi: 10.1016 / S0002-9440 (10) 64472-0). In some embodiments such as whole genome sequencing, isolated nuclei can be treated to dissociate nucleosomes from DNA while leaving the nucleus intact, and methods for generating nuclei free of nucleosomes can be used (WO 2018 / 018008).

[0110] In some embodiments, a large number of primary nucleic acids (e.g., from multiple cells) can be used to generate sequencing libraries as described herein. In other embodiments, a single cell or a cell nucleus can be used as a source of primary nucleic acids to obtain sequence information from a single cell and a cell nucleus. Many different single-cell library preparation methods are known in the art, including but not limited to Drop-seq, Seq-well, and single-cell combined indexing ("sci-") methods. Companies that provide single-cell products and related technologies include but are not limited to: Illumina, 10X genomics, Takara Biosciences, BD biosciences, Biorad, 1cellbio, isoplexis, CellSee, nanoselect, and Dolomite bio. Sci-seq is a method framework that uniquely labels the nucleic acid contents of a large number of single cells or cell nuclei using split pool barcodes. Typically, the number of cell nuclei or cells can be at least two. The upper limit depends on the actual limitations of the equipment used in other steps of the method as described herein (e.g., multi-well plates, index number). The number of usable cell nuclei or cells is not intended to be limited, but can be counted in the billions.

[0111] The target nucleic acid used in the methods and compositions of the present disclosure can be obtained by fragmentation. Random fragmentation refers to the fragmentation of polynucleotide molecules carried out from primary nucleic acid samples in a disordered manner by enzymatic, chemical or mechanical methods. This fragmentation method is known in the art, and standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3rd edition) are used. In addition, random fragmentation is designed to produce fragments that are independent of the sequence identity or position of the nucleotides containing and / or surrounding the fracture. In one or more embodiments, random fragmentation is to produce a length of about 50 base pairs to a length of about 1500 base pairs (for example, a length of about 50 to 700 base pairs, a length of about 50 to 400 base pairs) by mechanical means (such as atomization or ultrasonic treatment). In some preferred embodiments, the length of the fragment is about 100 to 300 base pairs, or about 100 to 200 base pairs.

[0112] In some embodiments, the DNA fragment is a DNA library fragment. Any of the many available library preparation schemes is compatible with the methods described herein. The library can be a whole genome library or a targeted library. Libraries include, but are not limited to, sequencing libraries. A variety of sequencing library methods are known to the skilled person (see, for example, Sequencing Methods Review, which is available on the World Wide Web at illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / sequencing-methods-review.pdf). For example, library preparation can be used with any of a variety of next-generation sequencing platforms, such as, for example, by synthesis platforms. ION TORRENT TM Sequencing is performed. For example, established ligase-dependent methods or transposon-based methods can be used (see, for example, Head et al., 2014, Biotechniques; 56(2):61 and Bruinsma et al., 2019, BMC Genomics; 19:722) and many kits for preparing sequencing libraries by these methods are commercially available from multiple suppliers.

[0113] DNA fragments, including DNA library fragments, can be prepared from input sample materials, so that adapter sequences are connected to the fragments to facilitate downstream workflow steps, such as degradation, amplification and / or sequencing of the second chain. For example, a universal amplification sequence (e.g., a sequence present in a universal adapter) can be placed at the end of each nucleotide fragment to facilitate amplification. Methods for connecting adapters to nucleic acids are known to those skilled in the art. For example, connection can be performed by using tag fragmentation of a transposase complex (WO 2016 / 130704) or by using standard library preparation techniques for connection (U.S. Patent Publication No. 2018 / 0305753). The addition of adapters can occur before or after the target nucleic acid is treated with cytidine deaminase and / or uracil deglycosylase.

[0114] The adapter sequence may include a 5' and / or 3' adapter sequence. The adapter may be connected to only one end of the DNA fragment, such as the 5' and / or 3' end, or to both ends. As used herein, the term "adapter" and its derivatives (e.g., universal adapters) generally refer to any linear oligonucleotide that can be connected to a target nucleic acid. The adapter may be a single-stranded or double-stranded DNA, or may include both a double-stranded region and a single-stranded region. The adapter may include: a universal sequence that is substantially identical or substantially complementary to at least a portion of a primer (e.g., a universal primer); an index (also referred to herein as a barcode or label), which is used to help downstream error correction, identification or sequencing; and / or a unique molecular identifier. In some embodiments, the adapter is not substantially complementary to the 3' end or 5' end of any target sequence present in the sample. In some embodiments, the adapter sequence may have one or more phosphorothioate bonds at the 5' end of the adapter sequence. In some embodiments, a suitable adapter length is in the range of about 6 to 100 nucleotides, about 12 to 60 nucleotides, or about 15 to 50 nucleotides. For example, the terms "adaptor" and "adapter" are used interchangeably. As used herein, the term "universal" when used to describe nucleotide sequences refers to sequence regions that are common to two or more nucleic acid molecules, where these molecules also have sequence regions that are different from each other. Non-limiting examples of universal capture sequences include sequences that are identical or complementary to the P5 and P7 primers. When referring to universal capture sequences or capture oligonucleotides, the terms "P5" and "P7" may be used. The terms "P5'" (P5 with a prime) and "P7'" (P7 with a prime) refer to the complementary sequences of P5 and P7, respectively. It should be understood that any suitable universal capture sequence or capture oligonucleotide can be used in the methods presented herein, and the use of P5 and P7 is merely an exemplary embodiment. The use of capture oligonucleotides such as P5 and P7 or their complementary sequences on a flow cell is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957. For example, any suitable forward amplification primer, whether immobilized or in solution, can be used in the methods presented herein for hybridization with complementary sequences and amplification of sequences. Similarly, any suitable reverse amplification primer, whether immobilized or in solution, can be used in the methods presented herein for hybridization with complementary sequences and amplification of sequences. Those skilled in the art will understand how to design and use primer sequences suitable for capturing and / or amplifying nucleic acids presented herein.

[0115] The DNA fragments including DNA library fragments may have an average chain length that is desired or applicable to the specific application of the method, composition or kit set forth herein. For example, the average chain length may be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides or 50 nucleotides. Alternatively or in addition thereto, the average chain length may be greater than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides or 100,000 nucleotides. The average chain length of the DNA fragment population may be within the range between any maximum and minimum values ​​set forth above.

[0116] In some embodiments, the DNA fragments comprising the DNA library fragments may have a shorter length, such as a length of about 50 nucleotides to about 500 nucleotides, a length of about 50 nucleotides to about 300 nucleotides, a length of about 50 nucleotides to about 250 nucleotides, a length of about 50 nucleotides to about 200 nucleotides, a length of about 50 nucleotides to about 100 nucleotides, a length of about 100 nucleotides to about 200 nucleotides, a length of about 100 nucleotides to about 250 nucleotides, a length of about 100 nucleotides to about 300 nucleotides, or a length of about 100 nucleotides to about 500 nucleotides. By minimizing the number of potential false positive uracils that may be present in any single DNA fragment, a shorter fragment length may be used to maximize the overall performance of enzymatic error correction. By minimizing the number of false positives in each fragment, the probability of successfully repairing the library fragment and mediating its downstream amplification may be increased, thereby maintaining library quality and diversity.

[0117] Cytosine deaminase

[0118] Using the methods described herein, a sample comprising single-stranded DNA (ssDNA) fragments can be contacted with a cytosine deaminase to deaminate methylated cytosines. In some embodiments, the sample comprising single-stranded DNA (ssDNA) fragments is a preparation of denatured library fragments. In some embodiments, the library fragments can include 5' and / or 3' adapter sequences.

[0119] As used herein, "cytidine deaminase enzyme" refers to an enzyme that deaminates cytosine and / or one or more cytosine derivatives. Deamination occurs at the amino group at the C4 position of cytosine or a cytosine derivative. For example, a cytidine deaminase can deaminate cytosine to form U, can deaminate mC to form T, and / or can deaminate hydroxymethylcytosine (hmC) to form hmU. A non-limiting example of an enzyme that can deaminate cytosine to form U, can deaminate mC to form T, and / or can deaminate hmC to form hmU is a catalytic polypeptide apolipoprotein B mRNA editing enzyme (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like, APOBEC). Non-limiting examples of such APOBECs include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4. As used herein, the term "methylcytosine" or "mC" refers to a cytosine that includes a methyl group (-CH3 or -Me). The methyl group may be located at position 5 of cytosine, in which case mC may be referred to as 5mC.

[0120] In some embodiments, when compared with a reference cytidine deaminase, the cytidine deaminase is a modified cytidine deaminase that is recombinantly engineered to include a substitution mutation at one or more residues. The modified cytidine deaminase may be based on a member of the AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3 subfamily (e.g., 3A subfamily, 3B subfamily, 3C subfamily, 3D subfamily, 3F subfamily, 3G subfamily, or 3H subfamily) or APOBEC4 subfamily. Those skilled in the art will readily appreciate that the modified or engineered cytidine deaminase described herein is not naturally occurring. In some embodiments, the modified or engineered cytidine deaminase exhibits a selective deamination of mC.

[0121] The altered cytidine deaminase may be one of three types of altered cytidine deaminases. One type of altered cytidine deaminase preferentially deaminates 5mC rather than C (i.e., converts 5mC to T at a rate higher than that of converting C to U), and is referred to herein as having "cytosine defective deaminase activity". The second type of altered cytidine deaminase preferentially deaminates C rather than 5mC (i.e., converts C to U at a rate higher than that of converting 5mC to T), and is referred to herein as having "5mC defective deaminase activity". The third type of altered cytidine deaminase preferentially deaminates C and 5mC to U and T, respectively, and has significantly reduced deamination degrees of 5hmC, 5fC, and 5caC. The third type is referred to herein as having "5hmC defective deaminase activity". Unless the context indicates otherwise, reference to an altered cytidine deaminase includes an altered cytidine deaminase having cytosine-deficient deaminase activity, an altered cytidine deaminase having 5mC-deficient deaminase activity, and an altered cytidine deaminase having 5mC-deficient deaminase activity.

[0122] The cytidine deaminase of change includes apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) and activation-induced cytidine deaminase (AID). Wild-type APOBEC and AID cytidine deaminase have the activity of deaminating cytidine (C) of DNA and / or RNA to form uridine (U). When compared with the wild-type enzyme, the cytidine deaminase of the change of the present disclosure has the C, 5mC and / or 5hmC deamination rate of change. The cytidine deaminase of the present disclosure may be referred to as "the cytidine deaminase of change", "recombinant cytidine deaminase", "mutant cytidine deaminase" or "modified cytidine deaminase" herein, and refers to any one of the cytidine deaminase of the change described herein, the cytidine deaminase of the change includes one or more changes from a reference (i.e., wild-type) amino acid sequence, and the change provides the unexpected properties of the deamination profile of the change, for example, changing its ability to deaminize a form of cytosine in preference to another form.

[0123] Whether a protein has cytidine deaminase activity can be determined by an in vitro assay. One example of an in vitro assay is based on digestion with the restriction enzyme SwaI. A protein that can deaminize 5mC to thymidine has cytidine deaminase activity.

[0124] The altered cytidine deaminase that preferentially deaminates 5mC over C (i.e., has cytosine-deficient deaminase activity) can have a catalytic efficiency for a 5mC substrate that is at least 10 times, at least 50 times, or at least 100 times greater than that for a C substrate. In one embodiment, the altered cytidine deaminase that preferentially deaminates 5mC over C has a catalytic efficiency for a 5mC substrate that is no more than 1500 times greater than that for a C substrate.

[0125] The catalytic efficiency of the altered cytidine deaminase that preferentially deaminates C rather than 5mC (i.e., has 5mC-deficient deaminase activity) for C substrates can be at least 10 times, at least 50 times, or at least 100 times greater than the catalytic efficiency for 5mC substrates. In one embodiment, the catalytic efficiency of the altered cytidine deaminase that preferentially deaminates C rather than 5mC for C substrates is no more than 1500 times greater than the catalytic efficiency for 5mC substrates.

[0126] When compared to a wild-type cytidine deaminase (an altered cytidine deaminase that deaminates C and 5mC to U and T, respectively, and has a significantly reduced degree of deamination of 5hmC (i.e., has 5hmC-deficient deaminase activity)), the extent of deamination of 5hmC by the altered cytidine deaminase disclosed herein is reduced by at least 80%, at least 90%, or at least 99% compared to the wild-type cytidine deaminase. In one embodiment, deamination of 5hmC by the altered cytidine deaminase disclosed herein is undetectable using an assay such as one based on SwaI.

[0127] In certain embodiments, the altered cytidine deaminase of the present disclosure is based on a member of the APOBEC protein family. The altered cytidine deaminase of the present disclosure "based on" a member of the APOBEC protein family means that, compared to the reference APOBEC sequence, the altered cytidine deaminase is an APOBEC protein comprising one or more of the substitution mutations described herein. The altered cytidine deaminase of the present disclosure "based on" a member of the APOBEC protein family may also comprise conservative and / or non-conservative mutations as described herein.

[0128] The APOBEC protein family includes subfamilies AID, APOBEC1, APOBEC2, APOBEC3 (including 3A, 3B, 3C, 3D, 3F, 3G, 3H) and APOBEC4. The modified cytidine deaminase of the present disclosure may be based on members of the AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3 subfamily (e.g., 3A subfamily, 3B subfamily, 3C subfamily, 3D subfamily, 3F subfamily, 3G subfamily or 3H subfamily) or APOBEC4 subfamily. The modified cytidine deaminase of the present disclosure may be based on members of the APOBEC protein family from vertebrates such as mammals. Examples of mammals include, but are not limited to, rodents, primates, rabbits, cattle (e.g., cows), swine (e.g., pigs) and equines (e.g., horses). An example of primates is humans and chimpanzees.

[0129] The APOBEC protein family is a member of a large cytidine deaminase superfamily that contains a canonical zinc-dependent deaminase (ZDD) signature motif embedded within a core cytidine deaminase fold. The fold consists of a five-stranded mixed β(b)-sheet surrounded by six α(a) helices in the order a1-b1-b2-a2-b3-a3-b4-a4-b5-a5-a6 (Salter et al., 2016, Trends Biochem Sci; 41(7):578–594. doi:10.1016 / j.tibs.2016.05.001; Salter et al., 2018, Trends Biochem Sci; 43(8):606-622 doi.org / 10.1016 / j.tibs.2018.04.013). The core structure of each cytidine deaminase domain of APOBEC protein contains a spatial arrangement of catalytic central residues of a highly conserved zinc binding motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1) (referred to herein as a ZDD motif, wherein X is any amino acid, and the subscript range of the number after X refers to the number of amino acids) (Salter et al., 2016, Trends Biochem Sci; 41(7): 578–594. doi: 10.1016 / j.tibs.2016.05.001). Without being limited by theory, H and two C residues coordinate with the Zn atom, and the E residue polarizes the water molecules near the Zn atom for catalysis (Chen et al., 2021, Viruses; 13: 497).

[0130] Some members of the APOBEC protein family (e.g., AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3C subfamily, APOBEC3H subfamily, and APOBEC4 subfamily) contain one copy of the ZDD motif. Other members of the APOBEC protein family (e.g., APOBEC3B subfamily, APOBEC3D subfamily, APOBEC3F subfamily, and APOBEC3G subfamily) contain two copies of the ZDD motif, but usually only the C-terminal copy is active (Salter et al., 2016, Trends Biochem Sci; 41 (7): 578–594.doi: 10.1016 / j.tibs.2016.05.001). Therefore, the modified cytidine deaminase disclosed herein contains one or two ZDD motifs. In one embodiment, the modified cytidine deaminase based on the member of the APOBEC3A subfamily contains the following ZDD motif: HXEX 24 SW(S / T)PCX[2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] M (SEQ ID NO: 2) (wherein X is any amino acid and the subscript number or number range after X refers to the number of amino acids) (Salter et al., 2016, Trends Biochem Sci; 41(7): 578–594).

[0131] In one embodiment, the altered cytidine deaminase disclosed herein is a member of the following subfamily: APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F and APOBEC3G, and may comprise one or more highly conserved sites that are part of the active site and within the ZDD motif SEQ ID NO: 1. These sites include tryptophan at position 98 and serine or threonine at position 99 (Kouno et al., 2017, Nat. Comm; 8: 15024).

[0132] In addition to the ZDD motif, members of the APOBEC protein family contain other highly conserved residues that are part of the active site but are not present as part of the ZDD motif SEQ ID NO:1. Members of the APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, and APOBEC3G subfamilies typically contain one or more of the following highly conserved sites that are part of the active site: arginine at position 28; histidine, asparagine, or arginine at position 29; serine or threonine, preferably threonine, at position 31; asparagine or aspartic acid at position 57; tyrosine or phenylalanine at position 130; asparagine or tyrosine at position 131; asparagine, tyrosine, or phenylalanine, preferably tyrosine, at position 132; and arginine or lysine at position 189 (Kouno et al., 2017, Nat. Comm; 8: 15024, DOI: 10.1038 / ncomms15024).

[0133] When compared to a reference cytidine deaminase, the modified cytidine deaminase of the present disclosure comprises a substitution mutation at one or more residues. The substitution mutation may be at the same position or a functionally equivalent position compared to the reference cytidine deaminase. The reference cytidine deaminase and the functionally equivalent positions are described in detail herein. Those skilled in the art will readily appreciate that the modified cytidine deaminase described herein is not naturally occurring.

[0134] The reference cytidine deaminase can be a member of the APOBEC protein family. Substantially any known member of the APOBEC protein family can be a reference cytidine deaminase. By using publicly available databases, such as the protein database (ncbi.nlm.nih.gov / protein) available at the National Center for Biotechnology Information, and searching for APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or when identifying members of the AID family (activation-induced cytidine deaminase), technicians can easily identify members of each subfamily. The wild-type reference cytidine deaminase has the activity of binding to single-stranded DNA (ssDNA) and deaminating the cytosine present on the ssDNA to convert it into uracil. In one embodiment, the wild-type reference cytidine deaminase has the activity of binding to single-stranded RNA (ssRNA) and deaminating the cytosine present on the ssRNA to convert it into uracil. Methods for determining whether a protein binds to ssDNA or ssRNA and deaminates the cytosine present are known to the skilled artisan.

[0135] In one embodiment, the altered cytidine deaminase has an amino acid sequence based on a reference sequence that is a member of the APOBEC protein family, including the ZDD motif H-[P / A / V]-EX [23–28] -PCX [2-4] -C (SEQ ID NO: 1) and at least one substitution mutation disclosed herein. Optionally, the altered cytidine deaminase comprises other active site residues disclosed herein. Non-limiting examples of reference cytidine deaminase proteins are shown in the table below.

[0136] In one embodiment, the altered cytidine deaminase has an amino acid sequence based on a reference sequence that is a member of the APOBEC3A subfamily and includes the ZDD motif HXEX 24 SW(S / T)PCX [2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] M (SEQ ID NO: 2) (wherein X is any amino acid, and the subscript number or number range after X refers to the number of amino acids) and at least one substitution mutation disclosed herein. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A). Optionally, the altered cytidine deaminase comprises other active site residues disclosed herein.

[0137] In one embodiment, the amino acid sequence of the altered cytidine deaminase comprises amino acids of a member of the APOBEC3A subfamily: X [16-26] -GRXXTXLCYXV-X 15 -GXXXN-X 12 -HAEXXF-X 14 -YXXTWXXSWSPC-X [2-4] -CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X 13 -LXXI-X [2-6] (SEQ ID NO: 3) (wherein X is any amino acid, and the subscript number or number range after X refers to the number of amino acids) or a subset thereof, and at least one substitution mutation disclosed herein. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine, such as a substitution mutation to alanine (A) or tryptophan (W).

[0138] In one embodiment, the amino acid sequence of the altered cytidine deaminase comprises amino acids of a member of the APOBEC3A subfamily: X 26 -GRXXTXLCYXV-X 15 -GX 16 -HAEXXF-X 14 -YXXTWXXSWSPC-X4-CA-X5-FL-X7-LXIFXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X 13 -LXXI-X6 (SEQ ID NO: 4) (wherein X is any amino acid, and the subscript number or number range after X refers to the number of amino acids present) or a subset thereof, and at least one substitution mutation disclosed herein. In one embodiment, the substitution mutation is a substitution mutation at the underlined tyrosine (Y), such as a substitution mutation to alanine (A) or tryptophan (W).

[0139] The substitution mutation can be at the same position or at a functionally equivalent position compared to the reference cytidine deaminase. "Functionally equivalent" means that the altered cytidine deaminase has an amino acid substitution at an amino acid position in the reference cytidine deaminase that is not present in the reference cytidine deaminase.

[0140] The enzyme has the same functional role as altered cytidine deaminase.

[0141] Typically, functionally equivalent substitution mutations in two or more different cytidine deaminases occur at homologous amino acid positions in the amino acid sequences of these cytidine deaminases. Therefore, the term "functionally equivalent" as used herein also encompasses mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutated amino acid is known. The positions of amino acid residues that are functionally equivalent and positionally equivalent in the amino acid sequences of two or more different cytidine deaminases can be identified based on sequence alignment and / or molecular modeling. For example, the tyrosine at residue 130 of the APOBEC3A protein of Homo sapiens, Pongopygmaeus, Nomascus leucogenys, Pan troglodytes, and Gorilla is equivalent to the tyrosine at residue 133 of the APOBEC3A protein from Macaca fascicularis in function and position. Functionally equivalent residues in cytidine deaminase can be readily identified by one skilled in the art.

[0142] In one embodiment, the altered cytidine deaminase has an amino acid sequence that is structurally similar to a reference cytidine deaminase disclosed herein. In one embodiment, the reference cytidine deaminase is a cytidine deaminase comprising an amino acid sequence listed in Table 1.

[0143] Table 1: Examples of APOBEC protein subfamily members .

[0144]

[0145]

[0146] UniProt, a database of protein sequence and functional information, is available at uniprot.org; GenBank, a collection of nucleotide sequences and their protein translations, is available at ncbi.nlm.nih.gov / protein / .

[0147] As used herein, an altered cytidine deaminase may be "structurally similar" to a reference cytidine deaminase if the amino acid sequence of the altered cytidine deaminase has a specified amount of sequence similarity and / or sequence identity compared to the reference cytidine deaminase.

[0148] The structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (e.g., a candidate altered cytidine deaminase described herein and a reference cytidine deaminase) to optimize the number of identical amino acids along the length of their sequences; in order to optimize the number of identical amino acids, gaps in either or both sequences are allowed when aligning, although the amino acids in each sequence must remain in their correct order. A candidate altered cytidine deaminase is a cytidine deaminase compared to a reference cytidine deaminase. A candidate altered cytidine deaminase having structural similarity to a reference cytidine deaminase and cytidine deaminase activity is an altered cytidine deaminase.

[0149] Unless otherwise indicated herein as modified, pairwise comparison analysis of amino acid sequences can be performed, for example, by the homology alignment algorithm of Smith and Waterman, 1981, Adv. Appl. Math; 2: 482, by the homology alignment algorithm of Needleman and Wunsch, 1907, J Mol Biol; 48: 443, by the search for similarity method of Pearson and Lipman, 1988, Proc Nat'l Acad Sci USA; 85: 2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.); or by visual inspection (see generally, Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented to 2004). One example of an algorithm suitable for determining structural similarity is the BLAST algorithm, which is described in Altschul et al., 1990, J Mol Biol; 215:403-410. Algorithms can be used to calculate percent sequence identity and percent sequence similarity between two sequences. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0150] In a comparison of two amino acid sequences, structural similarity can be expressed by a percentage of "identity" or can be expressed by a percentage of "similarity." "Identity" refers to the presence of identical amino acids. "Similarity" refers to the presence of not only identical amino acids, but also conservative substitutions. Therefore, in one embodiment, the amino acid sequence of a cytidine deaminase protein having sequence similarity to a reference sequence can contain conservative substitutions of the amino acids present in the reference sequence.

[0151] Conservative substitutions of amino acids in proteins can be selected from other members of the class to which the amino acid belongs. For example, it is well known in the field of protein biochemistry that amino acids belonging to a group of amino acids having a particular size or property (such as charge, hydrophobicity or hydrophilicity) can be substituted by another amino acid without changing the activity of the protein, particularly in regions of the protein that are not directly related to biological activity. For example, amino acids with nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine; amino acids with hydrophobic side chains include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; amino acids with polar side chains include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine and threonine; and amino acids with uncharged side chains include glycine, serine, cysteine, asparagine, glutamine, tyrosine and threonine.

[0152] Thus, as used herein, reference to a cytidine deaminase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs described herein, can include proteins having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference cytidine deaminase. Examples of altered cytidine deaminases having similarity to the reference amino acid sequence include those having, for example, at least 80%, at least 85%, at least 90%, or at least 95% similarity to SEQ ID NO: 5 and having an alanine at amino acid 130. Other examples of altered cytidine deaminases having similarity to a reference amino acid sequence include those having, e.g., at least 80%, at least 85%, at least 90%, or at least 95% similarity or identity to SEQ ID NO:6 and having an alanine at amino acid 130 and a histidine at amino acid 132.

[0153] Alternatively, as used herein, reference to a cytidine deaminase as described herein, such as reference to the amino acid sequence of one or more SEQ ID NOs described herein, can include proteins having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the reference cytidine deaminase. Examples of altered cytidine deaminases having identity to the reference amino acid sequence include those having, for example, at least 80%, at least 85%, at least 90%, or at least 95% similarity to SEQ ID NO: 5 and having an alanine (A) at amino acid 130. Other examples of altered cytidine deaminases having identity to a reference amino acid sequence include those having, for example, at least 80%, at least 85%, at least 90%, or at least 95% similarity or identity to SEQ ID NO:6 and having an alanine (A) at amino acid 130 and a histidine (H) at amino acid 132.

[0154] The cytidine deaminase of the change of the present disclosure can include a substitution mutation at a position of tyrosine (Y130) at position 130 in a member of the APOBEC3A subfamily that is functionally equivalent. Therefore, a member of the APOBEC3A subfamily and another candidate cytidine deaminase of the change from the APOBEC3A subfamily or a different APOBEC subfamily can be used to generate an alignment. In one embodiment, the candidate is selected from the APOPEC subfamily APOBEC1 or AID. An example of an algorithm that can be used to generate an alignment is Clustal O. In some APOBEC family proteins, the wild-type residue at a position functionally equivalent to Y130 is phenylalanine (F).

[0155] In another embodiment, the altered cytidine deaminase of the present disclosure comprises a ZDD motif HXEX that is functionally equivalent to a member of the APOBEC family, such as a member of the APOBEC3A subfamily. 24 SW(S / T)PCX [2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] Substitution mutation at the position of tyrosine (Y) of M (SEQ ID NO: 2). The underlined tyrosine (Y) of SEQ ID NO: 2 is the position functionally equivalent to tyrosine amino acid 130 of the wild-type APOBEC3A protein (SEQ ID NO: 12).

[0156] In one embodiment, a substitution mutation at a position functionally equivalent to Y130 increases cytidine deaminase activity and preferentially acts on 5mC compared to cytosine (i.e., has cytosine-deficient deaminase activity). The substitution mutation can be a mutation to alanine (A), glycine (G), phenylalanine (F), histidine (H), glutamine (Q), methionine (M), asparagine (N), lysine (K), valine (V), aspartic acid (D), glutamic acid (E), serine (S), cysteine ​​(C), proline (P) or threonine (T). For example, the altered cytidine deaminase may comprise SEQ ID NO: 9, wherein X is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T (and not Y), or may comprise SEQ ID NO: 10, wherein Z is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T (and not Y), preferably, in one embodiment, X or Z is A or L. In an exemplary aspect of this embodiment, the substitution mutation at a position functionally equivalent to Y130 is a mutation to alanine (A) (e.g., SEQ ID NO: 5). Specific examples of altered cytidine deaminase having increased activity compared to cytosine and preferentially acting on 5mC include SEQ ID NO: 5 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO: 5 and comprising Y130A.

[0157] The modified cytidine deaminase of the present disclosure having cytosine-deficient deaminase activity (i.e., converting 5mC to T at a higher rate than converting C to U) is optionally included in the position of two, three, four or five amino acids on the C-terminal side of the Y130 position, or a second substitution mutation functionally equivalent to the Y130 position. In one embodiment, the second mutation is at the position of two, three, four or five amino acids on the C-terminal side of the Y130 position, or functionally equivalent to tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H) or phenylalanine (F) at the Y130 position. In one embodiment, the second mutation is at the position of tyrosine (Y132) at position 132 in members of the APOBEC3A subfamily that is functionally equivalent. APOBEC protein, such as APOBEC3A protein, contains substitution mutations at both the first site (functionally equivalent to Y130) and the second site (two, three, four or five amino acids on the C-terminal side of the Y130 position), which increases the preferential activity acting on 5mC compared to the same APOBEC protein (such as an APOBEC3A protein containing a substitution mutation at Y130). In one embodiment, the substitution mutation at the second position is an amino acid with a positively charged side chain and selected from arginine (R), histidine (H), lysine (L), or a polar side chain and selected from glutamine (Q). In one embodiment, the substitution mutation at the second position is histidine (H), such as Y132 to histidine. The double mutant containing both the first mutation and the second mutation can be any combination of any substitution mutation at a position functionally equivalent to Y130 described herein and any second substitution mutation at a position of two, three, four or five amino acids on the C-terminal side of the Y130 position described herein. For example, the altered cytidine deaminase can be, for example, SEQ ID NO: 4, and has substitutions at Y130 and Y132, or at positions functionally equivalent to Y130 and Y132 as described herein. An example of an altered cytidine deaminase is SEQ ID NO: 11 comprising Y130X and Y132Z, wherein X is selected from (A), (L) or (W) (preferably (A)), and Z is selected from (R), (H), (L) or (Q), preferably (H). This encompasses examples including, but not limited to, Y130A and Y132R, Y130A and Y132H, Y130A and Y132L, Y130A and Y132Q, Y130L and Y132R, Y130L and Y132H, Y130L and Y132L, Y130L and Y132Q, Y130W and Y132R, Y130W and Y132H, Y130W and Y132L, Y130W and Y130Q, or any suitable combination thereof.In one embodiment, the double mutant comprises substitution mutations Y130A and Y132H. Specific examples of altered cytidine deaminases having two substitution mutations and preferentially acting on 5mC compared to APOBEC proteins having only a single substitution mutation at cytosine include SEQ ID NO: 6 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO: 6 and comprising Y130A and Y132H.

[0158] A person of ordinary skill in the art can confirm the 5mC preferential deaminase activity of the arginine, glutamine, histidine and lysine substitution mutations at the second position in the double mutants described above. For example, a double mutant can be constructed to produce such an altered cytidine deaminase, the altered cytidine deaminase having a first substitution mutation (at a position functionally equivalent to Y130) and a second arginine, glutamine, histidine or lysine substitution mutation (at the tyrosine position of the two amino acids on the C-terminal side of the Y130 position), and then assessing the deamination of C residues in one assay, and assessing the deamination of 5mC residues in a second assay. Using an assay (such as the SwaI-based assay described herein), the rates of 5mC deamination and C deamination can be compared to identify those double mutants that preferentially deaminize 5mC compared to C. One of ordinary skill in the art can similarly test double mutants having tyrosine at three, four, or five positions at the C-terminus of a position functionally equivalent to Y130, and confirm that substitution mutations at these positions to arginine, glutamine, histidine, or lysine, in combination with mutations at positions functionally equivalent to Y130 (such as Y130A), serve as double mutants that preferentially deaminate 5mC over C.

[0159] Some embodiments provided herein relate to substitution mutations that produce 5mC-deficient deaminase activity (i.e., converting C to U at a higher rate than converting 5mC to T). In one embodiment, compared to 5mC, a substitution mutation at a position functionally equivalent to Y130 increases cytidine deaminase activity and preferentially acts on cytosine, and is a mutation to an amino acid with a non-polar side chain or a hydrophobic side chain, such as leucine (L) or tryptophan (W). In an exemplary aspect of this embodiment, a substitution mutation at a position functionally equivalent to Y130 is a mutation to leucine. Other examples of mutations that produce increased preferential deamination activity for cytosine compared to 5mC include single mutants with Y132P and double mutants with substitution mutations at Y130V and Y132H or Y130W and Y132H. Specific examples of cytidine deaminases having increased cytidine deaminase activity and preferentially acting on altered cytosine compared to 5mC include SEQ ID NO: 7 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO: 7 and comprising Y130L.

[0160] In one embodiment, the substitution mutation is at a position functionally equivalent to Y130, and the substitution mutation produces 5hmC-deficient deaminase activity (i.e., preferentially deaminates C and 5mC to U and T, respectively, and has a significantly reduced degree of deamination of 5hmC). In an exemplary aspect of this embodiment, the substitution mutation at a position functionally equivalent to Y130 is a mutation to an amino acid with a non-polar side chain or a hydrophobic side chain, such as tryptophan (W). Specific examples of cytidine deaminases that have the ability to deaminate C and 5mC to U and T, respectively, but have a reduced ability to deaminate 5hmC, preferably without a detectable change in the ability to deaminate 5hmC include SEQ ID NO: 8 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity with SEQ ID NO: 8 and comprising Y130W.

[0161] In some embodiments, the altered cytidine deaminase comprises a substitution mutation at a position functionally equivalent to the tyrosine at position 130 (Y130) and / or position 132 (Y132) in a member of the APOBEC3A subfamily. In some embodiments, the altered cytidine deaminase exhibits selective deamination of mC.

[0162] In some embodiments, the altered cytidine deaminase is an altered APOBEC3A cytidine deaminase that is altered to include a substitution mutation at tyrosine at position 130 (Y130) and / or tyrosine at position 132 (Y132). In some embodiments, the altered APOBEC3A cytidine deaminase exhibits selective deamination of mC.

[0163] In some embodiments, the altered cytidine deaminase is a double mutant of APOBEC3A, with substitution mutations of Y130A / Y132H. In some embodiments, the altered APOBEC3A cytidine deaminase exhibits selective deamination of mC.

[0164] In some embodiments, the altered cytidine deaminase comprises an altered cytidine deaminase having an amino acid of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the altered APOBEC3A cytidine deaminase exhibits selective deamination of mC.

[0165] The altered cytidine deaminase described herein may comprise additional mutations. Typically, the additional mutations will not excessively alter the activity of the altered cytidine deaminase. One or more additional mutations may be conservative mutations.

[0166] The altered cytidine deaminase described herein can be a truncated protein. The truncated protein is a fragment of the altered cytidine deaminase disclosed herein that retains the ability to deaminize 5mC to thymidine. The truncated altered cytidine deaminase can include a deletion of 1 to 13 amino acids at the N-terminus of the protein, a deletion of 1 to 3 amino acids at the C-terminus of the protein, or a combination thereof.

[0167] In some embodiments, the altered cytidine deaminase includes any of those described in International Patent Application No. PCT / US2023 / 017846, filed on April 7, 2023 (“Altered Cytidine Deaminases and Methods of Use”), which is hereby incorporated by reference in its entirety.

[0168] Typically, methods for using cytidine deaminase include contacting a target nucleic acid (e.g., DNA or RNA) with an enzyme under conditions suitable for converting modified cytidine (such as 5mC) to thymidine or suitable for converting unmodified cytidine to uracil. Because the amplification of DNA does not maintain the modification state of cytidine (e.g., does not maintain the methylation state of 5mC), the use of cytidine deaminase typically occurs before the amplification of the target DNA. The target nucleic acid can be contacted with cytidine deaminase at essentially any time. For example, after separating genomic DNA or mRNA or cell-free DNA or mRNA, before or after fragmentation, or before or after tag fragmentation, the target nucleic acid can be contacted with cytidine deaminase. The skilled person will recognize that the target nucleic acid can be contacted with cytidine deaminase after adding universal sequences and / or adapters, provided that the universal sequences and / or adapters are not added by amplification.

[0169] Reaction conditions suitable for converting modified cytidine (such as 5mC) to thymidine by cytidine deaminase include, but are not limited to, substrates of target nucleic acids suspected of containing at least one modified cytidine, appropriate pH, reaction temperature, reaction time, and concentrations of cytidine deaminase and / or DNA or RNA substrates. It is expected that cytidine deaminase can function in substantially any buffer. Examples of useful buffers include, but are not limited to, citrate buffers, such as citrate buffers (Catalog #005000) available from Thermo Fisher Scientific; sodium acetate buffer, Bis Tris-propane HCl; and Tris-HCl Tris. Examples of other buffers include, but are not limited to, Bicine, DIPSO, glycylglycine, HEPES, imidazole, malonate, MES, MOPS, PB, phosphate, PIPES, SPG, succinate, TAPS, TAPSO, trincine. Cytidine deaminase typically functions at near-neutral pH (e.g., pH 7). In some embodiments, a reducing agent (such as dithiothreitol (DTT)) may be present. In some embodiments, divalent cations are not included.The deamination reaction may occur at a temperature of about 25°C to about 60°C, including but not limited to about 37°C, about 45°C, about 50°C, and about 60°C.

[0170] Some cytidine deaminases preferentially deaminize modified cytosine to thymidine at a faster rate than the deamination of cytosine to uracil.Therefore, in some embodiments, the reaction time can be used to complete the reaction operation, so that the difference between the deamination of the cytosine modified and the deamination of cytosine is maximized.In one embodiment, the reaction can be carried out for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes or at least 150 minutes, and carry out no more than 15 minutes, no more than 30 minutes, no more than 45 minutes, no more than 60 minutes, no more than 90 minutes, no more than 120 minutes, no more than 150 minutes or no more than 180 minutes.In some embodiments, the reaction can be run overnight.

[0171] In some embodiments, the deamination reaction may include a cytidine deaminase at a concentration of at least about 25 nanomolar (nM) to no more than about 5 micromolar (μM). For example, the concentration of the enzyme may be at least about 25nM, at least about 0.5, at least about 1μM, at least about 2μM, at least about 3μM, at least about 4μM or at least about 5μM and / or no more than 5μM, no more than 4μM, no more than 3μM, no more than 2μM, no more than 1μM or 0.5μM. In some embodiments, the deamination reaction may include nucleic acids of about 1ng to about 1μg input. In some embodiments, the deamination reaction may include nucleic acids of at least about 10pM to at least about 200nM.

[0172] Amplification

[0173] At different stages of the methods described herein, DNA fragments can be amplified. It should be understood that any of the amplification methods described herein or generally known in the art can be used together with universal or target-specific primers to amplify DNA fragments. Suitable amplification methods include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA), as described in U.S. Patent 8,003,354. The above-mentioned amplification methods can be used to amplify one or more nucleic acids of interest. For example, DNA fragments can be amplified using PCR (including multiplex PCR), SDA, TMA, NASBA, etc. In some embodiments, primers specific for polynucleotides of interest are included in the amplification reaction.

[0174] As used herein, "amplification" or "amplification reaction" and its derivatives generally refer to any action or process in which at least a portion of a nucleic acid molecule is replicated or copied to at least one other nucleic acid molecule. The other nucleic acid molecules optionally contain sequences that are substantially identical or substantially complementary to at least some portions of the target nucleic acid molecule. The target nucleic acid molecule may be single-stranded or double-stranded, and the other nucleic acid molecules may independently be single-stranded or double-stranded. Amplification optionally includes linear or exponential replication of nucleic acid molecules. In some embodiments, such amplification can be performed using isothermal conditions; in other embodiments, such amplification may include thermal cycling. In some embodiments, amplification is multiplex amplification, which includes simultaneously amplifying multiple target sequences in a single amplification reaction. In some embodiments, "amplification" includes amplifying at least some portions of DNA- and RNA-based nucleic acids, either alone or in combination. The amplification reaction may include any amplification process known to those of ordinary skill in the art.

[0175] As used herein, "amplification conditions" and their derivatives generally refer to conditions suitable for amplifying one or more nucleic acid sequences. This amplification can be linear or exponential. In some embodiments, amplification conditions may include isothermal conditions, or alternatively may include thermal cycling conditions, or a combination of isothermal conditions and thermal cycling conditions. In some embodiments, conditions suitable for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions refer to a reaction mixture sufficient to amplify nucleic acids (e.g., one or more target sequences) or amplify a target sequence (e.g., an amplified target sequence connected to one or more adapters) .

[0176] Generally speaking, amplification conditions include a catalyst for amplification or for nucleic acid synthesis, such as a polymerase; a primer having a certain degree of complementarity with the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs), which promote the extension of the primer once hybridized with the nucleic acid. Amplification conditions may require hybridization or annealing of the primer to the nucleic acid, extension of the primer, and a denaturation step in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, but not necessarily, amplification conditions may include thermal cycling; in some embodiments, amplification conditions include multiple cycles in which annealing, extension, and separation steps are repeated. Typically, amplification conditions include cations such as Mg++ or Mn++, and may also include various ionic strength modifiers.

[0177] As used herein, the term "polymerase chain reaction" (PCR) refers to KBMullis's method as described in U.S. Patent Nos. 4,683,195 and 4,683,202, which describes a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. The method of amplifying a polynucleotide of interest includes introducing a large amount of two oligonucleotide primers in excess into a DNA mixture containing a desired polynucleotide of interest, and then performing a series of thermal cycles in the presence of a DNA polymerase. The two primers are complementary to the chains of the double-stranded polynucleotide of interest corresponding to them. First, the mixture is denatured at a higher temperature, and then the primers are annealed with the complementary sequences in the polynucleotide molecules of interest. After annealing, the primers are extended with a polymerase to form a pair of new complementary chains. The steps of denaturation, primer annealing and polymerase extension can be repeated multiple times (referred to as thermal cycles) to obtain a high concentration of amplified fragments of the desired polynucleotide of interest. The length of the amplified fragment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers relative to each other, and therefore, the length is a controllable parameter. Because the process is repeated, the method is referred to as a "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are considered "PCR amplified." In a modified form of the above method, multiple different primer pairs (in some cases, one or more primer pairs per target nucleic acid molecule of interest) can be used to PCR amplify the target nucleic acid molecules, thereby forming a multiplex PCR reaction.

[0178] In some embodiments, a polymerase resistant to uracil may be used for amplification. In some embodiments, a polymerase intolerant to uracil may be used for amplification. In some embodiments, a polymerase intolerant to uracil may be used for the first round of amplification, and a polymerase resistant to uracil may be used for subsequent rounds of amplification.

[0179] Sequencing

[0180] In some embodiments, the DNA fragments obtained by amplification can be sequenced. Sequencing can be performed by any of a variety of known methods, including but not limited to any of a variety of high-throughput next-generation sequencing platforms (NGS) platforms, including but not limited to synthetic sequencing, ligation sequencing, nanopore sequencing, Sanger sequencing, etc. In some embodiments, sequencing is performed using the following method: by as described in the following literature by Commercial sequencing by synthesis methods U.S. Patent Application Publication No. 2007 / 0166705, U.S. Patent Application Publication No. 2006 / 0188901, U.S. Patent No. 7,057,026, Beijing Genomics Institute (BG), as described in Carnevali et al., 2012, J Comput Biol; 9(3): 279-92, or ION TORRENT as described in US2009 / 0026082A1; US2009 / 0127589A1; US2010 / 0137143A1; or US 2010 / 0282617 A1 TM Ion semiconductor sequencing methods, each of which is incorporated herein by reference.

[0181] Next generation sequencing (NGS) refers to sequencing methods that allow for massively parallel sequencing of clonally amplified molecules and single nucleic acid molecules. Non-limiting examples of NGS include sequencing by ligation and sequencing by synthesis using reversible dye terminators.

[0182] Preferred embodiments include sequencing by synthesis (SBS) techniques. SBS techniques generally involve enzymatic extension of a nascent nucleic acid chain by repeated addition of nucleotides to a template strand.

[0183] In some embodiments, the readout can be obtained by using an assay, for example, using a program as described at the following World Wide Web site: illumina.com / techniques / microarrays / methylation-arrays.html.

[0184] Reagent test kit

[0185] The present disclosure also provides a kit for performing the method as described herein, for reducing false positive uracil residues generated by deamination of unmethylated cytosine in an assay using cytosine deaminase to deaminate methylated cytosine. In some embodiments, the kit may include at least one or more of cytosine deaminase, primers, uracil-resistant polymerase, uracil-intolerant polymerase, dNTPs, uracil DNA glycosylase (UDG) and / or endonuclease in a suitable packaging material, in an amount sufficient for at least one reaction.

[0186] The kit may include one or more components. Examples of other components include, for example, a positive control polynucleotide or a negative control polynucleotide. Optionally, other reagents such as buffers and solutions are also included. Instructions for use of the packaging components are also typically included.

[0187] As used herein, the phrase "packaging material" refers to one or more physical structures for containing the contents of the test kit. The packaging material is constructed by known methods, preferably for providing an aseptic, contaminant-free environment. The packaging material has a label, and the label indicates the components of the uracil residues produced due to the deamination of unmethylated cytosine in the determination using cytosine deaminase to deaminate methylated cytosine. In addition, the packaging material includes instructions for how to use the material in the test kit to practice the RUBY method as described herein. As used herein, the term "packaging" refers to a solid matrix or material such as glass, plastic, paper, foil, etc., which can accommodate polypeptides within a fixed range. "Instructions for use" generally include a tangible expression describing the concentration of a reagent or at least one assay method parameter, such as the relative amount of a reagent to be mixed and a sample, the maintenance time period of a reagent / sample mixture, temperature, buffer conditions, etc.

[0188] Exemplary Aspects

[0189] The invention is defined in the claims. However, a non-exhaustive list of non-limiting exemplary aspects is provided below. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment or aspect described herein. Exemplary embodiments of the invention include, but are not limited to, the following:

[0190] Aspect 1: A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising:

[0191] providing a sample comprising DNA library fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5' and 3' library adaptors; and

[0192] Double-stranded DNA library fragments that do not contain uracil are generated by contacting the sample containing DNA library fragments with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis to subject the sample to at least one round of second strand synthesis.

[0193] Aspect 2 is a method according to aspect 1, further comprising: performing polymerase chain reaction (PCR) amplification on the sample containing double-stranded DNA library fragments that do not contain uracil using a uracil-resistant polymerase or a uracil-intolerant polymerase.

[0194] Aspect 3: A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising:

[0195] providing a sample comprising library DNA fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5' and 3' library adaptors; and

[0196] contacting the sample comprising DNA library fragments with uracil DNA glycosylase (UDG) and an endonuclease, thereby generating DNA library fragments cleaved at uracil residues; and

[0197] The sample comprising DNA library fragments cleaved at uracil residues is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising DNA library fragments cleaved at uracil residues with a polymerase, dNTPs, and primers complementary to 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA library fragments that do not contain uracil.

[0198] Aspect 4 is a method according to aspect 3, wherein the polymerase comprises a uracil-intolerant polymerase.

[0199] Aspect 5 is a method according to any one of aspects 1 to 4, wherein the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi.

[0200] Aspect 6: A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising:

[0201] providing a sample comprising original DNA library fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the original DNA library fragments comprise 5' and 3' library adaptors;

[0202] subjecting the sample comprising the original DNA library fragments to second strand synthesis by contacting the sample with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating DNA library fragments that do not contain uracil residues in the synthesized second strand;

[0203] contacting the sample comprising DNA library fragments not containing uracil residues in the synthesized second strand with an endonuclease to digest the original DNA library fragments, thereby generating a single-stranded synthesized second strand; and

[0204] The sample comprising the single-stranded synthesized second strand is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising the single-stranded synthesized second strand with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA library fragments that do not contain uracil.

[0205] Aspect 7 is a method according to aspect 6, wherein PCR amplifying the sample comprising the single-stranded synthesized second strand comprises contacting the sample comprising the single-stranded synthesized second strand with a uracil-resistant polymerase.

[0206] Aspect 8 is a method according to any one of aspects 1 to 7, wherein the DNA library fragments comprise single-stranded DNA library fragments.

[0207] Aspect 9 is a method according to any one of aspects 1 to 7, wherein the DNA library fragments comprise double-stranded DNA library fragments.

[0208] Aspect 10 is a method according to any one of aspects 1 to 9, wherein the cytosine deaminase comprises an altered cytosine deaminase.

[0209] Aspect 11 is a method according to aspect 10, wherein the altered cytosine deaminase is a member of the following subfamily: AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, APOBEC3G subfamily, APOBEC3G subfamily, APOBEC3H subfamily or APOBEC4 subfamily, or their alterations.

[0210] Aspect 12 is a method according to aspect 10, wherein the altered cytosine deaminase comprises an altered APOBEC3A.

[0211] Aspect 13 is a method according to any one of aspects 10 to 12, wherein the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in the wild-type APOBEC3A protein and / or an amino acid substitution mutation at a position functionally equivalent to Tyr132 in the wild-type APOBEC3A protein.

[0212] Aspect 14 is a method according to any one of aspects 10 to 13, wherein the altered cytidine deaminase comprises amino acid substitution mutations at positions functionally equivalent to (Tyr / Phe)130 and Tyr132 in the wild-type APOBEC3A protein.

[0213] Aspect 15 is a method according to any one of aspects 10 to 14, wherein the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in the wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp.

[0214] Aspect 16 is a method according to aspect 14 or 15, wherein the (Tyr / Phe)130 is Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:12.

[0215] Aspect 17 is a method according to any one of aspects 10 to 16, wherein the substitution mutation at the position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine or threonine.

[0216] Aspect 18 is a method according to any one of aspects 13 to 17, wherein the substitution mutation at the position functionally equivalent to Tyr130 comprises a mutation to Ala, Val or Trp.

[0217] Aspect 19 is a method according to any one of aspects 13 or 18, wherein the substitution mutation at the position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln or Lys.

[0218] Aspect 20 is a method according to any one of aspects 10 to 19, wherein the altered cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is greater than the rate at which cytosine (C) is converted to uracil (U) by deamination.

[0219] Aspect 21 is a method according to aspect 20, wherein the rate is at least 100 times greater.

[0220] Aspect 22 is a method according to any one of aspects 10 to 21, wherein the altered cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination, and its conversion rate is greater than the rate of conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

[0221] Aspect 23 is a method according to aspect 22, wherein conversion of 5hmC to 5hmU by deamination is undetectable.

[0222] Aspect 24 is a method according to any one of aspects 10 to 23, wherein the altered cytidine deaminase comprises a ZDD motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1).

[0223] Aspect 25 is a method according to any one of aspects 10 to 24, wherein the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX24SW(S / T)PCX[2-4]CX6FX8LX5R(L / I)YX[8-11]LX2LX

[10] M (SEQ ID NO:2), wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is a Tyr (Y) amino acid of the ZDD motif.

[0224] Aspect 26 is a method according to any one of aspects 10 to 25, wherein the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises X[16-26]-GRXXTXLCYXV-X15-GXXXN-X12-HAEXXF-X14-YXXTWXXSWSPC-X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X[2-6] (SEQ ID NO: 3).

[0225] Aspect 27 is a method according to any one of aspects 10 to 26, wherein the altered cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.

[0226] Aspect 28 is a method according to any one of aspects 1 to 27, wherein the length of the DNA library fragments is about 100 bp to about 300 bp.

[0227] Aspect 29 is a method according to any one of aspects 1 to 28, further comprising: sequencing the double-stranded DNA library fragments that do not contain uracil.

[0228] Aspect 30 is a method according to any one of aspects 1 to 29, further comprising: processing the uracil-free double-stranded DNA library fragments to generate a sequencing library.

[0229] Aspect 31 is a method according to aspect 30, wherein the method further comprises: sequencing the sequencing library.

[0230] Aspect 32 is a kit comprising cytosine deaminase and a uracil-intolerant polymerase.

[0231] Aspect 33 is a kit comprising cytosine deaminase, uracil DNA glycosylase (UDG) and AP endonuclease.

[0232] Aspect 34 is a kit according to aspect 33, wherein the AP endonuclease comprises endonuclease IV and / or AP endonuclease I.

[0233] Aspect 35 is a method for removing DNA fragments containing uracil residues from a sample, the method comprising:

[0234] providing a sample comprising DNA fragments, wherein the DNA fragments comprise 5' and 3' library adaptors; and

[0235] The sample containing the DNA library fragments is subjected to polymerase chain reaction (PCR) amplification by contacting the sample with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions to provide second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

[0236] Aspect 36 is a method according to aspect 35, further comprising: performing polymerase chain reaction (PCR) amplification on the sample containing double-stranded DNA library fragments that do not contain uracil using a uracil-resistant polymerase or a uracil-intolerant polymerase.

[0237] Aspect 37 is a method of removing DNA fragments containing uracil residues from a sample, the method comprising:

[0238] providing a sample comprising DNA fragments, wherein the DNA fragments comprise 5' and 3' library adaptors; and

[0239] contacting the sample comprising DNA fragments with uracil DNA glycosylase (UDG) and an endonuclease, thereby generating DNA fragments cleaved at uracil residues; and

[0240] A sample containing DNA fragments cleaved at uracil residues is subjected to polymerase chain reaction (PCR) amplification by contacting the sample containing DNA fragments cleaved at uracil residues with a polymerase, dNTPs, and primers complementary to 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

[0241] Aspect 38 is a method according to aspect 37, wherein the polymerase comprises a uracil-intolerant polymerase.

[0242] Aspect 39 is a method according to any one of aspects 35 to 38, wherein the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi.

[0243] Aspect 40 is a method of removing DNA fragments containing uracil residues from a sample, the method comprising:

[0244] Providing a sample comprising raw DNA fragments, wherein the raw DNA fragments comprise 5' and 3' library adapters;

[0245] subjecting the sample comprising the original DNA fragments to second strand synthesis by contacting the sample with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating DNA fragments that do not contain uracil residues in the synthesized second strand;

[0246] contacting the sample containing DNA fragments not containing uracil residues in the synthesized second strand with an endonuclease to digest the original DNA fragments, thereby generating a single-stranded synthesized second strand; and

[0247] The sample comprising the single-stranded synthesized second strand is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising the single-stranded synthesized second strand with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

[0248] Aspect 41 is a method according to aspect 40, wherein PCR amplifying the sample comprising the single-stranded synthesized second strand comprises contacting the sample comprising the single-stranded synthesized second strand with a uracil-resistant polymerase.

[0249] Aspect 42 is a method according to any one of aspects 35 to 41, wherein the DNA library fragments comprise single-stranded DNA library fragments.

[0250] Aspect 43 is a method according to any one of aspects 35 to 41, wherein the DNA library fragments comprise double-stranded DNA library fragments.

[0251] Aspect 44 is a method according to any one of aspects 35 to 43, wherein the length of the DNA library fragments is about 100 bp to about 300 bp.

[0252] Aspect 45 is a method according to any one of aspects 35 to 44, further comprising: sequencing the double-stranded DNA library fragments that do not contain uracil.

[0253] Aspect 46 is a method according to any one of aspects 35 to 45, further comprising: processing the uracil-free double-stranded DNA library fragments to generate a sequencing library.

[0254] Aspect 47 is a method according to aspect 46, wherein the method further comprises: sequencing the sequencing library.

[0255] The present invention is illustrated by the following examples.It is to be understood that the specific examples, materials, amounts and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0256] Example

[0257] Example 1

[0258] Uracil-intolerant polymerase and USER simultaneously reduce the false positive FP rate of methylation

[0259] NA12878 genomic DNA was combined with completely unmethylated lambda control DNA and enzymatic CpG methylated pUC19 control DNA and mechanically sheared to produce fragments of approximately 200 bp. The sheared DNA (50 ng) was then subjected to end repair, A-tailing and adapter ligation according to the standard library preparation procedure. The adapter-ligated DNA was denatured by incubation in 0.02N sodium hydroxide at 50°C for 10 minutes. Subsequently, the ssDNA sample was enzymatically deaminated at 37°C for 3 hours in 50mM Bis-Tris (pH 6.5), 1mM DTT, 0.2mg / mL BSA with engineered cytidine deaminase (750nM). After SPRI-based purification, additional treatment of some of these libraries was performed at 37°C for 30 minutes in rCutSmart buffer (NEB) by 1 unit USER (NEB), followed by additional SPRI purification. The libraries were then PCR amplified using unique dual-index primers, using either a uracil-tolerant polymerase (KAPA HiFi Uracil+, Roche) or a uracil-intolerant polymerase (KAPA HiFi, Roche) using 12 PCR cycles. The libraries were then sequenced on a NextSeq550 and downsampled to 5 million paired-end reads per sample and analyzed using the DRAGEN methylation pipeline.

[0260] Figure 4 The overall methylation levels of different gene sets when assayed using a uracil (+) tolerant polymerase, a uracil-intolerant (-) polymerase, or both USER treatment and uracil (-) polymerase are shown. Libraries amplified using a uracil-intolerant polymerase greatly reduced false positive methylation, as evidenced by a decrease in lambda CG methylation levels from 0.071 to 0.014 ( Figure 4 A small decrease in mC detection was also observed, as evidenced by lower pUC19 methylation (0.781 compared to 0.811).

[0261] Libraries treated with USER and subsequently amplified with a uracil-intolerant polymerase had even higher specificity, with a greatly reduced methylation level in the unmethylated lambda genome (0.00022). A reduction in overall mC detection was also observed in these samples. The library complexity of these differentially treated libraries was also compared using Picard Estimate Library Complexity Metrics. It was found that the estimated library size (a measure of library complexity) was slightly lower for those libraries amplified with a uracil-intolerant polymerase, and further reduced for libraries treated with USER and a uracil-intolerant polymerase (0.00022). Figure 5).

[0262] This example demonstrates that uracil discrimination reduces false positive methylation reports.

[0263] Example 2

[0264] Evaluation of alternative uracil-intolerant polymerases

[0265] Alternative polymerase pairs were evaluated in this workflow to characterize uracil discrimination levels and overall performance. NA12878 genomic DNA was combined with completely unmethylated lambda control DNA and enzymatic CpG methylated pUC19 control DNA and mechanically sheared to produce fragments of approximately 300 bp. The sheared DNA (50 ng) was then subjected to end repair, A-tailing, and adapter ligation according to standard library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02N sodium hydroxide at 50°C for 10 minutes. Subsequently, the ssDNA sample was enzymatically deaminated at 37°C for 30 minutes in 50mM Bis-Tris (pH 6.5), 1mM DTT, 0.2mg / mL BSA with cytidine deaminase (135nM). After SPRI-based purification, the library was divided into two separate reactions, in which they were amplified using 12 PCR cycles by a U-resistant polymerase or a U-intolerant polymerase of the same family. The polymerase families tested are listed in Table 2.

[0266] Table 2: Uracil-tolerant and Uracil-intolerant PCR polymerases evaluated in methylation assays .

[0267]

[0268] The libraries were then sequenced on a NextSeq 550 and downsampled to 5 million paired-end reads per sample and analyzed using the DRAGEN methylation pipeline. The analysis showed that all uracil-intolerant polymerases tested were able to reduce the false positive methylation rate reported on the lambda genome, with Q5 and Phusion polymerases showing greater reductions in false positive rates ( Figure 6 ).

[0269] Example 3

[0270] Methylation assays on human genomic DNA using a U-intolerant polymerase

[0271] In order to evaluate the performance of U-intolerant polymerases in methylation assays of human genomic DNA, NA12878 genomic DNA was combined with completely unmethylated lambda control DNA and enzymatic CpG methylated pUC19 control DNA, and mechanical shearing was performed to produce a fragment of about 300 bp. The sheared DNA (50 ng) was then subjected to end repair, A-tailing, and adapter ligation according to the standard Illumina library preparation procedure. The adapter-ligated DNA was denatured by incubation in 0.02N sodium hydroxide at 50°C for 10 minutes. Subsequently, the ssDNA sample was enzymatically deaminated at 37°C for 30 minutes in 50 mM Bis-Tris (pH 6.5), 1 mM DTT, 0.2 mg / mL BSA, 5 μg / mL RNAse A, 1 M betaine with cytidine deaminase (200 nM). The library was then PCR amplified using unique dual-index primers, using either a uracil-tolerant polymerase (Q5U, New England Biolabs) or a uracil-intolerant polymerase (Q5 HiFi, New England Biolabs) for 12 PCR cycles. The samples were sequenced on a NovaSeq 6000.

[0272] Analysis of sequencing data showed that the use of Q5 HiFi relative to Q5U resulted in a small increase in repeat reads and a corresponding small decrease in autosomal coverage ( FIG. 7A to FIG. 7D ). Fig. 7A Shown is the false positive rate observed in the lambda genome in this experiment. Figure 7B Duplicate readings are presented. Figure 7C Average autosomal coverage over the human genome is presented. Fig.7D Median average deviation (MAD) of coverage is presented. Coverage uniformity measured using the median average deviation (MAD) metric was slightly improved by using Q5HiFi (indicated by lower MAD values). Therefore, despite the removal of some reads by using a U-intolerant polymerase, the resulting libraries maintained good quality and the observed differences had little practical impact on performance.

[0273] To evaluate methylation performance against the human genome, methylpyrimidines were used to calculate regional methylation values ​​for CpG islands across the human genome. The EM-seq dataset on NA12878 was also analyzed for comparison. The methylation values ​​for each region were plotted against the methylation values ​​for each region used for EM-seq ( Figure 8 ). This analysis showed that the methylation values ​​generated using Q5 polymerase correlated well with those reported using EM-Seq, with an R-squared value of 0.971. For comparison, the R-squared for data generated using Q5U polymerase was 0.973.

[0274] This example demonstrates that uracil discrimination produces high quality and methylation reporters.

[0275] Example 4

[0276] Differential methylation analysis

[0277] A common application of methylation NGS assays is to identify differentially methylated regions (DMRs), which are genomic regions with different methylation levels across multiple DNA samples, which can be used to outline epigenetic changes in cancer and differences across tissue types (Chen et al., 2016, Briefings in Functional Genomics; 15(6): 485–90). To characterize the performance of the APOBEC methylation assay using a uracil-intolerant polymerase, two different human genomic DNA samples were used: NA12878 DNA and HeLa DNA. Both samples were supplemented with a small amount of control DNA samples (unmethylated λ DNA and enzymatic CpG methylated pUC19 DNA) and sheared to an average fragment size of approximately 300 bp. The sheared DNA (100 ng) was then subjected to end repair, A-tailing, and adapter ligation according to standard library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02N sodium hydroxide at 50°C for 10 minutes. Subsequently, the ssDNA samples were enzymatically deaminated in 50 mM Bis-Tris (pH 6.5), 1 mM DTT, 0.2 mg / mL BSA with APOBEC-Y130AY132H (750 nM) at 37°C for 30 minutes. The library was then PCR amplified using 10 PCR cycles using a unique dual-index primer, a uracil-tolerant polymerase (KAPA HiFi Uracil+, Roche) or a uracil-intolerant polymerase (KAPA HiFi, Roche). The comparison library was also processed using the EM-Seq conversion kit and amplified using Q5U PCR according to the manufacturer's recommendations. The samples were sequenced on the NovaSeq6000 and downsampled to 680 million paired-end reads for analysis according to the same procedure described above.

[0278] For differential methylation analysis, the program HOME for identifying DMRs was used (Srivastava et al., 2019, BMC Bioinformatics; 20(1):253). For each methylation assay, methylation between NA12878 and HeLa samples was compared. To evaluate the performance of APOBEC-based analysis relative to the comparative EM-seq method, the Jaccard statistic was calculated using bedtoolsjaccard under default settings. The Jaccard statistic evaluates the similarity between two sets of genomic intervals and ranges between 0 and 1, where 0 indicates no overlap and 1 indicates complete overlap (the World Wide Web URL is bedtools.readthedocs.io / en / latest / content / tools / jaccard.html). The Jaccard indices for both U-intolerant and U-tolerant polymerases were similar, indicating that U-intolerant polymerases did not interfere with the assay (Table 3). Visualization of the identified regions also shows the high similarity of the described method to EM-Seq ( Fig. 9 ).

[0279] Table 3: Jaccard index for comparison of DMRs identified between NA12878 and HeLa using EM-Seq data Make a comparison .

[0280] Workflow Jaccard Index

[0281] APOBEC assay + uracil-resistant polymerase 0.726

[0282] APOBEC assay + uracil-intolerant polymerase 0.736

[0283] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions in, for example, GenBank and RefSeq, amino acid sequence submissions in, for example, SwissProt, PIR, PRF, PDB, and translations of annotated coding regions from GenBank and RefSeq) are incorporated by reference. In the event of any inconsistency between the disclosure of the present application and the disclosure of any document incorporated herein by reference, the disclosure of the present application shall prevail. The above detailed description and examples are given only for a clear understanding of the present invention. They should not be construed as unnecessary limitations. The present invention is not limited to the exact details shown and described, as variations obvious to those skilled in the art will be included in the invention defined by the claims.

[0284] Sequence information

[0285] SEQ ID NO:1

[0286] Zinc binding motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C

[0287] SEQ ID NO:2

[0288] ZDD motif: HXEX 24 SW(S / T)PCX [2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] MSEQ ID NO:3

[0289] Amino acids altered in cytidine deaminases including members of the APOBEC3A subfamily: X [16-26] -GRXXTXLCYXV-X 15 -GXXXN-X 12 -HAEXXF-X 14 -YXXTWXXSWSPC-X [2-4] -CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X 13 -LXXI-X [2-6]

[0290] SEQ ID NO:4

[0291] Amino acids altered in cytidine deaminases including members of the APOBEC3A subfamily: X 26 -GRXXTXLCYXV-X 15 -GX 16 -HAEXXF-X 14 -YXXTWXXSWSPC-X4-CA-X5-FL-X7-LXIFXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X 13 -LXXI-X6

[0292] SEQ ID NO:5

[0293] Altered cytosine deaminase (SG1) - synthetic constructs:

[0294] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIADYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0295] SEQ ID NO:6

[0296] Altered cytosine deaminase (SG2) - synthetic constructs:

[0297] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIADHDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0298] SEQ ID NO:7

[0299] Altered cytosine deaminase - synthetic constructs

[0300] APOBECC3A has (Y130L)

[0301] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARILDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0302] SEQ ID NO:8

[0303] Altered cytosine deaminase - synthetic constructs

[0304] APOBEC3A has (Y130W)

[0305] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIWDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

[0306] SEQ ID NO:9

[0307] Altered cytosine deaminase (SG1 with Y130X)

[0308] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHV RLRIFAARIXDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (where X can be A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T, preferably A)

[0309] SEQ ID NO:10

[0310] Altered cytosine deaminase - synthetic constructs

[0311] X26-GRXXTXLCYXV-X15-G-X16-HAEXXF-X14-YXXTWXXSWSPC-X4-CA-X5-FL-X7-LXIFXXR(L / I)Z-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X6

[0312] (where Z is A, G, F, H, Q, M, N, K, V, D, E, S, C, P or T and the number after X refers to the number of amino acids present)

[0313] SEQ ID NO:11

[0314] Altered cytosine deaminase - synthetic constructs

[0315] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIXDZDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN (wherein X may be A, L or W, preferably A; and Z is selected from R, H, L or Q, preferably H).

[0316] SEQ ID NO:12

[0317] Wild-type human APOBEC3A protein (UniProt: P31941)

[0318] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

Claims

1. A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: providing a sample comprising DNA library fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5' and 3' library adaptors; and Double-stranded DNA library fragments that do not contain uracil are generated by contacting the sample containing DNA library fragments with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis to subject the sample to at least one round of second strand synthesis.

2. The method according to claim 1, further comprising: The sample containing the double-stranded DNA library fragments not containing uracil is subjected to polymerase chain reaction (PCR) amplification using a uracil-resistant polymerase or a uracil-intolerant polymerase.

3. A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: providing a sample comprising library DNA fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the DNA library fragments comprise 5' and 3' library adaptors; and contacting the sample comprising DNA library fragments with uracil DNA glycosylase (UDG) and an endonuclease, thereby generating DNA library fragments cleaved at uracil residues; and The sample comprising DNA library fragments cleaved at uracil residues is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising DNA library fragments cleaved at uracil residues with a polymerase, dNTPs, and primers complementary to 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA library fragments that do not contain uracil. The method of claim 3 , wherein the polymerase comprises a uracil-intolerant polymerase.

5. The method according to any one of claims 1 to 4, wherein the uracil-intolerant polymerase comprises KAPA HiFi, Ultra IIQ5 or Phusion HiFi.

6. A method for preventing false positive detection of 5-methylcytosine (5mC) and / or 5-hydroxymethylcytosine (5hmC) due to deamination of unmethylated cytosine in an assay using cytosine deaminase to selectively deaminate methylated cytosine, the method comprising: Providing a sample comprising original DNA library fragments, wherein cytosine deaminase deaminates methylated cytosine, wherein the original DNA library fragments comprise 5' and 3' library adaptors; subjecting the sample comprising the original DNA library fragments to second strand synthesis by contacting the sample with a uracil-intolerant polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating DNA library fragments that do not contain uracil residues in the synthesized second strand; contacting the sample comprising DNA library fragments that do not contain uracil residues in the synthesized second strand with an endonuclease to digest the original DNA library fragments, thereby generating a single-stranded synthesized second strand; as well as The sample comprising the single-stranded synthesized second strand is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising the single-stranded synthesized second strand with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA library fragments that do not contain uracil. 7 . The method of claim 6 , wherein PCR amplifying the sample comprising the single-stranded synthesized second strand comprises contacting the sample comprising the single-stranded synthesized second strand with a uracil-resistant polymerase.

8. The method according to any one of claims 1 to 7, wherein the DNA library fragments comprise single-stranded DNA library fragments.

9. The method according to any one of claims 1 to 7, wherein the DNA library fragments comprise double-stranded DNA library fragments.

10. The method of any one of claims 1 to 9, wherein the cytosine deaminase comprises an altered cytosine deaminase.

11. The method of claim 10, wherein the altered cytosine deaminase is a member of the following subfamily: AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3B subfamily, APOBEC3C subfamily, APOBEC3D subfamily, APOBEC3F subfamily, APOBEC3G subfamily, APOBEC3G subfamily, APOBEC3H subfamily or APOBEC4 subfamily, or an alteration thereof.

12. The method of claim 10, wherein the altered cytosine deaminase comprises an altered APOBEC3A.

13. The method according to any one of claims 10 to 12, wherein the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in the wild-type APOBEC3A protein and / or an amino acid substitution mutation at a position functionally equivalent to Tyr132 in the wild-type APOBEC3A protein.

14. The method of any one of claims 10 to 13, wherein the altered cytidine deaminase comprises amino acid substitution mutations at positions functionally equivalent to (Tyr / Phe)130 and Tyr132 in the wild-type APOBEC3A protein.

15. The method of any one of claims 10 to 14, wherein the altered cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in a wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp.

16. The method of claim 14 or 15, wherein the (Tyr / Phe)130 is Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:

12.

17. The method according to any one of claims 10 to 16, wherein the substitution mutation at the position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine or threonine.

18. The method according to any one of claims 13 to 17, wherein the substitution mutation at the position functionally equivalent to Tyr130 comprises a mutation to Ala, Val or Trp.

19. The method according to any one of claims 13 or 18, wherein the substitution mutation at the position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln or Lys.

20. The method of any one of claims 10 to 19, wherein the altered cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the rate at which cytosine (C) is converted to uracil (U) by deamination.

21. The method of claim 20, wherein the rate is at least 100 times greater.

22. The method of any one of claims 10 to 21, wherein the altered cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the rate at which 5-hydroxymethylcytosine (5hmC) is converted to 5-hydroxymethyluracil (5hmU) by deamination.

23. The method of claim 22, wherein conversion of 5hmC to 5hmU by deamination is undetectable.

24. The method of any one of claims 10 to 23, wherein the altered cytidine deaminase comprises the ZDD motif H-[P / A / V]-EX[23-28]-PCX[2-4]-C (SEQ ID NO: 1).

25. The method of any one of claims 10 to 24, wherein the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises a ZDD motif HXEX24SW(S / T)PCX[2-4]CX6FX8LX5R(L / I)YX[8-11]LX2LX[10]M (SEQ ID NO:2), wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is a Tyr (Y) amino acid of the ZDD motif.

26. The method of any one of claims 10 to 25, wherein the altered cytidine deaminase is a member of the APOBEC3A subfamily and comprises X[16-26]-GRXXTXLCYXV-X15-GXXXN-X12-HAEXXF-X14-YXXTWXXSWSPC-X[2-4]-CA-X5-FL-X7-LXIXXXR(L / I)Y-X8-GLXXLXXXG-X5-M-X4-FXXCWXXFV-X6-FXPW-X13-LXXI-X[2-6] (SEQ ID NO: 3).

27. The method of any one of claims 10 to 26, wherein the altered cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:

11.

28. The method of any one of claims 1 to 27, wherein the DNA library fragments are about 100 bp to about 300 bp in length.

29. The method according to any one of claims 1 to 28, further comprising: The double-stranded DNA library fragments not containing uracil are sequenced.

30. The method according to any one of claims 1 to 29, further comprising: The uracil-free double-stranded DNA library fragments are processed to generate a sequencing library.

31. The method according to claim 30, further comprising: The sequencing library is sequenced.

32. A kit comprising cytosine deaminase and a uracil-intolerant polymerase.

33. A kit comprising cytosine deaminase, uracil DNA glycosylase (UDG) and AP endonuclease.

34. The kit according to claim 33, wherein the AP endonuclease comprises endonuclease IV and / or AP endonuclease I.

35. A method for removing DNA fragments containing uracil residues from a sample, the method comprising: Providing a sample comprising DNA fragments, wherein the DNA fragments comprise 5' and 3' library adaptors; as well as By incubating the sample containing the DNA library fragments with a uracil-intolerant polymerase, The sample is subjected to polymerase chain reaction (PCR) amplification by contacting dNTPs with primers complementary to the 5' and 3' library adaptors under conditions to provide for second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

36. A method for removing DNA fragments containing uracil residues from a sample, the method comprising: Providing a sample comprising DNA fragments, wherein the DNA fragments comprise 5' and 3' library adaptors; as well as contacting the sample comprising DNA fragments with uracil DNA glycosylase (UDG) and an endonuclease, thereby generating DNA fragments cleaved at uracil residues; as well as A sample containing DNA fragments cleaved at uracil residues is subjected to polymerase chain reaction (PCR) amplification by contacting the sample containing DNA fragments cleaved at uracil residues with a polymerase, dNTPs, and primers complementary to 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

37. The method of claim 36, wherein the polymerase comprises a uracil-intolerant polymerase.

38. A method for removing DNA fragments containing uracil residues from a sample, the method comprising: Providing a sample comprising raw DNA fragments, wherein the raw DNA fragments comprise 5' and 3' library adapters; By incubating the sample containing the original DNA fragments with a uracil-intolerant polymerase, contacting the sample with dNTPs and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating DNA fragments that do not contain uracil residues in the synthesized second strand; contacting the sample containing DNA fragments that do not contain uracil residues in the synthesized second strand with an endonuclease to digest the original DNA fragments, thereby generating a single-stranded synthesized second strand; as well as The sample comprising the single-stranded synthesized second strand is subjected to polymerase chain reaction (PCR) amplification by contacting the sample comprising the single-stranded synthesized second strand with a polymerase, dNTPs, and primers complementary to the 5' and 3' library adapters under conditions for providing second strand synthesis, thereby generating double-stranded DNA fragments that do not contain uracil.

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