Construction and application of cell-free DNA methylation sequencing library

Abstract

The present invention relates to a method and application of a cell-free DNA methylation sequencing library, which can detect DNA methylation and fragmentomics simultaneously. In the method of the present invention, when constructing a sequencing library, the phosphodiester bond gap on the DNA double strand is first repaired, and then a linker sequence is linked to the double stranded DNA, and then a DNA methylation sequencing library is constructed by an enzymatic conversion method. The method provided by the present invention maintains the integrity of the DNA double strand, prevents DNA strand breakage, ensures that the fragmentomics information in the sequencing library is not damaged, and prevents the DNA methylation modification downstream of the gap from being erased in the end repair process, so that accurate DNA methylation information can be obtained.

Description

[Technical field]

[0001] The present application relates to biotechnology, and in particular to a method for epigenetic analysis of cell-free DNA. [Background technology]

[0002] Cell-free DNA (cfDNA) is generally DNA fragments released from inside cells into body fluids or the environment during cell metabolism, exocytosis or apoptosis and necrosis. Trace amounts of cell-free DNA are contained in materials such as plasma, serum, urine, saliva, and amniotic fluid, which reflect information on genetic mutations, diseases, aging, etc. in the body. Extraction, library construction, sequencing, and analysis of cell-free DNA have been widely applied in various aspects of clinical diagnosis and scientific research, such as cancer screening and diagnosis, prenatal diagnosis, preimplantation diagnosis, aging research, etc., as effective non-invasive / minimally invasive detection means. Epigenetic information contained in cell-free DNA, such as DNA methylomics / methylolomics characteristics and fragmentomics characteristics, are increasingly recognized as useful molecular markers for the detection, diagnosis, and / or monitoring of various diseases such as cancer.

[0003] DNA methylation is a phenomenon in which chemical modification of DNA occurs in vivo. Its chemical essence is that one original hydrogen atom at the 5th carbon atom of the pyrimidine ring of the cytosine base is replaced by a methyl group to form 5-methylcytosine. This DNA modification is an epigenetic mechanism that affects many life processes such as gene expression and regulation, cell division and differentiation, cell metabolism and growth, without changing the DNA sequence, and ultimately affects the growth and development of individual organisms, metabolic reproduction, disease development, and aging and death. When performing high-throughput sequencing (Next Generation Sequencing, NGS) of methylation modifications on DNA sequences, methylated / unmethylated cytosines must first be specifically converted to other bases in order to distinguish between methylation modifications.

[0004] Fragmentomics information is a set of information obtained by analyzing the fragmentation characteristics of cell-free DNA, including, for example, the distribution characteristics of cell-free DNA on the genome, fragment length and start / end positions, terminal base characteristics of fragments, non-smooth characteristics of fragments, etc. Research has shown that cell-free DNA tends to be cut in some genomic regions or elements, so fragmentomics information is one of the important epigenomic information that can reflect the status of chromatin opening and closing, nucleosome occupancy, transcription factor binding, epigenetic modification, etc., and is also a useful molecular marker for monitoring and diagnosing various diseases such as cancer [1]. When obtaining fragmentomics information by high-throughput sequencing, the integrity of DNA fragments must be maintained.

[0005] However, since cell-free DNA is formed by strong DNA enzymatic degradation inside / outside cells, it has several characteristics that make it difficult to detect: 1. The cfDNA content is low (e.g., only a few ng of cfDNA per mL of plasma). Therefore, detection methods must be compatible with starting amounts of cfDNA below 1 ng, and higher library construction efficiency is required to improve cfDNA detection sensitivity. 2. The cfDNA fragments are short (about 100bp to 200bp). First, short DNA fragments are easily lost during the purification process in library construction, and cannot withstand secondary cleavage due to chemical conversion. Second, whole genome library construction methods such as transposase, random primer, ligation and cleavage cannot be applied to short fragments, and the application of library construction methods by target amplification such as targeted PCR also has some difficulties and limitations. 3. cfDNA often has severe chemical and structural damage. Damage to cfDNA comes from various processes, including DNA damage caused by physiological and pathological conditions of cells, damage by nuclease digestion during cell death, damage caused by continuous attack by active substances such as free nucleases in the body fluid environment (e.g., blood circulation), damage caused by long-term storage in solutions such as plasma, and damage caused by repeated freezing and thawing. Studies have found that 97-98% of cfDNA has gaps, which are single-stranded phosphodiester bond cleavage gaps that occur in the DNA double strand [2]. When using single-stranded library methods, cfDNA is cleaved at the gaps after double-strand denaturation dissociation to generate fragments [3].

[0006] Existing DNA methylation sequencing library construction methods can be divided into four methods: (1) double-stranded library construction method by bisulfite conversion [4], (2) single-stranded library construction method by bisulfite conversion (e.g., Swift Biosciences kit Accel-NGS® Methyl-Seq DNA Library Kit, Cat. No. 30024), (3) double-stranded library construction method by enzymatic conversion method (e.g., EM-seq technology (WO2017075436A1) and its commercialized kit E7120 of New England Biolabs, Inc., TAPS technology in WO2019136413A1, Cabernet technology in WO2021077415), and (4) single-stranded library construction method by enzymatic conversion method (e.g., CN114032287A). However, none of the above four methods can completely solve the difficulties of cell-free DNA methylation detection.

[0007] Bisulfite conversion, a currently widely used method for detecting DNA methylation, requires DNA to be reacted for several hours under harsh conditions of high salt (9M), high acid (pH=5), and high temperature (50-90℃), which causes DNA to be denatured and cleaved, and most of it is decomposed and lost during the conversion reaction. Due to the large amount of DNA cleavage and loss, the bisulfite conversion method cannot meet the library construction requirements for low starting amounts of cfDNA, and it also exacerbates the short fragmentation and gap damage of cfDNA, making it difficult to develop a highly sensitive cell-free DNA methylation detection technique using the bisulfite conversion method.

[0008] In recent years, DNA methylation sequencing technologies using enzyme conversion methods, such as EM-seq, TAPS and Cabernet, have gradually developed and matured. The enzyme conversion method eliminates harsh chemical treatment processes, avoids DNA cleavage and degradation during conversion, and has higher library construction efficiency, higher sensitivity and higher library uniformity. However, none of the patents and products related to the conventional enzyme conversion method can solve all the problems in cell-free DNA methylation detection. Regarding the problem of low starting amount of cfDNA, the EM-seq technology (WO2017075436A1) and TAPS technology (WO2019136413A1) have a minimum starting amount of 5-10ng, which means that cell-free DNA cannot be sequenced with high sensitivity. Cabernet technology (WO2021077415) enables library construction from low starting amounts of DNA and enables highly sensitive library construction even for single cells, but it cannot be applied to short fragments of cell-free DNA using transposase. In particular, Cabernet technology has not yet provided a solution to the problem of cfDNA gaps.

[0009] Regarding the issue of chemical damage present in cfDNA, in conventional sequencing that does not detect methylation, the gaps are filled by the end repair process, which does not affect sequencing, and is not considered important. In conventional bisulfite methylation sequencing, the gaps are cut and lost by using a single strand to create a library, but since there are no errors in the methylation sequencing data itself, the problem is not considered important. The conventional method using the novel enzyme conversion method is double-stranded library construction, and indeed serious methylation loss is observed, which leads to serious methylation loss. For example, the methylation sequencing library data constructed by Erger et al. [5] using the double-stranded library construction method shows that the average methylation level continues to decrease from about 80% at the start of the library read to 40%, and then extensive methylation loss is observed in read 2, but the authors did not discuss this abnormality in their paper. In the DNA methylation sequencing library and its construction and detection methods disclosed by Takeishi Biology (CN114032287A), the authors observed an abnormal phenomenon in which the methylation level decreased with library reads. They attributed this to the simple loss of terminal single strands, and decided to use a single-stranded library construction method to avoid the problem, but this did not actually solve the problem, and the fragmentomics information could not be accurately obtained due to gap cleavage in the single-stranded state, and the single-stranded ligation efficiency was not high, resulting in low library productivity. Therefore, to date, the impact of the presence of single-stranded gaps on DNA methylation detection has not yet been recognized or resolved in the academic community.

[0010] In conclusion, there is currently no library construction method that can comprehensively solve the problems of low starting template amount, short fragments, and serious gaps in cell-free DNA methylation detection, and it is impossible to obtain a complete DNA library or accurately measure cell-free DNA methylation to obtain fragmentomics information. Summary of the Invention

[0011] The inventors have intensively studied the characteristics of cfDNA and the principles of library construction methods, analyzed the causes of problems in the cfDNA methylation detection process, and made a series of targeted improvements, thereby providing a novel cfDNA methylation sequencing method.

[0012] In one aspect, the present application provides a method for pre-treating a biological sample for constructing a sequencing library, comprising: S1: Repairing single-stranded gaps in DNA fragments contained in the sample with DNA ligase; and S2: optionally filling terminal gaps in the DNA fragments with a DNA polymerase, The sequencing library is for obtaining DNA methylation information data of the biological sample after sequencing, or for obtaining DNA methylation information and fragmentomics information data of the biological sample.

[0013] In some embodiments, the length of the DNA fragment is 50 bp to 1000 bp, preferably 100 bp to 500 bp, and more preferably 100 bp to 350 bp.

[0014] In some embodiments, the method further comprises, prior to step S1, fragmenting DNA molecules contained in the biological sample to generate the DNA fragments.

[0015] In some embodiments, the ligase has 3',5'-phosphodiester bond catalytic activity.

[0016] In some embodiments, the ligase is selected from HiFi-Taq ligase, T4 DNA ligase, Taq DNA ligase, E. coli DNA ligase, and T7 DNA ligase.

[0017] In some embodiments, the biological sample is selected from an animal body fluid, a cell culture fluid, or a natural biological sample, including, but not limited to, peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymphatic fluid, bronchoalveolar lavage fluid, amniotic fluid, cleavage cavity fluid, cell culture medium, embryo culture medium, microbial culture medium, soil leachate, and bone powder leachate.

[0018] In some embodiments, the DNA fragments include, but are not limited to, cell-free DNA, free DNA from outside the nucleus, and fragmented DNA, for example, cell-free DNA from a cancer subject.

[0019] In some embodiments, the content of DNA fragments in the sample is 10 ng or less, or 1 ng or less, for example 0.1 ng.

[0020] In some embodiments, the DNA methylation includes, but is not limited to, cytosine methylation and / or methylolation.

[0021] In another aspect, the present application provides a method for constructing a sequencing library, comprising: 1) preparing a biological sample and pretreating the biological sample using the above-mentioned pretreatment method to obtain a DNA fragment having no 3',5'-phosphodiester bond gap; 2) constructing the sequencing library using DNA fragments that do not have a 3',5'-phosphodiester bond gap; Including, The sequencing library is for obtaining DNA methylation information data of the biological sample after sequencing, or for obtaining DNA methylation information and fragmentomics information data of the biological sample.

[0022] In some embodiments, step 2) further comprises: a) blunting both ends of the DNA fragments and adding linker sequences, preferably linker sequences in which some or all of the cytosines are methylated and / or methylolated cytosines; and b) converting the cytosines in the DNA fragments to uracil by an enzymatic conversion method, wherein the uracil is recognized as a thymine in subsequent amplification and sequencing, and the methylated cytosine is recognized as a cytosine in subsequent amplification and sequencing.

[0023] In some embodiments, step 2) further comprises: a) blunting both ends of the DNA fragments and adding linker sequences; and b) converting methylated cytosines in the DNA fragments to uracil or dihydrouracil by an enzymatic conversion method, wherein the uracil or dihydrouracil is recognized as thymine in subsequent amplification and sequencing, and the unmethylated cytosines are recognized as cytosines in subsequent amplification and sequencing.

[0024] In some embodiments, the enzymatic conversion method is an EM-seq conversion method or a TAPS conversion method.

[0025] In some embodiments, the method further comprises adding carrier DNA to the biological sample after step a).

[0026] In some embodiments, the carrier DNA may be any DNA that does not contain the linker sequence, and preferably, the fragment size of the carrier DNA is 100 to 500 bp.

[0027] In another aspect, the present application provides a sequencing library obtained by the above method.

[0028] In another aspect, the present application provides a method for identifying a location of a single-stranded gap in a DNA fragment contained in a biological sample, the method comprising: 1) dividing the biological sample into two biological samples, biological sample A and biological sample B; 2) preparing a first sequencing library by treating biological sample A with the above pretreatment method, and preparing a second sequencing library by treating biological sample B with the above pretreatment method under conditions in which DNA ligase is not used in the pretreatment step; and 3) identifying the location of the single-stranded gap based on the difference in DNA methylation information obtained from the first sequencing library and the second sequencing library. The present invention provides a method comprising:

[0029] In another aspect, the present application provides a method for identifying a health status of a subject, comprising: 1) preparing a biological sample from a subject; 2) constructing a sequencing library by the above pretreatment method; and 3) Sequencing the sequencing library or sequencing after enrichment by hybrid capture. Including, The present invention provides a method for identifying the health status of a subject by obtaining DNA methylation information data of the biological sample or DNA methylation information data and fragmentmix information data of the biological sample based on sequencing data and comparing the data with normal DNA methylation information data and / or fragmentmix information data in a population.

[0030] The method provided herein can be used for epigenetic analysis of cell-free DNA. The method provided herein can also be used in the fields of genomics, medicine, diagnostics and epigenetics research. [Brief description of the drawings]

[0031] [Figure 1] 1 is a flow chart of the method for constructing a methylation library provided herein. [Diagram 2] 1 is a flowchart of the EM-seq transformation method. [Diagram 3]1 is a flow chart of the TAPS conversion method. [Figure 4] The results of library methylation bias analysis in the construction of a methylated library from human peripheral blood cell-free DNA using a method with (solid line) and without (dashed line) DNA ligase treatment are shown. The average CpG methylation rate (vertical axis) changes along the 5' to 3' direction of each library fragment (horizontal axis). [Diagram 5] This shows the regularity of the fragment distribution of cell-free DNA, with peaks at a period of approximately 170 bp. [Figure 6] The distribution of library fragments generated by different library construction methods is shown. Capillary gel electrophoresis was performed with the same library DNA input amount, and the distribution of DNA fragments in the range of 1 to 6000 bp was analyzed. As a result, the library of the method of the present invention (upper figure) shows a periodic DNA fragment peak profile that matches the characteristics of peripheral blood cell-free DNA. In contrast, the Swift Biosciences, Accel-NGS (registered trademark) Methyl-Seq DNA Library Kit kit (lower figure) has only one main peak, does not have periodic peaks, and contains a large amount of non-library impurity peaks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0033] As used herein, a "DNA ligase" is an enzyme for repairing a single-stranded gap (nick) in a double-stranded DNA molecule. A DNA ligase typically has 3',5'-phosphodiester bond catalytic activity. In one example, the DNA ligase can catalyze the formation of a phosphodiester bond between the 5' phosphate group and the 3' hydroxyl group in the gap. Generally, a DNA ligase cannot add a new nucleotide to the gap.

[0034] As used herein, the term "DNA polymerase" refers to an enzyme that can use a single DNA strand as a template to add dNTPs (deoxyribonucleotides) to the 3' end of a primer to synthesize a complementary strand when a primer is present. DNA polymerases can also have 3'-5' exonuclease activity, which normally performs proofreading during synthesis, and 5'-3' exonuclease activity, which performs excision repair.

[0035] As used herein, a "DNA fragment" is a short fragment of DNA, e.g., 50 bp to 700 bp in length, e.g., 100 bp to 500 bp, in particular 100 bp to 350 bp in length. The DNA fragments contained in a biological sample are usually heterogeneous, i.e., not identical in length, and accordingly the above length refers to the average length of these DNA fragments. These DNA fragments may have different sequences, e.g., originating from different regions of the genome of the same organism or from different organisms. The DNA fragments may have single-stranded gaps and may have blunt or non-blunt ends (3'- or 5'-overhangs).

[0036] As used herein, a "blunt-ended DNA fragment" is a double-stranded DNA fragment that does not have a 3' or 5' overhang at either end.

[0037] As used herein, "DNA methylation" refers to the modification of cytosine bases in a DNA molecule or DNA fragment to 5-methylcytosine (5mC). DNA methylation in vertebrates generally occurs at CpG sites (i.e., sites in the DNA sequence where guanine is linked immediately after cytosine) and converts cytosine to 5-methylcytosine by the catalysis of DNA methyltransferase. Many CpG sites in the human genome are already methylated, but some specific regions, such as CpG islands rich in cytosine (C) and guanine (G), are usually unmethylated. CpG methylation affects the transcriptional activity of associated genes, for example, methylation can suppress tumor suppressor genes, while demethylation stimulates the expression of some cancer genes, which in either case may lead to the development of cancer. In addition, a small number of cytosine bases may be modified to 5-methylolcytosine (5hmC), 5-formylation (5fC), or 5-carboxylation (5caC), even though the incidence is less than 5mC. In this specification, when referring to methylation, it may also refer to modification to 5hmC, 5fC, or 5caC, unless otherwise specified by the context.

[0038] As used herein, "DNA methylation information" refers to information on the methylation status in a DNA molecule or DNA fragment, including, but not limited to, a methylation site, a methylation level, a methylation method (5mC or 5hmC), and the like. As used herein, "methylation level" is also referred to as "methylation degree" and refers to the proportion (or frequency) of a specific methylation site in a sample that is modified by methylation. Whether a site is methylated or not can be detected by various methods. Conventional methods include chemical or enzymatic conversion methods, in which one of methylated cytosine and unmethylated cytosine is converted to uracil (U) or a base that is substantially equivalent to uracil in base pairing (e.g., dihydrouracil, DHU) in the conversion process. In the subsequent amplification process, the corresponding uracil pairs with adenine (A) as thymine (T), and finally, the cytosine or methylated cytosine at the methylation site appears as thymine in the detection result (e.g., sequencing result). By comparing with a reference sequence, it is possible to identify whether a cytosine in a DNA molecule or DNA fragment is methylated. The reference sequence may be a sequence derived from the same sample but without the above-mentioned conversion, or a corresponding sequence in a healthy population. As described below, 5mC and 5hmC can also be distinguished in several ways. Currently, DNA methylation information is widely used in the screening and diagnosis of cancer (e.g., lung cancer, breast cancer, liver cancer, colorectal cancer, etc.), especially in the early screening and diagnosis.

[0039] In this specification, "fragmentomics information" refers to a collection of information obtained by analyzing the fragmentation characteristics of cell-free DNA in a sample, including, for example, the distribution characteristics of cell-free DNA on the genome, fragment lengths and start and end positions, characteristics of the fragment terminal bases, non-smooth characteristics of fragments, etc. Recently, there have been several reports of its use in screening for cancer (e.g., lung cancer).

[0040] As used herein, "enzyme conversion method" refers to modifying cytosine or methylated cytosine by the catalytic action of a specific enzyme so that it can distinguish its methylation from unmethylation state or 5mC from 5hmC in subsequent detection. Several enzymatic conversion methods are known in the art, including, but not limited to, EM-seq conversion method and TAPS conversion method. In enzymatic conversion method, these enzymes are usually used in combination with some chemical reagents.

[0041] In this specification, the term "EM-seq conversion method" refers to a technique developed by New England Biolabs to distinguish between methylated and unmethylated cytosines by enzymatic conversion. 5mC and 5hmC are modified to 5-carboxycytosine (5caC) using dioxygenase TET (including TET1, TET2, TET3, etc.), and then cytidine deaminase is used to deaminate unmethylated cytosine and convert it to uracil. The cytidine deaminase used includes APOBEC protein family members such as APOBEC 3A. When DNA glycosyltransferase (GT) is utilized, 5mC and 5hmC can be further distinguished. The scheme of the EM-seq conversion method can be seen in FIG. 2. For more detailed information on the EM-seq conversion method, PCT Publication WO2017075436A can be seen, the entirety of which is incorporated herein by reference.

[0042] The "TAPS (TET-assisted pyridine borane sequencing) conversion method" is somewhat similar to the EM-seq conversion method, which also enzymatically modifies 5mC and 5hmC to 5-carboxycytosine (5caC), but does not perform a deamination step thereafter, and converts 5caC to dihydrouracil (DHU) using the reducing agent pyridine borane while retaining unmethylated cytosine. In the subsequent copying or amplification process, DHU is recognized by the polymerase as uracil and paired with A. In this way, 5mC and 5hmC modified sites are detected as T, and unmodified C is still detected as C. Similarly, when DNA glycosyltransferase (GT) or KRuO4 is used, 5mC and 5hmC can be further distinguished. The scheme of the TAPS conversion method can be seen in Figure 3. For more detailed information about the TAPS conversion method, see PCT Publication WO2019136413A, the entire contents of which are incorporated herein by reference.

[0043] As used herein, the term "health status" refers to the health condition of a subject, including whether or not the subject is suffering from a certain disease, the level of risk of suffering from a certain disease, suitability for a certain therapeutic agent or treatment, the prognosis of a disease, and the like. As used herein, a "subject" refers to an animal, e.g., a mammal, including, but not limited to, humans, rodents, monkeys, felines, canines, equines, bovines, hogs, sheep, goats, mammalian laboratory animals, mammalian livestock, mammalian sport animals, and mammalian pets. The subject may be male or female and may be of any age, including infants, toddlers, adolescents, adults, and elderly subjects. In some instances, the subject is a patient. In certain instances, the subject is a human, e.g., a human patient. The term may generally be used interchangeably with "patient," "test subject," "treatment subject," and the like.

[0044] In one aspect, the present invention provides a method for pretreating a sample to be subjected to construction of a sequencing library. This pretreatment includes repairing single-stranded gaps in DNA fragments contained in the sample, followed by normal end repair. Gap repair can be performed using DNA ligase, and end repair can be performed using DNA polymerase. The reason why gap repair must be performed prior to normal end repair is that the inventors have recognized and confirmed through research that the presence of these single-stranded gaps is an important cause of loss of methylation information, particularly in cell-free DNA (cfDNA), such as circulating tumor DNA (ctDNA). When only the normal repair process is performed, the DNA polymerase used performs strand displacement downstream of the post-gap due to its 5'-3' exonuclease activity and polymerase activity, resulting in the erasure of methylation information contained in the original strand. When end repair of DNA fragments is performed after repairing the gap, strand displacement does not occur, and the original methylation information can be retained.

[0045] In another aspect, the present invention provides a method for constructing a sequencing library, comprising subjecting a sample to the above pretreatment and then constructing a sequencing library. In some embodiments, the process of constructing the library also includes modifying cytosine (C) or methylated cytosine (including 5mC and / or 5hmC) therein by an enzymatic conversion method, thereby enabling a distinction between methylated and unmethylated cytosine, or between 5mC and 5hmC in a subsequent detection step, thereby forming a DNA methylation sequencing library. By installing this sequencing library in a computer and sequencing it, the corresponding methylation information data can be obtained. Those skilled in the art can understand that by employing the method of the present invention for sequencing, not only methylation information data but also fragmentomics information data can be obtained at the same time. This is because this library construction technology is a methylation NGS detection library construction based on cfDNA double-stranded fragments, and by differentiating the methylation status through enzymatic conversion after repairing single-stranded gaps, it retains the original fragmentomics information, including fragment length distribution characteristics, fragment lengths, etc., without substantially compromising the integrity of the original DNA fragments in the sample.

[0046] In another embodiment, the present invention makes it possible to further reduce the requirement for the DNA fragment content in the sample of the method of the present invention to a DNA content of 0.1 ng or less by using a carrier DNA.

[0047] In another aspect, the present invention identifies the position of a single-stranded gap in a DNA fragment contained in a biological sample based on the difference in methylation information caused by the addition or non-addition of DNA ligase. Since 5mC or 5hmC is not usually present exactly at the gap position, the position of the gap is often considered to be a gap position range, for example, the position of the gap is considered to be between a first position, which is a methylation position that can be detected with or without the addition of DNA ligase, and a second position, which is a downstream position where a difference in methylation is detected (for example, methylation is detected when DNA ligase is added, but not when ligase is not added).

[0048] In another aspect, the present invention makes it possible to assess the health status of a subject based on the acquired methylation information, and in particular, by combining the methylation information with fragment mix information, the health status of a subject can be more accurately assessed.

[0049] It should be understood that the technical problems that the present invention aims to solve include, but are not limited to, the following. 1. To provide a high-throughput second-generation sequencing library construction and NGS sequencing method for cell-free DNA that can accurately measure DNA methylation and fragmentomics information simultaneously. 2. Because it is a highly sensitive method, efficient NGS library construction can be performed even with low starting sample amounts of 1 ng or less. 3. It is a method that maintains the integrity of the cell-free DNA strand, ensuring that the fragmentomics information in the sequencing library is not destroyed. 4. Avoiding DNA breaks and loss of methylation information due to gap damage on cell-free DNA. 5. By simply modifying the technical route, it becomes possible to measure any methylation information, such as DNA methylolation, and fragmentomics information.

[0050] The method provided by the present invention can achieve the above-mentioned objectives by being used for, but not limited to, epigenetic analysis of cell-free DNA. The method includes one or more of the following features: 1. Using a specific DNA ligase to avoid loss of methylation due to DNA gap damage. In this method, one or more specific DNA ligases, such as Taq DNA ligase, are used for DNA having gaps to repair phosphodiester bond gaps on the template DNA double strand, thereby preventing DNA strand break loss and / or erasure of methylation information in the subsequent process. When the gap-repaired DNA template is converted into a double-stranded library by an enzymatic conversion method, serious erasure of methylation information does not occur at the 3' end of the library, and the detected methylation rate is restored to near the average methylation rate. 2. By using carrier DNA to assist in the protection of trace DNA, effective library construction and highly sensitive methylation sequencing of trace DNA can be realized. This method is an improvement based on the enzymatic conversion method, and is based on the Cabernet technology described in Patent Publication WO2021077415. After adding a library linker to the template DNA, carrier DNA with similar physicochemical properties but no linker sequence is added before the enzymatic conversion. In processes where DNA loss or decomposition is likely to occur, such as purification, the carrier DNA is the main part that is lost, and loss of the template DNA can be effectively avoided. In addition, by omitting the single-strand purification process after the conversion is completed and directly adding the PCR amplification reaction system, loss of the DNA template during the purification process is minimized. 3. Bisulfite conversion results in the loss of fragmentomics information, but this method, which applies enzyme conversion and double-stranded DNA library construction strategies, can accurately measure DNA methylation information and retain DNA fragmentomics information. Furthermore, treatment with DNA ligase does not affect the retention of fragmentomics information.

[0051] Methodology Overview (1) Extraction of cell-free DNA Cell-free DNA is DNA that is free outside the cell or outside the cell nucleus, and can be extracted from various biological materials such as body fluids, in vitro cell culture fluid, and natural environments, including, but not limited to, peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymph, alveolar lavage fluid, amniotic fluid, cleavage cavity fluid, cell culture fluid, embryo culture fluid, microbial culture medium, soil leachate, bone powder leachate, etc. Cell-free DNA can be obtained through extraction and purification.

[0052] The total amount of cell-free DNA used for downstream sequencing analysis can be as low as picogram or nanogram levels.

[0053] (2) DNA gap repair This step may involve the use of a specific ligase, which may be any one or more combinations of repair reagents and / or DNA ligases, such as, but not limited to, HiFi-Taq ligase, T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, E. coli DNA ligase, etc.

[0054] DNA ligase and / or DNA repair reagents repair gaps on the template DNA duplex, preventing DNA strand break loss and / or erasure of methylation information in subsequent processes.

[0055] (3) Addition of library linker sequences (adaptors) to both ends of the DNA A polymerase or the like is used to fill in the gaps at the double-stranded ends, and in some instances, a single A (adenine) base is added to the end to facilitate ligation of the linker.

[0056] In this step, DNA linkers having specific sequences that are partially or completely cytosine methylated or methylolated are ligated to both ends of a template DNA using DNA ligase.

[0057] In some instances, a biotin label may be added to the DNA linker sequence to facilitate subsequent purification.

[0058] In some instances, sample barcodes and single DNA molecule barcodes may be introduced into DNA linker sequences to label the origin of the DNA template, improving detection accuracy and allowing samples of different origins to be mixed in subsequent steps.

[0059] (4) Addition of Carrier DNA To prevent the loss of minute amounts of template DNA in the subsequent steps, carrier DNA with similar properties to the template DNA is added to the template DNA solution in an amount several times larger than the template DNA. This is the portion that is mainly lost in steps such as purification where DNA loss and degradation are likely to occur, and thus the loss of template DNA can be effectively avoided.

[0060] Since the carrier DNA does not have a specific DNA linker sequence, it is not detected in the subsequent library amplification and sequencing.

[0061] (5) Methylation conversion This process uses an enzymatic conversion method to specifically convert cytosine, methylated cytosine and / or methylolated cytosine on DNA, changing the base pairing rules of cytosine, methylated and / or methylolated cytosine so that they can correspond to and be differentiated as different bases during subsequent amplification and sequencing processes.

[0062] In this step, the "EM-seq" technology invented by New England Biolabs can be used. In another example, the TAPS technology can be used.

[0063] (6) Library amplification and sequencing During methylation conversion, DNA becomes single-stranded or partially single-stranded due to thermal denaturation and changes in base pairing rules. Single-stranded DNA has a weak affinity during the purification process, resulting in a large loss. This method omits the single-strand purification step and directly adds the PCR amplification reaction system, thereby maximally avoiding the loss of template DNA during the purification process.

[0064] The library is subjected to a PCR amplification reaction, and the amplification primer contains a sequence complementary to the linker sequence and the sequencer adaptation sequence, and a sample barcode may be introduced. The primers complementarily pair with the linker sequences added to both ends of the template DNA, and the desired sequencing library can be formed while amplifying.

[0065] After purification, the sequencing library is quantified and quality tested, and then sequenced using a sequencer.

[0066] (7) Analysis of sequencing results In the example of methylation conversion by EM-seq technology, cytosine is converted to thymine and methylated cytosine is left as cytosine, whereas in the example of methylation conversion by TAPS technology, methylated cytosine is converted to thymine and cytosine remains unchanged. DNA methylation information can be obtained by alignment with a reference genome. In this method, library linkers are added when the cell-free DNA is in a double-stranded state before methylation conversion, so DNA fragment information is maintained, and fragment mix information such as the original DNA fragment length, fragment distribution, upstream and downstream breakpoint positions, and terminal base pattern of the cell-free DNA can be obtained by alignment with a reference genome. The above analysis process can be seen in Figure 1.

[0067] Other applications of the method of the present invention DNA methylolation and fragment mix sequencing of cell-free DNA In mammals, the content of DNA methylolation is lower than that of DNA methylation, but has different biological significance.

[0068] If it is necessary to measure only methylolation without distinguishing between DNA methylation and methylolation during the above methylation conversion process, 5mC and 5hmC can be distinguished, for example, by not adding TET oxidase during the conversion process and maintaining other enzyme reactions.

[0069] Targeted PCR-based enrichment method Enriching target fragments of interest can save sequencing costs and increase the detection sensitivity of target genes.

[0070] In step (6) of the above "Overview of the Method", PCR is performed using primers complementary to the linkers at both ends of the template DNA to amplify the entire template DNA. If it is necessary to concentrate the target gene fragment, it can be amplified using the method of targeted PCR. Target primers containing a sequence complementary to the target gene fragment after methylation conversion are designed and introduced into the PCR reaction system to amplify the target gene sequence.

[0071] The target primer may or may not include some or all of a sequencer-compatible linker sequence.

[0072] Only the upstream or downstream primer can be input to obtain fragments with the target primer at one end and the linker sequence added in step (3) of the "Method Overview" above at the other end.

[0073] The upstream primer and the downstream primer may be linked at their termini and constructed on a single DNA strand to form a locked primer.

[0074] The default linker sequence primer may be introduced first, and the target primer may be introduced after amplification, or they may be introduced simultaneously, or the default linker sequence primer may not be introduced and only the target primer may be used.

[0075] Hybrid capture enrichment method In step (6) of the "Overview of the Method" above, hybrid capture enrichment may be performed before or after amplification. It is necessary to design a probe containing a sequence complementary to the target gene fragment after methylation conversion.

[0076] Enrichment by hybrid capture involves mixing the library with a single-stranded DNA / RNA probe labeled with biotin or the like, and by thermal denaturation and renaturation, the library complementary to the target sequence hybridizes to the probe, and the hybridized probe is captured using a method such as streptavidin-coupled magnetic beads to obtain an enriched library.

[0077] No gap repair is performed, and gap position information is obtained. If the ligase treatment in step (2) of the "Overview of the Method" above is omitted and the gap is repaired using an appropriate polymerase or modified nucleotides, the methylation information downstream of the gap will be changed. The gap position can be obtained by analyzing the sequence where methylation has been deleted / changed.

[0078] The present invention discloses a highly sensitive DNA methylation sequencing technology for single-stranded gapped DNA, which firstly repairs DNA gap damage to ensure that DNA methylation signals are not lost or misread, then the technology uses double-stranded DNA as a template to construct a library, overcoming the problem of low efficiency in single-stranded library construction, and then uses a highly optimized and adjusted enzyme conversion method to convert the base pairing rules of the methylation information, combined with the protection of the template DNA during the reaction and purification process by carrier DNA, finally obtains a high-quality and highly sensitive methylation information sequencing library with an extremely low starting amount of DNA as a template, and retains the fragmentomics information of DNA.

[0079] The method of the present invention has the following advantages, but is not limited to these: 1. The minimum starting amount of DNA is low, less than 0.1 ng. 2. Library production can be improved by four times. 3. The alignment rate of the library is improved from 40% to 70%. 4. The length of consecutive DNA methylation losses at the 3' end of the library is reduced from 170 bp to within 40 bp.

[0080] The method of the present invention can be applied to various liquid biopsy diagnostic products such as early cancer screening, cancer gene detection, prenatal diagnosis, preimplantation diagnosis, genetic diagnosis, etc., and also provides powerful technical support for scientific research in related fields.

[0081] The present invention will now be further described with reference to specific examples. EXAMPLES

[0082] Example 1: In this example, the process of preparing a sequencing library is explained using peripheral blood cfDNA as an example.

[0083] 1.1 cfDNA extraction Extraction of cfDNA can be performed using any standard means known in the art.

[0084] 5 mL of peripheral blood was collected using a 5 mL EDTA venipuncture vacuum blood collection tube (with purple cap), and within 4 hours, plasma separation was performed by low-speed centrifugation at 1600 x g for 10 min using a horizontal rotor at room temperature. The upper layer of plasma was carefully removed and transferred to a 1.5 mL tube. The sample was centrifuged at 6000 x g for 10 min at 4 ° C in a high-speed centrifuge, and the supernatant was transferred to a new 1.5 mL tube and stored in a -80 ° C refrigerator or on dry ice. cfDNA can be extracted from the separated plasma using a nucleic acid extraction or purification kit (QIAGEN, QIAamp (registered trademark) Circulating Nucleic Acid, part number 55114), and 5 to 20 ng of cfDNA can usually be obtained per 1 ml of plasma.

[0085] 1.2 cfDNA gap repair A ligase reaction system was set up, using Taq DNA ligase as an example, and the reagents in Table 1 below were added to the purified cfDNA solution. Among them, the sonicated spike-in DNA was prepared by mixing equal volumes of whole genome CpG methylated pUC19 DNA (NEB E7122) and whole genome CpG unmethylated lambda DNA (NEB E7123), which was cut to 300bp by ultrasonication, and then diluted to 0.2ng / μL for use. [Table 1]

[0086] The mixture was mixed uniformly for a short period of time, centrifuged, and then reacted in a PCR device at 37°C to 60°C for 10 to 30 minutes.

[0087] 1.3 End repair and linker ligation The End Prep reaction system was set up, and the reagents (Table 2) from the kit NEBNext Ultra II End Repair / dA-Tailing Module (NEB E7546L) were added sequentially to the repair product from the previous step. [Table 2]

[0088] The mixture was mixed uniformly for a short period of time, centrifuged, and then reacted in a PCR device at 20°C for 30 minutes and at 65°C for 30 minutes.

[0089] After the reaction was completed, a linker sequence was added in which all cytosines were methylated. To complement the subsequent library construction primers, the recommended pair of linker sequences are: 5'- A [5mC] A [5mC] T [5mC] TTT [5mC] [5mC] [5mC] TA [5mC] A [5mC] GA [5mC] G [5mC] T [5mC] TT [5mC] [5mC] GAT [5mC] T-3' (SEQ ID NO:1) and 5'-phosphate-GAT [5mC] GGAAGAG [5mC] A [5mC] A [5mC] GT [5mC] TGAA [5mC] T [5mC] [5mC] AGT [5mC] A-3' (SEQ ID NO:2). Primers were diluted to a total concentration of 15 μM, added in 1.25 μL, and mixed to homogeneity.

[0090] Then, the following reagents (Table 3) from the kit NEBNext Ultra II Ligation Module (NEB E7595L) were added sequentially. [Table 3]

[0091] The mixture was mixed uniformly for a short period of time, centrifuged, and then reacted in a PCR device at 20°C for 15 minutes.

[0092] 1.4 Addition and purification of carrier DNA 1 μL of carrier DNA was added to the ligation product from the previous step, mixed uniformly for a short period of time, and centrifuged.

[0093] Carrier DNA was prepared by cutting lambda DNA (NEB N3011) to 300 bp using ultrasound and then diluting it to 25 ng / μL for use.

[0094] DNA was purified using 1.8 volumes of SPRI or Ampure XP magnetic beads and eluted with water to obtain purified DNA.

[0095] 1.5 Enzymatic conversion of DNA methylation The purified DNA obtained in the previous step was subjected to enzymatic conversion including DNA TET oxidation reaction, glycosylation protection, DNA thermal denaturation, and APOBEC deamination reaction using the NEBNext (registered trademark) Enzymatic Methyl-seq Conversion Module (NEB, E7125L) kit. After the APOBEC deamination reaction, the DNA was directly subjected to the next reaction without purification.

[0096] 1.6 PCR amplification of libraries To the liquid after the APOBEC deamination reaction in the previous step, a PCR primer having a library linker and an index label was added, for example, 5 μL of NEBNext (registered trademark) Multiplex Oligos for Illumina primer set was added. Furthermore, 2x Q5U master mix (NEB M0597L) was added in the same volume as the existing liquid. After mixing uniformly for a short time and centrifuging, the reaction was carried out in a PCR device at 98 ° C for 30 s; 98 ° C for 10 s, 62 ° C for 30 s, 72 ° C for 90 s (7 cycles); 65 ° C for 5 min, and then stored at 4 ° C.

[0097] DNA was purified using 1.1 volumes of SPRI or Ampure XP magnetic beads, eluted with 1x TE buffer to obtain purified DNA, and stored at -20°C.

[0098] 1.7 Library Sequencing and Analysis The constructed libraries were subjected to concentration quantification and fragment distribution quality inspection, and then both ends were sequenced using an Illumina sequencer. The sequencing data was aligned and analyzed using Bismark software (the methylation rates of the top 10 bases in read 1 and the top 40 bases in read 2 were not included in the methylation analysis results to avoid bases with biased methylation rate distribution), and whole genome methylation data was obtained. Information such as the distribution characteristics in the genome of the library analyzed bioinformatically, fragment length and start / end positions, and base distribution characteristics at the 5' end, became fragment mix information. [Table 4]

[0099] The sequencing results contain tens of millions to billions of the above information, and the amount of sequencing varies depending on the choice of sequencer and chip. EXAMPLES

[0100] Example 2 Comparison of DNA gap repair To evaluate whether the present method can compensate for the drawback of methylation loss in the conventional enzymatic conversion library construction method, a parallel controlled study was performed.

[0101] After extracting cell-free DNA from the plasma of healthy subjects, the subjects were divided into two groups and experiments were conducted. Experimental group: DNA ligase treatment (see step 1.2: DNA gap repair reaction in Example 1) Control group: No DNA ligase treatment (control group, DNA ligase replaced with water)

[0102] The two groups of cell-free DNA were subjected to the same double-stranded library construction steps.

[0103] Terminal modification was performed using the NEBNe.t Ultra II End Repair / dA-Tailing Module reagent (NEB E7546L), and linker ligation was performed using the NEBNe.t Ultra II Ligation Module reagent (NEB E7595L) and the NEBNe.t® EM-seq® Adaptor reagent (NEB E7165).

[0104] The template DNA after linker addition was purified with magnetic beads and enzymatically converted using the NEBNe.t (registered trademark) Enzymatic Methyl-seq Conversion Module (NEB, E7125L) kit. The DNA after conversion was subjected to PCR library amplification using 2. Q5U master mi. (NEB M0597L).

[0105] The constructed library was sequenced at both ends of 150 bp using an Illumina sequencer, and alignment and analysis were performed using Bismark software. The methylation bias (M-bias) of the library was statistically analyzed and the average methylation rate of each read position was plotted along the 5'-3' direction of the library fragment to evaluate whether the methylation level of the library was uniform. The results are shown in Figure 4.

[0106] As shown in the figure, after library construction using methylated double-stranded DNA without gap repair of cell-free DNA, the average methylation level of DNA in the sequencing library showed increasingly significant methylation loss (dotted line in the figure) from about 130 bp in read 1 along the 5'-3' direction of the library fragment to read 2, which was particularly prominent in the sequencing data of read 2, losing 40% of the true methylation rate on average. In the experimental group, the methylation loss in rear 2 was repaired by DNA ligase repair treatment (solid line in the figure) (the decrease in the methylation rate of the last 30 bp at the 3' end of the library was due to the loss of the 3' end of the cell-free DNA double-stranded template itself).

[0107] This data indicates that the methods provided herein can compensate for methylation loss due to gap lesions in cell-free DNA. EXAMPLES

[0108] Example 3 Fragment mix information can be obtained along with methylation information. To evaluate the superiority of the present method over conventional methods, the present technology and a commercialized kit were tested side by side. Experimental group: the technical method of the present invention (for specific procedures, see Example 1) Control group: Swift Biosciences (Kit manufacturer part number: 33024 Accel-NGS(R) Methyl-Seq DNA Library Kit, for specific operation details, please refer to the kit operation manual)

[0109] After extracting cell-free DNA from plasma of healthy subjects, library construction tests were performed using the same starting amount of cell-free DNA (3 ng) and the same number of PCR amplification rounds (7 rounds) by two different methods. After sequencing and analyzing the libraries, the alignment efficiency was evaluated using Bismark software. The results are shown in Table 5. [Table 5]

[0110] Specifically, the DNA yield of the constructed library was quantified by Qubit, and compared with the Swift kit, the library yield by the technical method of the present invention was 83.4 ng, which is much higher than the library yield by the Swift method. The alignment rate of the sequencing library by the technical method of the present invention was 70%, which is about twice as high as that of the swift library (44%), indicating high library effectiveness. The duplication rate of the sequencing library constructed by this method was 15%, which is significantly lower than the duplication rate of the swift library (22%), indicating fewer overlapping fragments in the library and higher quality. According to this method, fragmentomics information is retained (see Figures 5 and 6). The DNA fragments before library construction were analyzed using an Agilent fully automated pulsed field electrophoresis fragment analysis system (Agilent Femto Pulse). As a result, as shown in Figure 5, the cell-free DNA itself has a characteristic peak profile with a period of about 170 bp, which is a typical fragmentation characteristic due to nucleosome occupancy. The fragments after library construction were analyzed using an Agilent fragment analysis system (Agilent 5200 Fragment Analyzer System) (see Figure 6). Compared with the library constructed using the Swift kit, the sequencing library constructed by the library construction sequencing method of the present invention contains a fragmentation distribution characteristic of cell-free DNA, and fragmentomics data can be better analyzed and obtained.

[0111] As shown for the first time in the present invention, serious methylation defects due to double-stranded library construction are caused by both single-strand gaps and deletion of terminal single strands. Deletion of terminal single strands is a loss of original information and cannot be restored. In addition, single-strand gaps erase the methylation of most reads, and the principle is that in the double-stranded library construction process, it is first necessary to end-repair the double-stranded DNA template to form a DNA double strand with a blunt end or a 3'-A sticky end, and then link a linker sequence. Meanwhile, the DNA synthesis enzyme used for end repair has an activity that starts synthesis from the gap (Extension from Nick), and displaces or enzymatically degrades the original DNA template strand downstream from the gap position along the 5'-3' direction of DNA, synthesizing a replacement strand with an artificially provided unmodified base, resulting in the deletion and deletion of continuous DNA methylation information. In addition, such methylation deletion cannot be restored later by means of bioinformatics or the like, and due to the randomness of the gap position, it is not possible to accurately determine the effective part of the methylation information by a consistent criterion. The pretreatment method provided herein repairs the gaps prior to library construction, resolving the problem of extensive methylation loss (Figure 4).

[0112] References: 1. Lo, YMD, Han, DSC, Jiang, P. & Chiu, RWK Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies. Science 372, (2021). 2. Sanchez, C., Roch, B., Mazard, T., Blache, P., Dache, Z., Pastor, B., Pisareva, E., Tanos, R., & Thierry, A. R. (2021). Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics. JCI insight, 6(7), e144561. https: / / doi.org / 10.1172 / jci.insight.144561. 3. (Sanchez, C., Snyder, M. W., Tanos, R., Shendure, J., & Thierry, A. R. (2018). New insights into structural features and optimal detection of circulating tumor DNA determined by single-strand DNA analysis. NPJ genomic medicine, 3, 31. https: / / doi.org / 10.1038 / s41525-018-0069-0). 4. Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 2008; 133:523-36. 5. Erger, F., Norling, D., Borchert, D., Leenen, E., Habbig, S., Wiesener, M. S., Bartram, M. P., Wenzel, A., Becker, C., Toliat, M. R., Nurnberg, P., Beck, B. B., & Altmuller, J. (2020). cfNOMe - A single assay for comprehensive epigenetic analyses of cell-free DNA. Genome Medicine, 12(1), 54. https: / / doi.org / 10.1186 / s13073-020-00750-5.

Claims

1. A method for pre-treating a biological sample for constructing a sequencing library, comprising: S1: Repairing single-stranded gaps in DNA fragments contained in the sample with DNA ligase; and S2: filling terminal gaps in the DNA fragments with a DNA polymerase; The sequencing library is for obtaining DNA methylation information data of the biological sample after sequencing, or for obtaining DNA methylation information and fragmentomics information data of the biological sample, In step S1, the single-stranded gap is a 3',5'-phosphodiester bond gap; method.

2. The method of claim 1, wherein the length of the DNA fragment is between 50 bp and 1000 bp.

3. The method of claim 1, further comprising, prior to step S1, fragmenting DNA molecules contained in the biological sample to generate the DNA fragments.

4. The method of claim 1, wherein the ligase has 3',5'-phosphodiester bond catalytic activity.

5. 5. The method of claim 1 or 4, wherein the ligase is selected from HiFi-Taq ligase, T4 DNA ligase, Taq DNA ligase, E. coli DNA ligase and T7 DNA ligase.

6. The method of claim 1 , wherein the biological sample is selected from a cell culture medium or a natural biological sample.

7. 2. The method of claim 1, wherein the biological sample is selected from peripheral blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, lymphatic fluid, bronchoalveolar lavage fluid, amniotic fluid, cleavage cavity fluid, cell culture medium, embryo culture medium, microbial culture medium, soil leachate, and bone powder leachate.

8. The method of claim 7, wherein the DNA fragments are selected from cell-free DNA and fragmented DNA.

9. The method according to claim 1, wherein the content of DNA fragments in the sample is 10 ng or less.

10. The method of claim 1, wherein the DNA methylation comprises cytosine methylation and / or methylolation.

11. 1. A method for constructing a sequencing library, comprising: 1) preparing a biological sample and pretreating the biological sample using the method according to any one of claims 1 to 10 to obtain DNA fragments that do not have 3',5'-phosphodiester bond gaps; and 2) constructing the sequencing library using the DNA fragments; Including, The sequencing library is for obtaining DNA methylation information data of the biological sample after sequencing, or for obtaining DNA methylation information and fragmentomics information data of the biological sample; method.

12. step 2) further comprises: a) blunting both ends of the DNA fragments and adding linker sequences in which some or all of the cytosines are methylated cytosines and / or methylolated cytosines; and b) converting the cytosines in the DNA fragments to uracils by an enzymatic conversion method, wherein the uracils are recognized as thymines in subsequent amplification and sequencing, and the methylated cytosines are recognized as cytosines in subsequent amplification and sequencing, or Step 2) further comprises: a) blunting both ends of the DNA fragment and adding a linker sequence; and b) converting methylated cytosine in the DNA fragment to uracil or dihydrouracil by an enzymatic conversion method, wherein the uracil or dihydrouracil is recognized as thymine in subsequent amplification and sequencing, and the unmethylated cytosine is recognized as cytosine in subsequent amplification and sequencing. The method of claim 11.

13. The method according to claim 12, wherein the enzymatic conversion method is EM-seq conversion method or TAPS conversion method.

14. 13. The method of claim 12, further comprising adding carrier DNA to the biological sample after step a).

15. The method of claim 14, wherein the carrier DNA does not contain the linker sequence and the fragment size of the carrier DNA is 100-500 bp.

16. 1. A method for identifying the location of a single-stranded gap in a DNA fragment contained in a biological sample, comprising: 1) dividing the biological sample into two biological samples, biological sample A and biological sample B; 2) preparing a first sequencing library by treating a biological sample A with the method according to any one of claims 1 to 10, and preparing a second sequencing library by treating a biological sample B with the method according to any one of claims 1 to 10 under conditions in which the DNA ligase is not used in step S1; and 3) identifying the location of the single-stranded gap based on the difference in DNA methylation information obtained from the first sequencing library and the second sequencing library. Including, The method, wherein said single-stranded gap is a single-stranded 3',5'-phosphodiester bond gap.

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