Whole genome methylation library building and sequencing method

Abstract

The invention provides a method for repairing the tail end of a DNA (deoxyribonucleic acid) fragment, a method for constructing a whole genome methylation sequencing library, a whole genome methylation sequencing method, a method for determining whole genome methylation information and a kit. The method for repairing the tail end of the DNA fragment comprises the following steps: carrying out 5'tail end phosphorylation, 3 'tail end dephosphorylation, protruded single strand 3'tail end excision and protruded single strand 5' tail end excision on fragmented DNA. According to the method, 3 '-terminal overhangs and 5'-terminal overhangs of a fragmented DNA library with the tail ends subjected to phosphorylation and dephosphorylation are cut off in the tail end repairing process, a double-chain DNA library with a blunt tail end is formed, no exogenous basic group is introduced in the process, original methylation modification of the library is reserved, and therefore it is avoided that due to introduction errors of library building, the methylation modification efficiency of the library is greatly improved, and the methylation modification efficiency of the library is greatly improved. And the accuracy of whole genome methylation sequencing is improved.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a method for constructing a whole genome methylation library and sequencing. Background Art

[0002] Abnormal changes in epigenetic modifications are always associated with the occurrence of many diseases, such as cancer, diabetes, Alzheimer's disease and other age-related diseases. Therefore, epigenetic modifications are considered to be potential biomarkers for the diagnosis and treatment of clinical diseases. Among the many epigenetic modifications, methylated cytosine (5mC) is one of the most studied epigenetic modifications. 5mC contains a methyl group at the 5th position of the cytosine base, which protrudes into the major groove of DNA, providing a potential recognition site (or obstacle) for protein binding without changing Watson-Crick base pairing. This chemical derivative of cytosine has functional effects on cells and plays an important role in a series of biological processes such as gene silencing, gene imprinting, and X chromosome suppression. Therefore, DNA methylation sequencing can provide mechanistic insights into the occurrence and treatment of diseases and is expected to be applied to the clinical diagnosis of diseases.

[0003] The whole genome methylation library construction method for next-generation sequencing requires steps such as sample fragmentation, end repair, A-tailing, adapter ligation, base conversion, and PCR amplification. In the end repair step, the polymerase will remove the 3' end overhangs of the interrupted sample and extend and fill the 5' end overhangs, making the library a flat-ended double-stranded library. When the polymerase fills the 5' end overhangs, the sample will introduce exogenous bases (bases introduced by non-methylated dNTPs). In conventional whole genome library construction, this step will not introduce errors, but in methylation library construction, the introduced non-methylated dNTPs will be treated as the original bases of the library in the subsequent base conversion step. In other words, the end repair step erases the original methylation modification at the 5' end of the library, thus introducing errors. In the sequencing results, the methylation information at both ends of the reads will be unreliable due to the erroneous introduction of non-methylated dNTPs, resulting in data waste.

[0004] Therefore, the current methylation library construction method still needs to be improved. Summary of the invention

[0005] The present invention aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present invention proposes an improved method for end repairing DNA fragments, a method for constructing a whole genome methylation sequencing library, a method for whole genome methylation sequencing of DNA samples, a method for determining whole genome methylation information of DNA samples, a kit for end repairing DNA fragments, and a kit for constructing a whole genome methylation sequencing library, by cutting off the 3' end overhangs and 5' end overhangs of the fragmented DNA library whose ends are phosphorylated and dephosphorylated during the end repair process, a double-stranded DNA library with blunt ends is formed, and the process does not introduce exogenous bases, retains the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of whole genome methylation sequencing.

[0006] In one aspect of the present invention, the present invention proposes a method for end repair of DNA fragments. According to an embodiment of the present invention, the method comprises the following steps: phosphorylating the 5' end of the fragmented DNA, dephosphorylating the 3' end, cutting off the 3' end of the protruding single strand, and cutting off the 5' end of the protruding single strand. During the end repair process, the 5' end is phosphorylated and the 3' end is dephosphorylated to facilitate subsequent enzyme reactions. Cutting off the 3' end of the protruding single strand and cutting off the 5' end of the protruding single strand to form a flat-ended double-stranded DNA structure avoids the introduction of exogenous bases due to the extension and completion of the 5' end of the protruding single strand, retains the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0007] In another aspect of the present invention, the present invention proposes a method for constructing a whole genome methylation sequencing library. According to an embodiment of the present invention, the method comprises the following steps: (i) fragmenting a DNA sample; (ii) performing end repair on the fragmented DNA according to the aforementioned method for performing end repair on the DNA fragment; (iii) adding base A to the reaction product of step (ii) to obtain a DNA fragment with a sticky end A; (iv) connecting the DNA fragment with a sticky end A to a connector to obtain a connection product; (v) treating the connection product with bisulfite to convert non-methylated cytosine into uracil to obtain a converted target fragment; and (vi) performing PCR amplification on the converted target fragment to obtain an amplified product. Thus, by using the method according to an embodiment of the present invention, exogenous bases are not introduced during end repair, and the original methylation modification of the library is retained, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0008] In another aspect of the present invention, the present invention proposes a method for performing whole genome methylation sequencing on a DNA sample. According to an embodiment of the present invention, the sequencing method comprises the following steps: (i) obtaining a sequencing library of the DNA sample according to the method for constructing a whole genome methylation sequencing library as described above; and (ii) sequencing the sequencing library. Thus, the sequencing data obtained by the sequencing method according to an embodiment of the present invention is of high quality and the sequencing result is highly accurate.

[0009] In another aspect of the present invention, the present invention proposes a method for determining the whole genome methylation information of a DNA sample. According to an embodiment of the present invention, the method comprises the following steps: (i) obtaining a sequencing library of the DNA sample according to the method for constructing a whole genome methylation sequencing library as described above; (ii) sequencing the sequencing library to obtain a sequencing result; and (iii) performing data analysis on the sequencing result to determine the methylation information of the DNA sample. Thus, the whole genome methylation information can be accurately obtained using the method according to an embodiment of the present invention.

[0010] In another aspect of the present invention, the present invention proposes a kit for performing end repair on DNA fragments. According to an embodiment of the present invention, the kit includes the following reagents: 5' terminal phosphorylase; 3' terminal dephosphorylase; enzyme with 5' terminal exonuclease activity; enzyme with 3' terminal exonuclease activity. Thus, using the kit according to an embodiment of the present invention, the fragmented DNA can be end-repaired to phosphorylate the 5' end of the fragmented DNA, dephosphorylate the 3' end, excise the 3' end of the protruding single strand, and excise the 5' end of the protruding single strand, thereby avoiding the introduction of exogenous bases due to the extension and completion of the 5' end of the protruding single strand, retaining the original methylation modification of the library, thereby avoiding the introduction of errors due to library construction and improving the accuracy of methylation sequencing.

[0011] In another aspect of the present invention, the present invention proposes a kit for constructing a whole genome methylation sequencing library. According to an embodiment of the present invention, the kit includes the following reagents: the reagents in the kit for end repair of DNA fragments described above; the reagent for adding base A to the end; the reagent for connecting the adapter; the reagent for bisulfite treatment; the reagent for PCR amplification. Thus, when using the kit according to an embodiment of the present invention, no exogenous bases are introduced during end repair, and the original methylation modification of the library is retained, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0012] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0014] Figure 1 Shows a graph of analysis of sequencing results of a control group according to one embodiment of the present invention;

[0015] Figure 2 An analysis diagram of sequencing results of an experimental group according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0016] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0017] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0018] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0019] The present invention proposes a method for performing end repair on DNA fragments, a method for constructing a whole-genome methylation sequencing library, a method for performing whole-genome methylation sequencing on a DNA sample, a method for determining whole-genome methylation information of a DNA sample, a kit for performing end repair on DNA fragments, and a kit for constructing a whole-genome methylation sequencing library, which will be described in detail below.

[0020] Method for end repair of fragmented DNA

[0021] In one aspect of the present invention, the present invention provides a method for end repairing DNA fragments. According to an embodiment of the present invention, the method comprises the following steps: phosphorylating the 5' end of the fragmented DNA, dephosphorylating the 3' end, cutting off the 3' end of the protruding single strand, and cutting off the 5' end of the protruding single strand.

[0022] During the end repair process, the 5' end is phosphorylated and the 3' end is dephosphorylated to facilitate subsequent enzyme reactions. The 3' end of the protruding single strand and the 5' end of the protruding single strand are removed to form a blunt-ended double-stranded DNA structure, avoiding the introduction of exogenous bases due to the extension and completion of the 5' end of the protruding single strand, retaining the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0023] According to an embodiment of the present invention, 5' end phosphorylation and 3' end dephosphorylation are performed by at least one 5' end phosphorylase and 3' end dephosphorylase; and excision of the protruding single strand 3' end and excision of the protruding single strand 5' end are performed by at least one enzyme with exolytic activity.

[0024] It should be noted that the 5' terminal phosphorylase and 3' terminal dephosphorylase used for 5' terminal phosphorylation and 3' terminal dephosphorylation can be the same enzyme or different enzymes; the enzyme with exolytic activity for removing the protruding single strand 3' end and removing the protruding single strand 5' end can be the same enzyme or different enzymes.

[0025] According to an embodiment of the present invention, the 5' terminal phosphorylase includes T4 polynucleotide kinase (T4 PNK); the 3' terminal dephosphorylase includes at least one of T4 polynucleotide kinase (T4 PNK), shrimp alkaline phosphatase (rSAP) and Antarctic phosphatase (AnP). Among them, T4 PNK can simultaneously play the role of 3' terminal dephosphorylation and 5' terminal phosphorylation, with better effect, and can reduce the types of enzymes used.

[0026] According to an embodiment of the present invention, the enzyme with exonuclease activity includes at least one of T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, DNA polymerase I (DNA Polymerase I (E.coli)), Vent DNA polymerase, Mung Bean Nuclease, Nuclease P1, Exonuclease I, Exonuclease VII, Exonuclease T and RecJf exonuclease. Among them, DNA polymerase I (DNA Polymerase I (E.coli)), T7 DNA polymerase, Klenow fragment, T4 DNA polymerase, Vent DNA polymerase, exonuclease I and exonuclease T only cut off the protruding single-stranded 3' end of the fragmented DNA; RecJf exonuclease only cuts off the protruding single-stranded 5' end of the fragmented DNA; mung bean nuclease, exonuclease VII and nuclease P1 can simultaneously cut off the protruding single-stranded 3' and 5' ends of the fragmented DNA, with better effects, which can reduce the types of enzymes used.

[0027] According to an embodiment of the present invention, the reaction reagents used in the end repair further include: a buffer solution, an ATP solution and nuclease-free pure water.

[0028] According to an embodiment of the present invention, the buffer solution uses Tris-HCl or acetate as a buffer system. In some embodiments, the buffer solution includes: 10-200mM sodium ions, 0-20mM magnesium ions and 0.0001-5mM zinc ions, and the pH value is 4.5-8.0. Thus, it can be ensured that multiple enzymes can react normally at the same time. In some embodiments, the sodium ion concentration in the buffer solution can be 30mM, 50mM, 80mM, 100mM, 120mM, 150mM, 180mM, etc., the magnesium ion concentration can be 5mM, 10mM, 15mM, etc., and the zinc ion concentration can be 0.1mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, etc.

[0029] According to an embodiment of the present invention, the temperature of the end repair is 20-40° C. and the time is 10-20 minutes. Thus, it can be ensured that multiple enzymes can react normally at the same time.

[0030] Method for constructing a whole-genome methylation sequencing library

[0031] In another aspect of the present invention, the present invention proposes a method for constructing a whole genome methylation sequencing library. According to an embodiment of the present invention, the method comprises the following steps: (i) fragmenting a DNA sample; (ii) performing end repair on the fragmented DNA according to the method for performing end repair on the DNA fragment as described above; (iii) adding base A to the reaction product of step (ii) to obtain a DNA fragment with a sticky end A; (iv) connecting the DNA fragment with a sticky end A to a connector to obtain a connection product; (v) treating the connection product with bisulfite to convert non-methylated cytosine into uracil to obtain a converted target fragment; and (vi) performing PCR amplification on the converted target fragment to obtain an amplified product. Thus, by using the method according to an embodiment of the present invention, no exogenous bases are introduced during end repair, and the original methylation modification of the library is retained, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0032] According to an embodiment of the present invention, step (ii) of the method for constructing a whole genome methylation sequencing library further comprises the following steps: (ii-1) purifying the reaction product after end repair to remove or inactivate the enzyme with exo-active activity, thereby preventing the enzyme with exo-active activity from interfering with the subsequent A addition reaction.

[0033] According to an embodiment of the present invention, the purification process includes: incubating the end-repaired reaction product with magnetic beads, collecting the magnetic beads and eluting, collecting the eluate, and obtaining purified DNA, thereby removing the enzyme with exonuclease activity to prevent it from interfering with the subsequent A addition reaction.

[0034] According to an embodiment of the present invention, the method for constructing a whole genome methylation sequencing library further comprises: (vii) performing enzyme digestion and cyclization treatment on the amplified product to obtain a double-stranded cyclized product; and (viii) preparing the double-stranded cyclized product into DNA nanoballs.

[0035] It should be noted that the present invention does not strictly limit the specific experimental conditions for adding base A to the 3' end, linker connection, bisulfite treatment, PCR amplification, enzyme digestion (double-stranded enzyme digestion) and cyclization (double-stranded cyclization) of the amplified product, and preparation of DNA nanospheres, and can be flexibly selected by combining the existing technology in the field with conventional technical means.

[0036] It should be noted that the features and advantages described above for the method of end repair of DNA fragments are also applicable to the method for constructing a whole genome methylation sequencing library, and will not be described in detail here.

[0037] Methods for whole-genome methylation sequencing of DNA samples

[0038] In another aspect of the present invention, the present invention proposes a method for performing whole genome methylation sequencing on a DNA sample. According to an embodiment of the present invention, the method for performing whole genome methylation sequencing on a DNA sample comprises the following steps: (i) obtaining a sequencing library of the DNA sample according to the method for constructing a whole genome methylation sequencing library as described above; and (ii) sequencing the sequencing library. Thus, the sequencing data obtained by the sequencing method according to an embodiment of the present invention is of high quality and the sequencing result is highly accurate.

[0039] It should be noted that the features and advantages described above for the method for constructing a whole-genome methylation sequencing library are also applicable to the method for performing whole-genome methylation sequencing on DNA samples, and will not be described in detail here.

[0040] Methods for determining genome-wide methylation information of DNA samples

[0041] In another aspect of the present invention, the present invention proposes a method for determining the whole genome methylation information of a DNA sample. According to an embodiment of the present invention, the method for determining the whole genome methylation information of a DNA sample comprises the following steps: (i) obtaining a sequencing library of a DNA sample according to the method for constructing a whole genome methylation sequencing library as described above; (ii) sequencing the sequencing library to obtain a sequencing result; and (iii) performing data analysis on the sequencing result to determine the methylation information of the DNA sample. Thus, the whole genome methylation information of a DNA sample can be accurately obtained using the method according to an embodiment of the present invention, which is of great significance for epigenetic research.

[0042] It should be noted that the features and advantages described above for the method for constructing a whole-genome methylation sequencing library are also applicable to the method for determining the whole-genome methylation information of a DNA sample and will not be described in detail here.

[0043] Kit for end repair of DNA fragments

[0044] In another aspect of the present invention, the present invention proposes a kit for performing end repair on DNA fragments. According to an embodiment of the present invention, the kit includes the following reagents: 5' terminal phosphorylase; 3' terminal dephosphorylase; enzyme with 5' terminal exonuclease activity; enzyme with 3' terminal exonuclease activity. Thus, using the kit according to an embodiment of the present invention, the fragmented DNA can be end-repaired to phosphorylate the 5' end of the fragmented DNA, dephosphorylate the 3' end of the protruding single strand, and remove the 5' end of the protruding single strand, thereby avoiding the introduction of exogenous bases due to the extension and completion of the 5' end of the protruding single strand, retaining the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0045] According to an embodiment of the present invention, the 5' terminal phosphorylase comprises T4 polynucleotide kinase.

[0046] According to an embodiment of the present invention, the 3'-terminal dephosphorylation enzyme comprises at least one of T4 polynucleotide kinase (T4 PNK), shrimp alkaline phosphatase (rSAP) and Antarctic Phosphatase (AnP).

[0047] According to an embodiment of the present invention, the enzyme with exonuclease activity includes at least one of T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, DNA polymerase I, Vent DNA polymerase, mung bean nuclease, exonuclease I, exonuclease VII, RecJf exonuclease, nuclease P1 and exonuclease T. Thus, the protruding single-stranded 3' end and 5' end of the fragmented DNA are excised to form a double-stranded DNA with a flat end, avoiding the introduction of exogenous bases due to the extension and filling of the protruding single-stranded 5' end, retaining the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0048] It should be noted that the 5' terminal phosphorylase and the 3' terminal dephosphorylase may be the same enzyme or different enzymes; the enzyme having 5' terminal exonuclease activity and the enzyme having 3' terminal exonuclease activity may be the same enzyme or different enzymes.

[0049] According to an embodiment of the present invention, the kit further comprises at least one of the following reagents: a buffer solution, an ATP solution and nuclease-free pure water.

[0050] According to an embodiment of the present invention, the buffer uses Tris-HCl or acetate as a buffer system, and in some embodiments, the buffer includes: 10-200 mM sodium ions, 0-20 mM magnesium ions and 0.0001-5 mM zinc ions, and the pH value is 4.5-8.0. Thus, it can be ensured that multiple enzymes can react normally at the same time.

[0051] It should be noted that the features and advantages described above for the method of end repair of DNA fragments are also applicable to this kit and will not be described in detail here.

[0052] In addition, the present invention proposes the use of the aforementioned kit for performing end repair on DNA fragments in performing end repair on DNA fragments, constructing a whole genome methylation sequencing library, performing whole genome methylation sequencing on DNA samples, and determining the whole genome methylation information of DNA samples. The present invention also proposes the use of the aforementioned kit for performing end repair on DNA fragments in preparing products for performing end repair on DNA fragments. Thus, the use of the kit of the present invention avoids the introduction of exogenous bases due to the extension and completion of the 5' end of the protruding single strand, retains the original methylation modification of the library, thereby avoiding errors introduced by library construction and improving the accuracy of methylation sequencing.

[0053] Kit for constructing whole-genome methylation sequencing libraries

[0054] In another aspect of the present invention, the present invention proposes a kit for constructing a whole genome methylation sequencing library. According to an embodiment of the present invention, the kit includes the following reagents: the reagents in the kit for end repair of DNA fragments as described above; the reagent for adding base A to the end; the reagent for connecting the adapter; the reagent for bisulfite treatment; the reagent for PCR amplification. Thus, when using the kit according to an embodiment of the present invention, no exogenous bases are introduced during end repair, and the original methylation modification of the library is retained, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0055] According to an embodiment of the present invention, the kit further includes at least one of the following reagents: a reagent for DNA sample fragmentation; a reagent for purifying the end-repair reaction product; a reagent for enzyme digestion and cyclization; a reagent for making DNA nanospheres.

[0056] It should be noted that the “reagents for fragmenting DNA samples”, “reagents for performing DNA sample fragmentation”, “reagents for adding base A to the end”, “reagents for connecting adapters”, “reagents for performing bisulfite treatment”, “reagents for performing PCR amplification”, “reagents for enzyme cleavage treatment” and “reagents for (double-stranded DNA) cyclization treatment” can be conventional reagents in the field, and those skilled in the art can flexibly select them according to actual needs.

[0057] It should be noted that the features and advantages described above for the kit for constructing a whole-genome methylation sequencing library are also applicable to this kit and will not be described in detail here.

[0058] In addition, the present invention proposes the use of the aforementioned kit for constructing a whole genome methylation sequencing library in constructing a whole genome methylation sequencing library, performing whole genome methylation sequencing on a DNA sample, and determining the whole genome methylation information of a DNA sample. The present invention also proposes the use of the aforementioned kit for constructing a whole genome methylation sequencing library in preparing a product for constructing a whole genome methylation sequencing library. Thus, the kit of the present invention does not introduce exogenous bases during end repair, retains the original methylation modification of the library, thereby avoiding errors introduced due to library construction and improving the accuracy of methylation sequencing.

[0059] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0060] Example 1

[0061] In this example, commercial standard DNA was used for library construction and sequencing, which was divided into an experimental group and a control group. The difference mainly lies in the end repair step. The specific experimental steps are as follows:

[0062] 1. DNA fragmentation

[0063] The DNA was derived from a commercial standard (NA12878) and was ultrasonically sheared using a Covaris shearer. After fragment screening, fragmented DNA with a main band of 350 bp was obtained.

[0064] 2. End repair

[0065] 2.1 Experimental group: The reaction system was configured as follows: Mung Bean Nuclease was purchased from New England BioLabs (NEB), catalog number M0250L, and Mung Bean Nuclease Reaction Buffer was a matching buffer (containing 30 mM NaCl, 50 mM sodium acetate, 1 mM ZnSO4); T4 PNK was purchased from BGI, catalog number 01E016MM.

[0066] Fragmented DNA 50ng(xμL) Mung Bean Nuclease 0.5μL Mung Bean Nuclease Reaction Buffer 5μL T4 PNK 0.5μL 100mM ATP 1μL Nuclease-Free Water (43-x)μL common 50μL

[0067] The reaction was carried out according to the following procedure

[0068] Heat cover 45℃ 30℃ 15min 4℃ Hold

[0069] The reaction product was terminated with 5 μL 0.1% SDS, and the above samples were added with TE to 100 μL and then incubated with 100 μL magnetic beads (AMPure XP Beads, Beckman Agencourt AMPure XP Magnetic Beads, Product No. A63881). 43 μL TE was used for elution, and 41 μL was used for subsequent reaction.

[0070] 2.2 Control group

[0071] The reaction system was configured as follows: ER Buffer Mix, ER Enzyme Mix, and ER Stop Buffer were all from the MGIEasy Whole Genome Methylation Library Preparation Kit.

[0072] Fragmented DNA 50ng(xμL) ER Buffer Mix 7.3μL ER Enzyme Mix 2.7μL TE Buffer (30-x)μL common 40μL

[0073] The ER Buffer Mix contains dNTPs; the ER Enzyme Mix contains enzymes that phosphorylate the 5' end and dephosphorylate the 3' end, an enzyme that cuts the 3' end, and a DNA polymerase.

[0074] The reaction was carried out according to the following procedure

[0075] Heat cover 45℃ 25℃ 30min 4℃ Hold

[0076] After the reaction, the product was centrifuged instantly to collect the reaction solution to the bottom of the tube. 6 μL ER Stop Buffer was pipetted into the PCR tube and vortexed 3 times for 3 seconds each time. The reaction solution was centrifuged instantly to collect the reaction solution to the bottom of the tube and allowed to stand for 2 minutes.

[0077] Perform the end repair termination reaction as follows:

[0078] Heat cover 85℃ 75℃ 20min 4℃ Hold

[0079] The reaction product was centrifuged instantly to collect the reaction solution at the bottom of the tube.

[0080] 3. Add A at the end

[0081] 3.1 Experimental group: The reaction system was configured as follows: 10×PNK Buffer was purchased from BGI, catalog number 01E016MM; AT Buffer Mix and AT Enzyme Mix were both from the MGIEasy Whole Genome Methylation Library Preparation Kit.

[0082] sample 41μL 10×PNK Buffer 5μL AT Buffer Mix 3.8μL AT Enzyme Mix 0.2μL common 50μL

[0083] The reaction was carried out according to the following procedure

[0084] Heat cover 75℃ 65℃ 15min 4℃ Hold

[0085] The reaction product was centrifuged instantly to collect the reaction solution at the bottom of the tube.

[0086] 3.2 Control group: The reaction system was configured as follows. AT Buffer Mix and AT Enzyme Mix were both from the MGIEasy Whole Genome Methylation Library Preparation Kit.

[0087]

[0088]

[0089] The reaction was carried out according to the following procedure

[0090] Heat cover 75℃ 65℃ 15min 4℃ Hold

[0091] The reaction product was centrifuged instantly to collect the reaction solution at the bottom of the tube.

[0092] 4. Adapter Ligation Reaction

[0093] Add 5 μL of WGBS Adapter to the PCR tube of the sample obtained in the previous step and shake to mix. Then, prepare the reaction system in a 200 μL PCR tube according to the table below, where Fast Ligation Buffer, Ad Ligase, and Ligation Enhancer are all from the Fast PCR-FREE Enzyme Digestion Library Preparation Kit.

[0094] Sample 55μL Ligation Buffer Mix 23.4μL DNA Ligase 1.6μL common 80μL

[0095] The reaction was carried out according to the following procedure

[0096] Heat cover 35℃ 23℃ 30min 4℃ Hold

[0097] The reaction product was incubated with 40 μL magnetic beads (AMPure XP Beads, Agencourt AMPure XP Magnetic Beads from Beckman Company, Catalog No. A63881), eluted with 21 μL TE, and 19 μL was taken for subsequent reaction.

[0098] 5. The subsequent library construction steps (including BS conversion treatment, PCR amplification, PCR product quality inspection, double-stranded restriction digestion, double-stranded circularization, double-stranded circularization product purification, and WGBS DNB preparation) were performed according to the instructions of the MGIEasy Whole Genome Methylation Library Preparation Kit (16RXN) (Cat. No. 1000005251).

[0099] 6. Sequencing

[0100] The qualified DNBs were sequenced on the sequencing platform DNBSEQ-2000, sequencing type PE150, and sequencing depth 30×.

[0101] 7. Data Analysis

[0102] Perform analysis using analysis software.

[0103] The results are as follows Figure 1 and Figure 2 As shown, it can be seen that the inventive method can greatly improve the GC_bias of library sequencing and improve the accuracy of whole genome methylation second-generation sequencing.

[0104] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for end repair of a DNA fragment, characterized in that: The method comprises the following steps: The fragmented DNA is subjected to 5' end phosphorylation, 3' end dephosphorylation, excision of the protruding single strand 3' end and excision of the protruding single strand 5' end.

2. The method according to claim 1, characterized in that The 5' terminal phosphorylation and 3' terminal dephosphorylation are carried out by using at least one 5' terminal phosphorylase and 3' terminal dephosphorylase; The excision of the protruding single strand 3' end and the excision of the protruding single strand 5' end are treated with at least one enzyme having exo-cleavage activity; Optionally, the temperature of the end repair is 20 to 40° C., and the time is 10 to 20 minutes.

3. The method according to claim 2, characterized in that The 5' terminal phosphorylase includes T4 polynucleotide kinase; Optionally, the 3' terminal dephosphorylase comprises at least one of T4 polynucleotide kinase, shrimp alkaline phosphatase and Antarctic phosphatase; Optionally, the enzyme having exonuclease activity comprises at least one of T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, DNA polymerase I, Vent DNA polymerase, mung bean nuclease, nuclease P1, exonuclease I, exonuclease VII, exonuclease T and RecJf exonuclease; Optionally, the reaction reagents used in the end repair further include: buffer, ATP solution and nuclease-free pure water; Preferably, the buffer is Tris-HCl or acetate as a buffer system; Preferably, the buffer comprises: 10-200 mM sodium ions, 0-20 mM magnesium ions and 0.0001-5 mM zinc ions, and the pH value is 4.5-8.

0.

4. A method for constructing a whole-genome methylation sequencing library, characterized in that: The following steps are involved: (i) fragmenting the DNA sample; (ii) performing end repair on the fragmented DNA according to the method according to any one of claims 1 to 3; (iii) adding base A to the reaction product of step (ii) to obtain a DNA fragment having a sticky end A; (iv) connecting the DNA fragment having the sticky end A to a linker to obtain a connection product; (v) treating the ligation product with bisulfite to convert the unmethylated cytosine into uracil to obtain a converted target fragment; as well as (vi) performing PCR amplification on the converted target fragment to obtain an amplified product; Optionally, further comprising the following steps: (vii) digesting and cyclizing the amplified product to obtain a double-stranded cyclized product; (viii) preparing the double-stranded cyclization product into DNA nanoballs.

5. The method according to claim 4, characterized in that The step (ii) further comprises the following steps: (ii-1) purifying the reaction product after the end repair to remove the enzyme with exo-active activity or inactivate it; Preferably, the purification process comprises: The reaction product after end repair is incubated with magnetic beads, the magnetic beads are collected and eluted, and the eluate is collected to obtain purified DNA.

6. A method for whole genome methylation sequencing of a DNA sample, characterized in that: The following steps are involved: (i) obtaining a sequencing library of the DNA sample according to the method of claim 4 or 5; as well as (ii) sequencing the sequencing library.

7. A method for determining the whole genome methylation information of a DNA sample, characterized in that: The following steps are involved: (i) obtaining a sequencing library of the DNA sample according to the method of claim 4 or 5; (ii) sequencing the sequencing library to obtain sequencing results; as well as (iii) performing data analysis on the sequencing results to determine the methylation information of the DNA sample.

8. A kit for end repair of DNA fragments, characterized in that: The following reagents are included: 5' end phosphorylase; 3' end dephosphorylase; An enzyme having 5' end exonuclease activity; and An enzyme with 3' end exonuclease activity.

9. The kit according to claim 8, characterized in that The 5' terminal phosphorylase includes T4 polynucleotide kinase; Optionally, the 3' terminal dephosphorylase comprises at least one of T4 polynucleotide kinase, shrimp alkaline phosphatase and Antarctic phosphatase; Optionally, the enzyme having exonuclease activity comprises at least one of T4 DNA polymerase, T7 DNA polymerase, Klenow fragment, DNA polymerase I, Vent DNA polymerase, mung bean nuclease, exonuclease I, exonuclease VII, RecJf exonuclease, nuclease P1 and exonuclease T; Optionally, the kit further comprises at least one of the following reagents: Buffer, ATP solution, and nuclease-free water; Preferably, the buffer is Tris-HCl or acetate as a buffer system; Preferably, the buffer comprises: 10-200 mM sodium ions, 0-20 mM magnesium ions and 0.0001-5 mM zinc ions, and the pH value is 4.5-8.

0.

10. A kit for constructing a whole-genome methylation sequencing library, characterized in that: The following reagents are included: The reagent in the kit according to claim 8 or 9; Reagents for adding base A to the termini; Reagents for ligating adapters; Reagents for carrying out bisulfite treatment; Reagents for performing PCR amplification; Optionally, the kit further comprises at least one of the following reagents: Reagents for fragmenting DNA samples; Reagents for purifying end-repair reaction products; Reagents for enzyme digestion and cyclization; Reagents used to make DNA nanospheres.

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