Library building method and application of genomic library and methylated library

Abstract

The invention provides a library building method and application of a genomic library and a methylated library. The construction method comprises the following steps: carrying out linker connection on a DNA template subjected to terminal repair, and carrying out PCR (Polymerase Chain Reaction) amplification on a connection product to obtain a mixture of a copy chain and an original chain; separating the mixture of the copy chain and the original chain, and respectively performing genome library construction and methylation library construction by using the obtained copy chain group and the original chain group to obtain a genome library and a methylation library; wherein the 3'end of the linker sequence connected with the 3 'end of the DNA template is modified by biotin. According to the present invention, the 3'end of the DNA template is connected with the biotin-modified linker, such that the effective separation of the copy chain and the original chain is achieved, the same DNA template can be adopted to respectively construct the genome library and the methylation library, and the information of the genome and the methylation genome can be comprehensively and accurately obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of gene library construction, and in particular to a method for constructing a genomic library and a methylation library and applications thereof. Background Art

[0002] In recent years, advances in high-throughput sequencing technology have enabled us to more comprehensively detect genetic and epigenetic information in DNA. These technological advances have given us greater potential in early diagnosis and personalized medicine. For example, by analyzing a patient's genomic data, doctors can develop targeted treatment plans to improve treatment outcomes. Non-invasive prenatal diagnostic analysis has also demonstrated that DNA methylation signals can determine the fetal origin of DNA fragments. Cell-free DNA (cfDNA) methylation sequencing can be used to detect pregnancy complications associated with placental function. In addition, the length of cfDNA fragments can also be used to distinguish between fetal and maternal DNA. Combining genetic and epigenetic studies provides us with a richer biological perspective and new ideas for precision medicine and disease prevention.

[0003] At present, the DNA methylation detection method based on high-throughput sequencing technology is the main method of epigenetic analysis. It converts unmethylated cytosine (C) into uracil (U) through bisulfite, thereby distinguishing methylated cytosine from unmethylated cytosine and analyzing the DNA methylation level of the sample. However, since bisulfite can convert more than 90% of C in the sequencing data, it is impossible to determine whether the change from cytosine (C) to thymine (T) is caused by bisulfite or a mutation carried by the gene, which will directly affect the genetic analysis of the change from C to T of the gene. Therefore, if the mutation information and methylation information of the sample need to be analyzed simultaneously, a large number of samples are usually required for genome testing and methylation group testing respectively. However, this requirement is difficult to meet for research and clinical applications with limited sample sizes such as rare tissues, cfDNA, and single cells.

[0004] To solve this problem, Xiaomin Chen et al. used the TET-assisted pyridine borane sequencing (TAPS) method to reduce the impact of analyzing C to T variation by converting methylated C (accounting for less than 10% of the total genome C) to U and retaining most of the C, thereby achieving the purpose of simultaneously analyzing the genome and methylation group. However, the conversion efficiency of TAPS is low and it is impossible to convert all methylated C to U. Pei Wang et al. developed the MCP technology to simultaneously detect the genetic and epigenetic information of samples. This method uses restriction endonucleases to cut specific sequences, while methylated specific sequences are not cut, thereby achieving the purpose of distinguishing methylated sequences from non-methylated sequences, while avoiding the problem of being unable to analyze C to T variation due to bisulfite conversion. However, restriction endonucleases can only recognize specific sequences and cannot analyze the degree of methylation of all sites in the sample at the single base level. Therefore, there is an urgent need for a method that can simultaneously and comprehensively detect the genetic information and epigenetic information of all sites in a sample. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for constructing a genomic library and a methylation library and an application thereof, so as to solve the problem in the prior art that it is impossible to accurately obtain genomic and methylation information at the same time.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for constructing a genomic library and a methylation library is provided, the construction method comprising: connecting a DNA template after end repair with a linker, and performing PCR amplification on the connection product to obtain a mixture of copy chains and original chains; separating the mixture of copy chains and original chains, and using the obtained copy chain group and original chain group to construct a genomic library and a methylation library respectively, to obtain a genomic library and a methylation library; wherein the 3' end of the linker sequence connected to the 3' end of the DNA template is modified with biotin.

[0007] Furthermore, the cytosine in the linker sequence is 5-methylcytosine.

[0008] Furthermore, the biotin-modified linker includes a linker sequence, a U base and biotin connected in sequence from the 5' end to the 3' end.

[0009] Furthermore, separating the mixture of the copy chain and the original chain includes: using magnetic beads modified with streptavidin to mix and separate and purify the mixture of the copy chain and the original chain in turn, and the obtained first supernatant is the copy chain group; using deionized water to resuspend the precipitate, cutting off the biotin of the linker in the original chain, and obtaining the original chain group; wherein the magnetic beads modified with streptavidin are magnetic beads that have been rinsed with magnetic bead buffer and resuspended.

[0010] Further, resuspending the precipitate with deionized water and cutting off the biotin of the linker in the original chain includes: resuspending the precipitate with deionized water, adding nuclease, purifying by magnetic absorption, and discarding the supernatant; resuspending the precipitate of the enzyme cleavage product with TE buffer, adding USER II, and purifying by magnetic absorption, and the obtained second supernatant is the original chain group.

[0011] Furthermore, the magnetic beads modified with streptavidin include M270 magnetic beads; preferably, the magnetic bead buffer includes: 1M NaCl, 10mM Tris-HCl, pH 8.0, 1mM EDTA, pH 8.0, 0.05% Tween-20, and 0.5% SDS.

[0012] Furthermore, constructing the methylation library includes: using bisulfite to convert the C bases in the DNA in the original chain group that are not methylated into U bases; using the converted product as a template, performing PCR amplification to obtain the methylation library.

[0013] Furthermore, the first supernatant and the methylation library were purified using AMPure XP magnetic beads.

[0014] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a kit is provided, which comprises: a biotin-modified linker, a nuclease and M270 magnetic beads; preferably, the kit also comprises: USER II, AMPureXP magnetic beads, a magnetic bead buffer and a TE buffer; preferably, the magnetic bead buffer comprises: 1M NaCl, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20, and 0.5% SDS.

[0015] Furthermore, the biotin-modified linker comprises a linker sequence and a biotin modification at the 3' end of the linker sequence, wherein the cytosine in the linker sequence is 5-methylcytosine; preferably, the biotin in the biotin-modified linker is connected to the linker sequence via a U base.

[0016] By applying the technical solution of the present invention, a biotin-modified linker is connected to the 3' end of the DNA template to achieve effective separation of the copy chain and the original chain, and the same DNA template can be used to construct a genomic library and a methylation library, respectively, so that the information of the genome and the methylation genome can be obtained more comprehensively and accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A flow chart showing the construction of the genome and methylation genome of the present application is shown;

[0019] Figure 2 The result diagram showing the average depth of the original chain library and the copy chain library obtained in Example 1 of the present application is shown;

[0020] Figure 3 A result diagram showing the average depth of the control group and the experimental group obtained in Example 2 of the present application is shown;

[0021] Figure 4 The collection diagram of unique reads of the control group and the experimental group obtained in Example 2 of the present application is shown;

[0022] Figure 5 The result diagram of the methylation consistency analysis of the control group and the experimental group in Example 3 of the present application is shown;

[0023] Figure 6 The result diagram of the methylation consistency analysis of the control group 1 and the experimental group in Example 4 of the present application is shown. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0025] As mentioned in the background technology, conventional mutation and methylation analysis methods in the prior art require a large number of samples, which are difficult to analyze in limited sample quantities such as rare tissues, cfDNA, and single cells; and although TAPS can achieve the purpose of simultaneously analyzing the genome and methylation group, the conversion efficiency is low and it is impossible to convert all methylated C to U; MCP technology cannot analyze the methylation level of all sites in the sample at the single base level. Therefore, this application intends to provide a library construction method that can comprehensively and accurately obtain genome and methylation genome information.

[0026] In the first typical embodiment of the present application, a method for constructing a genomic library and a methylation library is provided, and the construction method includes: connecting the DNA template after end repair with a linker, and performing PCR amplification on the connection product to obtain a mixture of a copy chain and an original chain; separating the mixture of the copy chain and the original chain, and using the obtained copy chain group and the original chain group to construct a genomic library and a methylation library, respectively, to obtain a genomic library and a methylation library; wherein the 3' end of the linker sequence connected to the 3' end of the DNA template is biotin-modified. The present application uses a biotin-modified linker to connect to the DNA template, which can separate the original chain from the copy chain to obtain a template suitable for constructing a genomic and methylated genome, respectively.

[0027] In order to avoid the appearance of excess converted uracil during bisulfite treatment during subsequent methylation library construction, in a preferred embodiment, the cytosine in the linker sequence is 5-methylcytosine, which can avoid the cytosine in the linker sequence being recognized as unmethylated cytosine in the DNA template.

[0028] In order to completely remove the biotin modification in the linker, in a preferred embodiment, the linker with biotin modification includes a linker sequence, a U base and biotin in sequence from the 5' end to the 3' end. Subsequently, an enzyme that specifically recognizes and cuts the U base is used to remove the biotin modification in the linker.

[0029] The original chain and the copy chain are separated by using magnetic beads that can specifically bind to biotin modification. In a preferred embodiment, the separation of the copy chain and the original chain mixture includes: using magnetic beads modified with streptavidin to sequentially mix and separate and purify the copy chain and the original chain mixture, and the first supernatant obtained is the copy chain group; using magnetic bead buffer to resuspend the precipitate, cutting off the biotin of the linker in the original chain, and obtaining the original chain group; wherein the magnetic beads modified with streptavidin are magnetic beads that have been rinsed with magnetic bead buffer and resuspended. wherein streptavidin can specifically bind to biotin, and the copy chain without biotin modification is present in the supernatant.

[0030] In order to remove the residual copy chain and biotin modification in the separated original chain, in a preferred embodiment, the precipitate is resuspended with a magnetic bead buffer, and the biotin of the linker in the original chain is cut off, including: resuspending the precipitate with deionized water, adding nuclease, purifying by magnetic absorption, and discarding the supernatant; resuspending the precipitate of the enzyme cleavage product with TE buffer, adding USERII, and purifying by magnetic absorption, and the obtained second supernatant is the original chain group.

[0031] In order to efficiently combine biotin and streptavidin, in a preferred embodiment, the magnetic beads modified with streptavidin include M270 magnetic beads; preferably, the magnetic bead buffer includes: 1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20, 0.5% SDS.

[0032] The methylation library is constructed by removing the original chain modified by biotin. In a preferred embodiment, the construction of the methylation library includes: using bisulfite to convert the C base in the DNA in the original chain group into a U base; using the converted product as a template, performing PCR amplification to obtain the methylation library.

[0033] In order to further obtain PCR amplification products with better purity, in a preferred embodiment, the first supernatant and the methylation library are purified using AMPureXP magnetic beads.

[0034] In a second typical embodiment of the present application, a kit is provided, which comprises: a biotin-modified linker, a nuclease and M270 magnetic beads; preferably, the kit also comprises: USER II, AMPure XP magnetic beads, a magnetic bead buffer and a TE buffer; preferably, the magnetic bead buffer comprises: 1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20, 0.5% SDS.

[0035] In order to further efficiently remove the modification in the linker and construct a methylation library with better quality, in a preferred embodiment, the linker with biotin modification includes a linker sequence and a biotin modification located at the 3' end of the linker sequence, wherein the cytosine in the linker sequence is 5-methylcytosine; preferably, the biotin in the linker with biotin modification is connected to the linker sequence through a U base.

[0036] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0037] Material:

[0038] 5x Annealing Buffer (Biyuntian, catalog number D0251)

[0039] Library construction kit (Novozymes, catalog number ND607)

[0040] Paired-end index primer (Novozymes, catalog number NB34401)

[0041] Methylation library construction kit (Yisheng Biotechnology, catalog number 12214)

[0042] Methylation Conversion Kit (ZYMO RESEARCH, catalog number D5006)

[0043] AMPure XP magnetic beads (Beckman, catalog number A63881)

[0044] Enzyme-free water (Life, catalog number 4387936)

[0045] M270 magnetic beads (invitrogen, catalog number 65306)

[0046] Exonuclease III (NEB, catalog number M0206)

[0047] USER II (NEB, catalog number M5508)

[0048] Structural variation 5% ctDNA standard (Jingliang Technology, catalog number GW-OCTM001)

[0049] 5M NaCl (Bomed, catalog number SH411-01)

[0050] 10% SDS (Bomed, catalog number SH412-01)

[0051] 1M Tris-HCl Buffer (pH 8.0) (Coollab, catalog number SL3080)

[0052] 0.5M EDTA Solution (pH 8.0) (Coollab, catalog number SL3070)

[0053] Tween-20 (Merida, catalog number M1665)

[0054] Example 1: Evaluation of the effect of separating and purifying the original chain by streptavidin magnetic beads (using Figure 1 The process shown)

[0055] 20 ng of cfDNA sample was taken for library construction, and the copy chain library and the original chain library were captured and sequenced. The effect of separating and purifying the original chain in the present application scheme was evaluated based on the sequencing results.

[0056] 1) Joint preparation:

[0057] The DNA fragments shown in SEQ ID NO: 1 and 2 were mixed in equal molar amounts, incubated at 95° C. for 2 minutes in a 1× annealing buffer, and then slowly cooled to room temperature to obtain a linker.

[0058] SEQ ID NO: 1: 5'-GATmCGGAAGAGmCAmCAmCGTmCTGAAmCTmCmCAGTmCUTTTT-TEG-Bio-3'.

[0059] SEQ ID NO: 2: 3'-TmCTAGmCmCTTmCTmCGmCAGmCAmCATmCmCmCTTTmCTmCAmCA-5'.

[0060] 2) End repair & adding "A" base

[0061] Vortex end prep mix4 and centrifuge briefly before adding to 20 ng cfDNA. Repair plus "A" system is as follows: 1:

[0062] Table 1:

[0063] Components Volume (μl) cfDNA 50 End prep mix4 15 Total volume 65

[0064] Place the PCR tube in the PCR instrument. The reaction conditions are as shown in Table 2:

[0065] Table 2:

[0066] temperature time Heating cover 105℃ On 20℃ 15min 65℃ 15min 4℃ Hold

[0067] 3) Connector connection

[0068] Prepare the ligation reaction mixture system according to Table 3 below:

[0069] Table 3:

[0070] Components Volume (μl) End preparation products 65 Rapid ligation buffer2 25 Rapid DNA ligase 5 DNA adapter 5 Total volume 100

[0071] Place the PCR tube in a PCR instrument. The ligation reaction conditions are shown in Table 4 below:

[0072] Table 4:

[0073] temperature time Heating cover 105℃ Off 20℃ 15min 4℃ Hold

[0074] Use 60ul xp magnetic beads for purification and recovery to obtain the library before PCR amplification.

[0075] 4) PCR amplification reaction

[0076] The pre-PCR amplification library after the adapter ligation is subjected to PCR amplification, and a PCR amplification reaction mixture is prepared according to the following Table 5:

[0077] Table 5:

[0078] Components Volume (μl) Ligation reaction purification product 20 PCR primer mix 5 HIFI amplification mix 25 total 50

[0079] Place the PCR tube in a PCR instrument. The PCR amplification reaction conditions are shown in Table 6 below:

[0080] Table 6:

[0081]

[0082]

[0083] 5) Separation of the separation chain and the original chain

[0084] Activation of M270 magnetic beads: Vortex and resuspend the stock solution of M270 magnetic beads to obtain a magnetic bead suspension. Transfer 20μl of the magnetic bead suspension to a 1.5ml test tube for each reaction. Wash twice with 500μl beads buffer (1M NaCl, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20, 0.5% SDS), and resuspend in 150μl beads buffer for later use.

[0085] 150μl of magnetic bead suspension was transferred to a 1.5ml centrifuge tube as the unit for each separation. The ligation product after PCR amplification was added to the magnetic bead suspension, vortexed briefly and shaken at room temperature for 20 minutes, and placed on a magnetic stand. The supernatant was aspirated and added with 200ul xp magnetic beads for purification and recovery. The resulting PCR product was the copy chain library.

[0086] The above precipitate was washed once with 200 μl beads buffer, then washed twice with deionized water, resuspended with 19.5 μl 1x NEBuffer 1, and 0.5 μl Exonuclease III was added, vortexed to mix, and reacted at 37°C for 30 min.

[0087] Place the product treated with Exonuclease III on a magnetic rack, discard the supernatant, wash twice with deionized water, add 19ul TE buffer to resuspend the magnetic beads, add 1ul USER II, vortex to mix, react at 37℃ for 15min, and react at 65℃ for 10min.

[0088] The product treated with USER II is placed on a magnetic rack, and the supernatant is aspirated. The obtained product is the original chain nucleic acid with biotin and magnetic beads removed.

[0089] 6) Construction of the original chain library

[0090] The original chain separated as above was used as a template to construct the library. The PCR amplification reaction was the same as step 4). After the PCR was completed, 50ul xp magnetic beads were used to purify and recover the amplified product. The obtained nucleic acid was the original chain library.

[0091] 7) The two libraries obtained above were respectively subjected to in-depth analysis using customized panel capture sequencing.

[0092] The analysis results are as follows Figure 2 As shown, the average depth of the original chain library is 1607x, and the average depth of the copy chain library is 1714x. This shows that the original chain can be effectively separated using the method of the present application, and the separation efficiency is 93%, which meets the downstream detection requirements.

[0093] Example 2: Evaluation of the effect of original strand separation on copy strand genetic analysis

[0094] Take 20 ng of cfDNA sample and perform the same experimental operation as steps 1)-4) in Example 1. Before performing step 5), the PCR reaction product is divided into two equal parts, one of which is purified and recovered by xp magnetic beads and recorded as the control group, and the other part is subjected to the same experimental operation as step 5) in Example 1, and the supernatant obtained after separation of the original chain is purified and recovered by xp magnetic beads and recorded as the experimental group. The control group and the experimental group are respectively subjected to library construction operations, and the sequencing data results of the two groups are analyzed.

[0095] The library analysis results of the control group and the experimental group are shown in Figure 3 As shown, the average depth of the control group library is 1779x, ​​and the average depth of the experimental group library is 1750x. In the control group library, 1902356 unique reads were identified, and 1872553 unique reads were identified in the experimental group library. Among them, 1807239 unique reads were identified in both libraries, accounting for about 95% of each library. 95117 unique reads (5.0%) were only present in the control group library, and 65314 unique reads (3.6%) were only present in the experimental group library ( Figure 4 ).

[0096] It can be seen that there is no significant difference in the average depth of the two groups of libraries, indicating that the separation of the original chain using the method of the present application will not cause the loss of nucleic acid fragments. The two groups of libraries have 95% of the same unique reads, indicating that the separation of the original chain using the method of the present application will not affect the genetic analysis.

[0097] Example 3: Feasibility assessment of separated original strands for methylation analysis

[0098] 40 ng cfDNA was divided into two equal parts, one of which was recorded as the control group, and the adapter connection was performed using the Yisheng methylation library construction kit. The other was recorded as the experimental group, and the connection product containing only the original chain was obtained using steps 1-5 in Example 1.

[0099] 1) The two ligation products were methylated and recovered using the zymo methylation conversion kit (EZ DNA Methylation-GoldKit D5006).

[0100] 2) Perform PCR amplification on the methylation conversion products to construct a methylation library.

[0101] Prepare the PCR amplification reaction mixture according to Table 7 below:

[0102] Table 7:

[0103] Components Volume (μl) Methylation conversion and purification of products 20 PCR primer mix 5 HIFI amplification mix 25 total 50

[0104] Place the PCR tube in a PCR instrument. The reaction conditions are shown in Table 8 below:

[0105] Table 8:

[0106]

[0107] 50ul XP magnetic beads were used for purification and recovery to obtain the methylation libraries of the control group and the experimental group.

[0108] 3) The above two methylation libraries were analyzed by capture sequencing using a customized panel.

[0109] The analysis results are as follows Figure 5 As shown, the quantification of all CpGs between the control group and the experimental group was highly consistent at the single base level (P<0.01, r=0.93), indicating that the original chain separated by the method of the present invention can be used for methylation detection and will not affect the quantification of methylation at the single base level.

[0110] Example 4: Technical verification and analysis of simultaneous detection of genome and methylation information in the same sample

[0111] 60 ng of the structural variation 5% ctDNA standard was equally divided into 3 groups, 2 control groups and 1 experimental group. Control group 1 was constructed using the standard methylation library construction process, control group 2 was constructed using the standard genome library construction process, and the experimental group was constructed using the method of the present invention to obtain the genome library and the methylation group library, respectively. The library sequencing data results were analyzed.

[0112] The control group 1 used a methylation library construction kit to prepare the ligation product and obtained the control 1 methylation library after transformation. The control group 2 used a genome library construction kit to prepare the control 2 genome library. The experimental group used the same method as Example 1 to obtain the experimental genome library and the experimental methylation library.

[0113] 4) The above libraries were captured and sequenced using a customized panel to analyze the differences between different libraries. The genomic variation is shown in Table 9. It can be seen that all the variations in the standard can be detected, and the detection frequency is not much different from that of the control group 2.

[0114] Table 9:

[0115] Mutation type mutation Expected frequency Control group 2 Experimental Group SNV AKT1:E17K 5％ 4.8％ 5.2％ SNV PIK3CA:E545K 5％ 5.3％ 4.9％ Insertion EGFR:A767_V769dup 5％ 3.9％ 5.1％ Deletion EGFR: E746_A750del 5％ 6.2％ 5.7％ Fusion CD74(6)-ROS1(34) 5％ 4.6％ 6.1％ Fusion EML4(6)-ALK(20) 5％ 5.0％ 6.3％

[0116] like Figure 6 As shown, the quantification of all CpGs between the control group 1 and the experimental group was highly consistent at the single base level (P<0.01, r=0.92). In summary, the method of the present application can accurately and effectively analyze the methylation level and genomic variation of the same sample.

[0117] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: by connecting a biotin-modified linker to the 3' end of the DNA template, the copy chain and the original chain are effectively separated, and the same DNA template can be used to construct a genomic library and a methylation library respectively, so that the information of the genome and the methylation genome can be obtained more comprehensively and accurately, which is more conducive to analyzing and obtaining accurate genetic information and epigenetic information.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a genomic library and a methylation library, characterized in that: The construction method comprises: The DNA template after end repair is connected to the adapter, and the connection product is amplified by PCR to obtain a mixture of the copy chain and the original chain; Separating the mixture of the copy chain and the original chain, and using the obtained copy chain group and the original chain group to construct a genomic library and a methylation library, respectively, to obtain a genomic library and a methylation library; The 3' end of the linker sequence connected to the 3' end of the DNA template is modified with biotin.

2. The construction method according to claim 1, characterized in that: The cytosine in the linker sequence is 5-methylcytosine.

3. The construction method according to claim 1, characterized in that: The biotin-modified linker includes the linker sequence, U base and biotin connected in sequence from the 5' end to the 3' end.

4. The construction method according to claim 1, characterized in that: Separating the mixture of the copy strand and the original strand comprises: Using streptavidin-modified magnetic beads and the mixture of the copy chain and the original chain to sequentially mix and separate and purify, the obtained first supernatant is the copy chain group; Resuspending the precipitate with deionized water, and cutting off the biotin of the linker in the original chain to obtain the original chain group; Wherein, the magnetic beads modified with streptavidin are magnetic beads that have been rinsed with a magnetic bead buffer and resuspended.

5. The construction method according to claim 4, characterized in that: The method of resuspending the precipitate with deionized water and cutting off the biotin of the linker in the original chain comprises: Resuspend the precipitate with the deionized water, add exonuclease, perform magnetic purification, and discard the supernatant; The precipitate of the enzyme-digested product was resuspended in TE buffer, USERII was added, and magnetic purification was performed. The obtained second supernatant was the original chain group.

6. The construction method according to claim 4, characterized in that: The magnetic beads modified with streptavidin include M270 magnetic beads; Preferably, the magnetic bead buffer comprises: 1M NaCl, 10mM Tris-HCl, pH 8.0, 1mM EDTA, pH 8.0, 0.05% Tween-20, and 0.5% SDS.

7. The construction method according to claim 1, characterized in that: Constructing the methylation library comprises: Using bisulfite to convert the C bases in the DNA of the original chain group that are not methylated into U bases; The transformed product is used as a template to perform PCR amplification to obtain the methylation library.

8. The construction method according to claim 5, characterized in that: The first supernatant was purified using AMPure XP magnetic beads.

9. A kit, characterized in that: The kit comprises: a biotin-modified adapter, an exonuclease and M270 magnetic beads; Preferably, the kit further comprises: USER II, AMPure XP magnetic beads, magnetic bead buffer and TE buffer; Preferably, the magnetic bead buffer comprises: 1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0), 0.05% Tween-20, and 0.5% SDS.

10. The kit according to claim 9, characterized in that The biotin-modified linker comprises a linker sequence and the biotin modification located at the 3' end of the linker sequence, wherein the cytosine in the linker sequence is 5-methylcytosine; Preferably, in the biotin-modified linker, the biotin is connected to the linker sequence via a U base.

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