Construction method and application of CpG island-enriched DNA methylation library

Abstract

The invention discloses a construction method and application of a CpG island-enriched DNA methylation library. The construction method of the DNA methylation library comprises the following steps: carrying out primary end repair on a DNA fragment and adding an A basic group at a 3'end to obtain an end-repaired DNA fragment, connecting the end-repaired DNA fragment with a closed fragment, sequentially carrying out dephosphorylation and purification to obtain a product, carrying out enzyme digestion on the product, carrying out secondary end repair and adding the A basic group at the 3 'end, and finally obtaining the DNA methylation library. And connecting the product of which the secondary end is repaired and the 'A' basic group is added with a methyl-modified sequencing joint, carrying out methylation conversion and library amplification on the DNA fragment connected with the joint, and purifying to obtain the DNA methylation library. According to the DNA methylation library construction method provided by the invention, a relatively high CpG island region and a relatively high sequencing depth can be covered by only using a small amount of sequencing data, and the method not only can obtain relatively comprehensive methylation information, but also can reduce the sequencing amount so as to reduce the detection cost.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for constructing a DNA methylation library enriched in CpG islands and an application thereof. Background Art

[0002] DNA methylation is an important form of epigenetic modification, that is, the addition of a methyl (-CH3) group to the cytosine (C) base of the DNA molecule under the catalysis of the enzyme. DNA methylation mainly occurs on CpG dinucleotide sequences and is involved in important biological processes such as gene expression regulation, gene silencing, cell differentiation, embryonic development, X chromosome inactivation, and tumorigenesis.

[0003] DNA methylation variation occurs in the early stages of carcinogenesis and is a type of epigenetic change that changes dynamically with tumor progression. It is usually hypomethylated oncogenes and hypermethylated tumor suppressor genes. It is highly specific and has the advantage of tissue traceability. Among them, 5mC is the main type of DNA methylation, accounting for 1% of the human genome, and is almost always in CpG islands. The length of CpG islands is about 300-3000bp, overlapping with 60% of human genome promoters and almost 100% of the promoters of conserved genes. Therefore, DNA methylation detection and analysis of CpG islands is becoming an important means of cancer screening, early diagnosis and treatment.

[0004] At present, the commonly used detection methods for DNA methylation include whole genome methylation sequencing (WGBS), reduced genome methylation sequencing (RRBS), targeted bisulfite sequencing (Target-BS) and methylation-specific PCR (MSP). These four methods have their own advantages and disadvantages. WGBS obtains the most comprehensive methyl information, but the sequencing data is large and the cost is high; RRBS enriches CpG islands, and the amount of methyl information obtained is second, but the enrichment effect is limited, and a deeper sequencing coverage is required, and the required sequencing volume and sequencing cost are also high; the detection area of ​​Target-BS can be customized, with high repeatability, but the detection area is limited; MSP accurately detects specific regions of specific genes, with a simple process and high sensitivity, but the detection sites are few and the throughput is low. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a method for constructing a DNA methylation library enriched in CpG islands. The library constructed by the DNA methylation library construction method provided by the present invention can cover a higher CpG island region and sequencing depth with only a small amount of sequencing data. This method can not only obtain more comprehensive methylation information, but also reduce the amount of sequencing, thereby reducing the detection cost.

[0006] The present invention also provides a sequencing method for a methylation library.

[0007] The invention also provides a methylation determination method.

[0008] The present invention also provides a nucleic acid molecule.

[0009] The present invention also provides a kit.

[0010] The present invention also provides an application.

[0011] According to a first aspect of the present invention, a method for constructing a DNA methylation library enriched in CpG islands is proposed, and the method for constructing a DNA methylation library comprises the following steps:

[0012] The DNA fragment is subjected to a primary end repair and an "A" base is added to the 3' end to obtain an end-repaired DNA fragment. The end-repaired DNA fragment is connected to the closed fragment and then sequentially dephosphorylated and purified to obtain a product. The product is digested with enzymes and subjected to a secondary end repair and an "A" base is added to the 3' end. The product of the secondary end repair and "A" base is connected to a methyl-modified sequencing adapter. The DNA fragment connected to the adapter is subjected to methylation conversion and library amplification, and a DNA methylation library is obtained after purification.

[0013] In some embodiments of the invention, the blocking fragment is a double-stranded DNA fragment;

[0014] The 3' end of one end of the double-stranded DNA fragment has a T base protruding, and the 5' end is connected to a phosphate group; the 5' end of the other end of the double-stranded DNA fragment has no phosphate group, or the original phosphate group at the 5' end is replaced by a special modification group, and the original hydroxyl group at the 3' end is replaced by a special modification group.

[0015] In some embodiments of the present invention, the special modification group is a modification group that can block the extension from the 5' end to the 3' end and the cleavage from the 3' end to the 5' end of the DNA polymerase.

[0016] In some embodiments of the present invention, the special modifying group is NH2, NH2C3, NH2C6, NH2C 12 , NH2C6dT, NH2C7, CHCH, CHO, CHCH-dT, Invert dT and Invert dG.

[0017] In some embodiments of the present invention, the DNA fragments include at least one of cell-free DNA (cfDNA) and fragmented genomic DNA (gDNA).

[0018] In some embodiments of the present invention, the fragmented gDNA is mechanically sheared or digested with a restriction endonuclease.

[0019] In some embodiments of the invention, the mechanical disruption comprises ultrasonic disruption.

[0020] In some embodiments of the present invention, the terminal modification enzyme used in the secondary end repair includes KlenowExo enzyme.

[0021] In some embodiments of the present invention, the method for preparing the blocked fragment comprises: diluting the first oligonucleotide and the second oligonucleotide to 15-25 μmol / L respectively with 1× DNA annealing buffer and mixing them in equal volumes, placing the mixed solution in a PCR instrument for denaturation at 90°C to 100°C for 1-3 minutes, and slowly cooling to 20°C to 30°C to obtain the blocked fragment.

[0022] In some embodiments of the present invention, the phosphatase used for dephosphorylation is alkaline phosphatase.

[0023] In some embodiments of the present invention, the restriction endonuclease used for the enzyme cleavage includes at least one of MspI, HpaII, BstBI, AclI, TaqI, HpyCH4IV and AluI.

[0024] In some embodiments of the present invention, the sequencing adapter is ligated to the product using a DNA ligase, and the DNA ligase includes T4 DNA ligase and / or Taq DNA ligase.

[0025] In some embodiments of the present invention, the sequencing adapter is a Y-shaped double-stranded DNA obtained by annealing two partially complementary adapter oligonucleotides, the nucleotide sequences of the adapter oligonucleotides are as shown in SEQ ID NO: 20 and SEQ ID NO: 21, all the cytosine nucleotides in the two adapter oligonucleotides are methylated, and the 5' end of the bottom nucleic acid of the Y-shaped adapter is connected to a phosphate group.

[0026] In some embodiments of the present invention, the method for preparing the sequencing adapter comprises: diluting two adapter oligonucleotides to 15-25 μmol / L respectively with 1× DNA annealing buffer and mixing them in equal volumes, placing the mixed solution in a PCR instrument for denaturation at 90° C. to 100° C. for 1-3 min, and slowly cooling to 20° C. to 30° C. to obtain a sequencing adapter.

[0027] In some embodiments of the present invention, the methylation conversion reagent used in the methylation conversion includes at least one of sodium bisulfite, bisulfite and TET enzyme.

[0028] In some embodiments of the present invention, the amplification primers used for library amplification include primers shown in SEQ ID NO:22 and SEQ ID NO:23.

[0029] In some embodiments of the present invention, the reaction procedure for library amplification includes: hot cover 100°C to 110°C; 95°C to 100°C, 40 to 50s; 95°C to 100°C, 5 to 15s, 55°C to 65°C, 40 to 50s, 65°C to 80°C, 20 to 40s (14 to 17 cycles); 65°C to 80°C, 3 to 7min.

[0030] According to the second aspect of the present invention, a method for sequencing a methylation library is provided, the sequencing method comprising: sequencing the DNA methylation library constructed by the DNA methylation library construction method described in the first aspect of the present invention.

[0031] According to a third aspect of the present invention, a methylation determination method is provided, the methylation determination method comprising: constructing a DNA methylation library according to the DNA methylation library construction method described in the first aspect of the present invention;

[0032] Sequencing the DNA methylation library to obtain sequencing data;

[0033] The sequencing data are subjected to bioinformatics analysis to obtain methylation results.

[0034] According to a fourth aspect of the present invention, a nucleic acid molecule is provided, wherein the nucleic acid molecule is a double-stranded DNA molecule obtained by annealing two complementary oligonucleotides; the oligonucleotides include a first oligonucleotide and a second oligonucleotide;

[0035] In addition to the complementary part, the 3' end of the first oligonucleotide has one more T base than the second oligonucleotide, and the 5' end has no phosphate group or is connected with a special modification group;

[0036] The 5' end of the second oligonucleotide is modified with a phosphate group, and the 3' end is connected with a special modification group.

[0037] In some embodiments of the present invention, the special modifying group is NH2, NH2C3, NH2C6, NH2C 12 , NH2C6dT, NH2C7, CHCH, CHO, CHCH-dT, Invert dT and Invert dG.

[0038] In some embodiments of the present invention, the length of the first oligonucleotide and the second oligonucleotide is 10 to 25 bp.

[0039] In some embodiments of the present invention, the GC content of the first oligonucleotide and the second oligonucleotide is 30-70%.

[0040] In some embodiments of the present invention, the nucleic acid molecule does not contain a restriction endonuclease cleavage site.

[0041] In some embodiments of the present invention, the sequence of the nucleic acid molecule has no highly similar sequence to the sequencing primer.

[0042] In some embodiments of the invention, the nucleotide sequence of the first oligonucleotide is shown as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or SEQ ID NO:18.

[0043] In some embodiments of the invention, the nucleotide sequence of the second oligonucleotide is shown as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:19.

[0044] In some embodiments of the present invention, the annealing comprises the following steps: diluting the first oligonucleotide and the second oligonucleotide to 15-25 μmol / L respectively with 1× DNA annealing buffer and mixing them in equal volumes, placing the mixed solution in a PCR instrument for denaturation at 90°C to 100°C for 1-3 min, and slowly cooling to 20°C to 30°C to obtain the nucleic acid molecule.

[0045] According to a fifth aspect of the present invention, a kit is provided, the kit comprising the nucleic acid molecule as described in the fourth aspect of the present invention.

[0046] In some embodiments of the present invention, the kit further comprises at least one of a methylation conversion reagent, a DNA ligase, a restriction endonuclease, a sequencing adapter, and a PCR amplification primer.

[0047] In some embodiments of the present invention, the methylation conversion reagent comprises at least one of sodium bisulfite, bisulfite and TET enzyme.

[0048] In some embodiments of the present invention, the DNA ligase comprises T4 DNA ligase and / or Taq DNA ligase.

[0049] In some embodiments of the present invention, the restriction endonuclease comprises at least one of Mspl, Hpall, BstBI, Acll, Taql, HpyCH4IV and AluI.

[0050] In some embodiments of the present invention, the sequencing adapter is a Y-shaped double-stranded DNA obtained by annealing two partially complementary adapter oligonucleotides, the nucleotide sequences of the adapter oligonucleotides are as shown in SEQ ID NO: 5 and SEQ ID NO: 6, all the cytosine nucleotides in the two adapter oligonucleotides are methylated, and the 5' end of the bottom nucleic acid of the Y-shaped adapter is connected to a phosphate group.

[0051] In some embodiments of the present invention, the method for preparing the sequencing adapter comprises: diluting two adapter oligonucleotides to 15-25 μmol / L respectively with 1× DNA annealing buffer and mixing them in equal volumes, placing the mixed solution in a PCR instrument for denaturation at 90° C. to 100° C. for 1-3 min, and slowly cooling to 20° C. to 30° C. to obtain a sequencing adapter.

[0052] In some embodiments of the present invention, the PCR amplification primers include primers shown in SEQ ID NO:7 and SEQ ID NO:8.

[0053] According to the sixth aspect of the present invention, the use of the nucleic acid molecule according to the fourth aspect of the present invention or the kit according to the fifth aspect of the present invention in (1) or (2) is provided:

[0054] (1) Construction of DNA methylation library;

[0055] (2) Methylation sequencing.

[0056] The present invention has at least the following beneficial effects:

[0057] The present invention provides a method for constructing a DNA methylation library enriched in CpG islands. The method first uses a synthetic closed fragment to close the ends of the fragmented gDNA or cfDNA, and then performs restriction endonuclease digestion, so that the non-CpG island region fragments cannot form a library required for sequencing due to the end closure and cannot perform a ligation reaction, and the CpG island region fragments can be normally constructed, sequenced and analyzed after digestion, and finally obtain the methyl information of the genome and CpG island. The DNA methylation library construction method provided by the present invention can effectively enrich the CpG island region in the sample to be tested, and the CpG island region of more than 10Mb can be covered when the sequencing amount is only 2Gb, the coverage is more than 40%, the coverage of the number of CpG islands is more than 80%, and the total CpG island average sequencing depth is more than 2.5×; the sequencing amount required to obtain high sequencing depth methylation information is smaller, which can reduce the detection cost and improve the detection cost performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0059] Figure 1 A schematic diagram of the process of constructing a DNA methylation library provided by the present invention. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] In the description of the present invention, "several" means more than one, "many" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing the technical features, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0062] Example 1

[0063] This example provides a method for constructing a DNA methylation library enriched in CpG islands. gDNA extracted from whole blood samples is used as the library construction material. The gDNA is extracted using a commercial kit (Golden Mager, GMB-MS-F48) and is extracted according to the instructions provided by the supplier. The specific steps for constructing the DNA methylation library are as follows:

[0064] 1) gDNA fragmentation:

[0065] Use ultrasound to shear gDNA for 30 minutes with an ultrasound power of 25kHz. After working for 20 seconds, pause for 10 seconds. Detect the double-stranded concentration and fragment size of the sheared DNA, and take 5ng and 40ng of DNA fragments for subsequent operations.

[0066] 2) End repair and A tailing:

[0067] Add the DNA fragment obtained in step 1) into a 0.2 mL PCR tube, then add 3.5 μL of End Prep Mix (Norvozyme, N203), and add water to 15 μL; run the following reaction program in a PCR instrument: 20°C, 15 min; 65°C, 15 min to obtain end-repaired DNA fragments.

[0068] 3) Sequence design, oligonucleotide synthesis and annealing of blocking fragments:

[0069] ① Design principles of the blocking fragment: length 10-25bp; GC content between 30% and 70%; no highly similar sequence to the sequencing primer; no restriction endonuclease cleavage site sequence; the two blocking oligonucleotides can be partially complementary or completely complementary; the second blocking oligonucleotide has an additional thymine at the 3' end complementary to the first blocking oligonucleotide.

[0070] ② Blocked oligonucleotide synthesis: Synthesized by purification with HPLC or higher purity; Special modifications are added to both ends of the oligonucleotides, a phosphate group (pho) that is conducive to AT ligation reaction is added to the 5' end of the second blocked oligonucleotide, and a modification that blocks the ligation reaction is added to the 3' end; Optional modifications to the 5' end of the first blocked oligonucleotide include: adding a modification that blocks the ligation reaction or removing the phosphate group at the 5' end;

[0071] The specific blocking oligonucleotide sequence can be selected from any one of the following groups (a) to (j):

[0072] (a) first blocking oligonucleotide: 5'-GTCGCGGGAACAGAGGTGT-3' (SEQ ID NO: 1), second blocking oligonucleotide: 5'-pho-CACCTCTGTTCCCGCGAC-NH2C6-3' (SEQ ID NO: 2);

[0073] (b) first blocking oligonucleotide: 5'-CAAGTCGCGGGAACAGAGGTGT-3' (SEQ ID NO: 3), second blocking oligonucleotide: 5'-pho-CACCTCTGTTCCCGCGACTACAAC-CHCH-3' (SEQ ID NO: 4);

[0074] (c) first blocking oligonucleotide: 5'-TCGTCGGCAGGTGTATAAGAGACAGT-3' (SEQ ID NO: 5), second blocking oligonucleotide: 5'-pho-CTGTCTCTTATACACCTGCCGACG*A-inverted dT-3' (SEQ ID NO: 6);

[0075] (d) first blocking oligonucleotide: 5'-TCGTCGGCAGGTGTATAAGAGACAGT-3' (SEQ ID NO: 5), second blocking oligonucleotide: 5'-pho-CTGTCTCTTATACACCTGCCGACG*A-NH2C6-3' (SEQ ID NO: 7);

[0076] (e) first blocking oligonucleotide: 5'-NH2C6-GTCGCGGGAACAGAGGTGT-3' (SEQ ID NO: 8), second blocking oligonucleotide: 5'-pho-CACCTCTGTTCCCGCGA*C-inverted dT-3' (SEQ ID NO: 9);

[0077] (f) first blocking oligonucleotide: 5'-NH2C6-CGAAATCGGTAGACGCTACGT-3' (SEQ ID NO: 10), second blocking oligonucleotide: 5'-pho-CGTAGCGTCTACCGATTTC*G-inverted dT-3' (SEQ ID NO: 11);

[0078] (g) first blocking oligonucleotide: 5'-NH2C6-CAACGACGTGAACACTACAACT-3' (SEQ ID NO: 12), second blocking oligonucleotide: 5'-pho-GTTGTAGTGTTCACGTCGTT*G-inverted dT-3' (SEQ ID NO: 13);

[0079] (h) first blocking oligonucleotide: 5'-NH2C6-AAGGCGGGAAACGACT-3' (SEQ ID NO: 14), second blocking oligonucleotide: 5'-pho-GTCGTTTCCCGCCT*T-inverted dT-3' (SEQ ID NO: 15);

[0080] (i) first blocking oligonucleotide: 5'-NH2C6-CAAGTCGCGGGAACAGAGGTGT-3' (SEQ ID NO: 16), second blocking oligonucleotide: 5'-pho-CACCTCTGTTCCCGCGACTACAA*C-NH2C6-3' (SEQ ID NO: 17);

[0081] (j) first blocking oligonucleotide: 5'-NH2C6-GTCGCGGGAACAGAGGTGT-3' (SEQ ID NO: 18), second blocking oligonucleotide: 5'-pho-CACCTCTGTTCCCGCGA*C-NH2C6-3' (SEQ ID NO: 19);

[0082] Here, * indicates thio modification.

[0083] ③ Preparation of blocked fragments: In this example, oligonucleotides as shown in SEQ ID NO: 16 and SEQ ID NO: 17 were selected for the synthesis of blocked fragments. The first and second blocked oligonucleotides were diluted to 20 μmol / L respectively with 1× DNA annealing buffer, and then the two dilutions were mixed in equal volumes, with a total volume of no more than 200 μL; after mixing, the mixture was placed in a PCR instrument for denaturation at 95°C for 2 min, and the temperature was slowly lowered to 25°C (the cooling process took at least 45 min) to obtain blocked fragments. The product can be stored temporarily at 4°C for subsequent experiments, and can be frozen at -20°C for long-term storage.

[0084] 4) Ligation of blocked fragments: Prepare the reaction system shown in Table 1 in a PCR tube; run the following reaction program in a PCR instrument: close the hot lid, 30°C, 10 min, to obtain blocked DNA fragments. T4 DNA Ligase (Rapid) was purchased from Novozymes, catalog number N103.

[0085] Table 1 Reaction system for connecting closed fragments

[0086] Reagents Volume (μL) Step 2) The resulting end-repaired DNA fragments 15 Step 3) Obtained closed fragment 1 DNA Ligase Buffer 9 T4 DNA Ligase (Rapid) 2 Nuclease-free water 3

[0087] 5) 5' dephosphorylation and purification:

[0088] Prepare the reaction system shown in Table 2 in a PCR tube; run the following reaction program in a PCR instrument: 37°C, 10 min; 80°C, 2 min. After the reaction is completed, 50 μL of Ampure XP Beads (Beckman, A63880, the same below) is used for purification, and eluted into 13 μL of EB eluent to obtain a purified product.

[0089] Table 2 Reaction system for 5' dephosphorylation

[0090] Reagents Volume (μL) Step 4) The resulting closed DNA fragment 30 <![CDATA[rCutSmart TM Buffer]]> 2 Quick CIP (NEB, M0525) 1 Nuclease-free water 17

[0091] 6) Restriction endonuclease digestion:

[0092] 0.5 μL of MspI (NEB, R0106) and 1.5 μL of 10×CutSmart buffer were added to the purified product obtained in step 5), mixed and centrifuged instantly, and transferred to a PCR instrument and incubated at 37° C. for 30 min to obtain the enzyme digestion product.

[0093] 7) Methylated linker connection:

[0094] ① End repair and A-tailing: Prepare the reaction system shown in Table 3 in a PCR tube; run the following reaction program in a PCR instrument: 37°C, 30 min; 75°C, 20 min to obtain the end-repaired fragments.

[0095] Table 3 End repair reaction system

[0096]

[0097] ② Preparation and connection of methylation sequencing adapters:

[0098] The methylation sequencing adapter used is a Y-shaped double-stranded chain obtained by annealing two oligonucleotides. The oligonucleotide sequence used is as follows:

[0099] Adapter oligonucleotide 1: 5'-AC m AC m TC m TTTC m C m C m TAC m AC m GAC m GC m TC m TTC m C m TC m *T-3' (SEQ ID NO: 20),

[0100] Adapter oligonucleotide 2: 5'-pho-GATC m GGAAGAGC m AC m AC m GTC m TGAAC m TC m C m AGT C m -3' (SEQ ID NO: 21);

[0101] Among them, m represents methylation modification, and * represents thiolation modification.

[0102] Preparation method of methylation sequencing adapter: dilute adapter oligonucleotide 1 and adapter oligonucleotide 2 to 20 μmol / L respectively with 1× DNA annealing buffer (Solebol, D2810), and then mix the two dilutions in equal volumes, with the total volume not exceeding 200 μL; after mixing, put them into a PCR instrument for denaturation at 95°C for 2 minutes, and slowly cool to 25°C (the cooling process takes at least 45 minutes) to obtain the methylation sequencing adapter. The product can be stored at 4°C for a short time for subsequent experiments, and can be frozen at -20°C for long-term storage.

[0103] Prepare the reaction system shown in Table 4 in a PCR tube; run the following reaction program in a PCR instrument: hot lid closed, 25°C, 15 min, 4°C, hold. After the reaction is completed, purify with 42 μL of Ampure XP Beads and elute into 20 μL of EB eluent to obtain fragments with sequencing adapters.

[0104] Table 4 Reaction system for connecting methylation sequencing adapter

[0105]

[0106]

[0107] 8) Methylation conversion:

[0108] The fragments connected to the sequencing adapter obtained in step 7) were methylated and purified using a bisulfite methylation conversion kit (Novozyme, EM103) according to the instructions, and eluted with 20 μL of EB elution buffer to obtain methylation conversion products.

[0109] 9) Library amplification and purification:

[0110] High-fidelity DNA polymerase (Ibotek, RK20723) was used for library amplification. The amplification primers were synthetic primers with the following sequences:

[0111] i5 Primer: 5'-AATGATACGGCGACCACCGAGATCTACACnnnnnnnnACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3' (SEQ ID NO: 22);

[0112] i7 Primer: 5'-CAAGCAGAAGACGGCATACGAGATnnnnnnnnGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' (SEQ ID NO: 23);

[0113] Among them, nnnnnnnn represents 8 random bases and * represents thiolation modification.

[0114] Prepare the reaction system shown in Table 5 in a PCR tube; run the following reaction program in PCR: hot cover 105°C; 98°C, 45s; 98°C, 10s, 60°C, 45s, 72°C, 30s (14-17 cycles); 72°C, 5min. After PCR amplification, 50μL of Ampure XP Beads was used for purification and eluted into 13μL of EB eluent to obtain a DNA methylation library enriched in CpG islands that can be directly used for sequencing.

[0115] Table 5 Amplification reaction system

[0116] Reagents Volume (μL) Step 8) The obtained methylation conversion product 20 Gloria U 2X Mix for NGS 25 i5 Primer 2.5 i7 Primer 2.5

[0117] Example 2

[0118] This embodiment provides a method for constructing a DNA methylation library enriched for CpG islands, using gDNA extracted from whole blood samples as the library construction material. The only difference from Example 1 is that the fragmentation method in step 1) is replaced by enzymatic fragmentation instead of ultrasonic fragmentation, and the remaining steps are the same.

[0119] The steps of the above enzymatic method for breaking gDNA are as follows: add 3 μL of FEA Enzyme Mix and 1.5 μL of FEABuffer to gDNA, add water to 15 μL, mix and centrifuge briefly, incubate at 37°C for 20 minutes in a PCR instrument, and then incubate at 65°C for 30 minutes; detect the double-stranded concentration and fragment size of the DNA after breaking, and take 5ng and 40ng of DNA fragments for subsequent operations respectively.

[0120] Example 3

[0121] This example provides a method for constructing a DNA methylation library enriched for CpG islands, using 5 ng of cfDNA extracted from a plasma sample. The only difference from Example 1 is that the library construction materials are different, and the other steps are the same.

[0122] Comparative Example 1

[0123] In this comparative example, the library construction material used in Example 1 was constructed using degenerate representational bisulfite sequencing (RRBS), and RRBS included the following steps:

[0124] The gDNA was digested with MspI, and methyl conversion and library construction were performed using a methyl library construction kit (Agitech, B30061 and B30061) and a methyl conversion kit (Novozyme, EM103). After quality inspection, the constructed library was sequenced using Illumina's NovaSeq 6000 in PE150 mode; for specific experimental methods, please refer to the reference "Gnirke, Andreas, et al." Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling." Nature Protocols 6.4 (2011): 468-481.".

[0125] Test example

[0126] This test example tested the quality of the library obtained by the DNA methylation library construction method provided in Examples 1 to 3 and Comparative Example 1, and sequenced it on the Illumina NovaSeq 6000 sequencer in the PE150 sequencing mode. After bioinformatics analysis, the results are shown in Table 6.

[0127] Table 6 Sequencing results of DNA methylation libraries provided in Examples 1 to 3 and Comparative Example 1

[0128]

[0129]

[0130] It can be seen from Table 6 that compared with the coverage of CpG islands by the RRBS library (Comparative Example 1) with a sequencing volume of 5 Gb, the library constructed using the construction method provided by the present invention can obtain a larger CpG island coverage area and a higher average sequencing depth of CpG islands when the sequencing volume is 2 Gb, which greatly reduces the sequencing cost and cost.

[0131] In addition to the blocking sequences actually used in Examples 1 to 3, the present invention also tested the effects of other blocking sequences described in Example 1 in library construction, and the quality of the obtained library was consistent with that in Table 6 obtained from the test example test.

[0132] In summary, the library constructed using the DNA methylation library construction method provided by the present invention can cover a relatively high CpG island region and sequencing depth with only a small amount of sequencing data. When only 2Gb is sequenced, a CpG island region of more than 10Mb can be covered, with a coverage of more than 40%, a coverage of more than 80% for the number of CpG islands, and an average sequencing depth of more than 2.5× for the total CpG islands. The library construction method provided by the present invention can enrich CpG islands and reduce detection costs; the flow diagram of the DNA methylation library construction method provided by the present invention is as follows Figure 1 shown.

[0133] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a DNA methylation library enriched in CpG islands, characterized in that: The DNA methylation library construction method comprises the following steps: The DNA fragments are subjected to a primary end repair and "A" base is added to the 3' end to obtain the end-repaired DNA fragments. The end-repaired DNA fragments are connected to the closed fragments and then dephosphorylated and purified in sequence to obtain the products. After the products are digested by enzymes, secondary end repair and "A" base is added to the 3' end. The products of the secondary end repair and "A" base are connected to methyl-modified sequencing adapters. The DNA fragments connected to the adapters are subjected to methylation conversion and library amplification. After purification, the DNA methylation library is obtained.

2. The method for constructing a DNA methylation library according to claim 1, characterized in that: The blocking fragment is a double-stranded DNA fragment; The 3' end of one end of the double-stranded DNA fragment has a T base protruding, and the 5' end is connected to a phosphate group; the 5' end of the other end of the double-stranded DNA fragment has no phosphate group, or the original phosphate group at the 5' end is replaced by a special modification group, and the original hydroxyl group at the 3' end is replaced by a special modification group.

3. The method for constructing a DNA methylation library according to claim 2, characterized in that: The special modification group is a modification group that can block the extension of the 5' end to the 3' end and the cleavage of the 3' end to the 5' end of the DNA polymerase; Preferably, the special modifying group is NH2, NH2C3, NH2C6, NH2C 12 , NH2C6 dT, NH2C7, CHCH, CHO, CHCH-dT, Invert dT and Invert dG.

4. The method for constructing a DNA methylation library according to claim 1, characterized in that: The DNA fragments include at least one of free DNA and fragmented genomic DNA.

5. The method for constructing a DNA methylation library according to claim 1, characterized in that: The enzyme digestion uses a restriction endonuclease; Preferably, the restriction endonuclease is selected from at least one of MspI, HpaII, BstBI, AcII, TaqI, HpyCH4IV and AluI.

6. A methylation determination method, characterized in that: The methylation determination method comprises: constructing a DNA methylation library according to the DNA methylation library construction method according to any one of claims 1 to 5; Performing next-generation sequencing on the DNA methylation library to obtain sequencing data; The sequencing data are subjected to bioinformatics analysis to obtain methylation results.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule is a double-stranded DNA molecule obtained by annealing two complementary oligonucleotides; the oligonucleotides include a first oligonucleotide and a second oligonucleotide; In addition to the complementary part, the 3' end of the first oligonucleotide has one more T base than the second oligonucleotide, and the 5' end has no phosphate group or is connected with a special modification group; The 5' end of the second oligonucleotide is modified with a phosphate group, and the 3' end is connected with a special modification group.

8. The nucleic acid molecule according to claim 7, characterized in that The length of the nucleic acid molecule is 10 to 25 bp; Preferably, the GC content of the nucleic acid molecule is 30% to 70%; Preferably, the nucleic acid molecule does not contain restriction endonuclease cleavage sites; Preferably, the nucleic acid molecule does not contain a fragment that is highly similar to the sequencing primer sequence.

9. A kit, characterized in that: The kit comprises the nucleic acid molecule of claim 7 or 8.

10. Use of the nucleic acid molecule according to claim 7 or 8 or the kit according to claim 9 in (1) or (2): (1) Construction of DNA methylation library; (2) Methylation sequencing.

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