Construction method and application of methylated DNA-enriched high-throughput sequencing library

Abstract

The invention belongs to the technical field of DNA methylation library construction and DNA methylation detection, and particularly relates to a construction method and application of a high-throughput sequencing library for enriching methylated DNA. According to the construction method disclosed by the invention, two digestion steps of restriction endonuclease are added on the basis of an existing high-throughput sequencing library construction method, and selective library construction of methylated CpG can be realized in combination with a specially designed joint. Compared with an existing conventional construction method of the high-throughput sequencing library, only a small number of steps are added, and the whole steps are relatively simple; and the prepared library is directly subjected to high-throughput sequencing, so that the methylation state in a widely distributed interval on the whole genome can be obtained, and tedious base conversion and targeted enrichment steps are omitted.

Description

Technical Field

[0001] The invention belongs to the technical field of DNA methylation library construction and DNA methylation detection, and specifically relates to a method for constructing a high-throughput sequencing library enriched with methylated DNA and an application thereof. Background Art

[0002] DNA methylation is an extremely important epigenetic mechanism, which is closely related to cell function, differentiation, embryonic development, and the occurrence and development of tumors. DNA methylation in mammals mainly occurs at position 5 (5mC) of the C base in CpG dinucleotides. As a molecular marker, CpG methylation has the advantages of high richness and high specificity, and therefore has attracted more and more attention from researchers. For example, in the field of early cancer screening, using methylation signals as tumor markers can detect tumor-specific methylation signals from a small amount of free nucleic acids with extremely high sensitivity. This is because from the richness of molecular markers, abnormal methylation in tumors will affect a large number of genomic regions, while commonly used sequence variation markers are relatively rare; from the specificity of markers, methylation usually appears continuously, that is, adjacent CpGs have the same methylation state, and multiple CpGs are used together to determine whether a fragment is methylated. Compared with a point mutation fragment with only one position variation, it is less susceptible to sequencing noise.

[0003] There are many ways to detect 5mC, the most common of which is the bisulfite conversion method. Its basic principle is that bisulfite (usually sodium bisulfite) can catalyze the deamination of cytosine and convert it into uracil. In the subsequent PCR and Sanger sequencing, uracil is identified as thymine, but 5mC is not affected and is still detected as cytosine, which can distinguish unmethylated cytosine from 5mC. Bisulfite conversion can be combined with PCR detection technology for the detection of a single or a small number of CpG sites, and can also be combined with NGS technology to detect a large number of CpG sites at the same time. The converted library can be sequenced directly to analyze the methylation status on a genome-wide scale, or it can be combined with library enrichment technology, such as capturing the target region of interest through hybridization capture technology. In addition, 5mC detection can also use methylation-sensitive nucleases to cut off unmethylated fragments and retain methylated fragments. Methylation-sensitive nucleases are often combined with PCR. The methylation status of the site can be distinguished without conversion based on the principle that non-methylated fragments are disconnected and cannot be amplified after enzyme cleavage.

[0004] In some application scenarios, the current technology still has various shortcomings. For example, in early screening for methylated cancer, methylation detection of free DNA with low sample amounts is often involved. The large loss of template DNA during bisulfite conversion is a very serious problem. Although there are currently enzyme conversion technologies that replace bisulfite conversion, they have not yet replaced the bisulfite conversion technology as the gold standard in terms of conversion rate and conversion consistency. In addition, when there is no clear target site and it is hoped that a wide range of genomic regions can be analyzed simultaneously, the cost of the current targeted enrichment technology will increase with the increase of the target area, and there is a greater pressure on the detection cost. Therefore, there is an urgent need in the art for a DNA methylation library construction method that does not rely on complex base conversion steps, is suitable for low-quality samples, and can simultaneously detect the methylation status of a wide range of genomic regions. Summary of the invention

[0005] The object of the present invention is to provide a method for constructing a high-throughput sequencing library enriched for methylated DNA and its application, wherein the construction method can realize the selective construction of a library for methylated CpG, and the constructed library can be directly subjected to high-throughput sequencing without the need for cumbersome base conversion and targeted enrichment steps.

[0006] The present invention provides a method for constructing a high-throughput sequencing library enriched in methylated DNA, comprising the following steps:

[0007] 1) Connecting a first adapter to one end of the DNA to be constructed to obtain a first connection product;

[0008] 2) performing a first enzyme digestion on the first ligation product with a first restriction endonuclease to obtain a first enzyme digestion product containing a first linker, wherein the first restriction endonuclease has a CpG site in its cleavage site and is insensitive to 5mC;

[0009] 3) connecting the first enzyme cleavage product containing the first linker to a second linker to obtain a second ligation product, wherein the second linker is complementary to the cleavage end of the first enzyme cleavage product containing the first linker, and the second ligation product contains a recognition sequence for a second restriction endonuclease;

[0010] 4) performing a second digestion on the second ligation product with the second restriction endonuclease to obtain a second digestion product, wherein the second restriction endonuclease is sensitive to 5mC;

[0011] 5) performing PCR amplification on the second enzyme digestion product with a primer pair to obtain the high-throughput sequencing library enriched in methylated DNA; the upstream primer and the downstream primer of the primer pair respectively contain a linker; the linker includes a first linker and / or a second linker.

[0012] Preferably, the nucleotide sequences of the first linker and the second linker are different.

[0013] Preferably, the DNA to be library constructed includes extracellular free DNA and / or fragmented DNA.

[0014] Preferably, the ligation in step 1) and step 3) comprises double-stranded DNA ligation or single-stranded DNA ligation.

[0015] Preferably, the first restriction endonuclease comprises one or more of Msp I, Taq I and Sfu I.

[0016] Preferably, the second restriction endonuclease comprises Pvu I and / or Hpa II.

[0017] The present invention also provides the application of the construction method described in the above technical solution in DNA methylation detection.

[0018] The present invention also provides a method for detecting DNA methylation, comprising:

[0019] Construct a high-throughput sequencing library enriched for methylated DNA using the sample to be tested according to the construction method described in the above technical solution;

[0020] Performing high-throughput sequencing based on the high-throughput sequencing library enriched for methylated DNA to obtain high-throughput sequencing data;

[0021] The high-throughput sequencing data is subjected to bioinformatics analysis to obtain DNA methylation detection results.

[0022] Preferably, the first adapter and the second adapter used in the process of constructing the high-throughput sequencing library enriched for methylated DNA match the sequencing platform of the high-throughput sequencing.

[0023] The present invention also provides the use of the construction method described in the above technical solution or the detection method described in the above technical solution in the preparation of a product having one or more functions of screening, diagnosis and auxiliary treatment of DNA methylation-related diseases.

[0024] Beneficial effects:

[0025] The present invention provides a method for constructing a high-throughput sequencing library enriched with methylated DNA. The construction method adds two restriction endonuclease digestion steps on the basis of the existing high-throughput sequencing library construction method, and combines with a specially designed linker to achieve selective library construction for methylated CpG. Compared with the existing conventional high-throughput sequencing library construction method, only a small number of steps are added, and the overall steps are relatively simple; and the prepared library can be directly subjected to high-throughput sequencing to obtain the methylation status of the widely distributed intervals on the entire genome, eliminating the cumbersome base conversion and targeted enrichment steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 The schematic diagram is a schematic diagram of constructing a high-throughput sequencing library enriched for methylated DNA in the present invention;

[0028] Figure 2 This is a comparative diagram showing the coverage depth results of the 10Gb sequencing data of the Chinese library for the Msp I site in Example 1 and Comparative Example 1;

[0029] Figures 3-4 This is a graph showing the comparison of the results of methylation level detection between the Chinese library in Example 1 and the Chinese library in Comparative Example 1;

[0030] Figure 5 This is a graph showing the data percentage of LambdaDNA and puc19 plasmids under different enzyme digestion treatments in Example 2. DETAILED DESCRIPTION

[0031] The present invention provides a method for constructing a high-throughput sequencing library enriched in methylated DNA, comprising the following steps:

[0032] 1) Connecting a first adapter to one end of the DNA to be constructed to obtain a first connection product;

[0033] 2) performing a first enzyme digestion on the first ligation product with a first restriction endonuclease to obtain a first enzyme digestion product containing a first linker, wherein the first restriction endonuclease has a CpG site in its cleavage site and is insensitive to 5mC;

[0034] 3) connecting the first enzyme cleavage product containing the first linker to a second linker to obtain a second ligation product, wherein the second linker is complementary to the cleavage end of the first enzyme cleavage product containing the first linker, and the second ligation product contains a recognition sequence for a second restriction endonuclease;

[0035] 4) performing a second digestion on the second ligation product with the second restriction endonuclease to obtain a second digestion product, wherein the second restriction endonuclease is sensitive to 5mC;

[0036] 5) performing PCR amplification on the second enzyme digestion product with a primer pair to obtain the high-throughput sequencing library enriched with methylated DNA; the upstream primer and the downstream primer of the primer pair respectively contain a linker; the linker includes a first linker or a second linker.

[0037] The present invention connects a first adapter to one end of the DNA to be constructed to obtain a first connection product. As an embodiment, the DNA to be constructed in the present invention includes one or more of extracellular free DNA, fragmented FFPE specimen DNA and fragmented DNA. The present invention has no special limitation on the length of the fragmented DNA, and it can be applied to conventional sequencing platforms in the art.

[0038] As an embodiment, the first connector of the present invention can be a connector suitable for a high-throughput sequencing platform in the art; in a specific embodiment, the first connector can be a P7 connector; in another specific embodiment, the first connector includes a positive chain and a negative chain, and the nucleotide sequence of the positive chain is: 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 3), and the nucleotide sequence of the negative chain is: 5'-AACAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 4).

[0039] As an embodiment, the connection method of the present invention can be double-stranded DNA connection or single-stranded DNA connection.

[0040] After obtaining the first ligation product, the present invention performs a first enzyme digestion on the first ligation product with a first restriction endonuclease to obtain a first enzyme digestion product containing a first connector, wherein the enzyme digestion site of the first restriction endonuclease includes a CpG site and the first restriction endonuclease is insensitive to 5mC.

[0041] As an embodiment, the first restriction endonuclease of the present invention can be but not limited to Msp I, Taq I and Sfu I.

[0042] After obtaining the first enzyme cleavage product containing the first connector, the present invention connects the first enzyme cleavage product containing the first connector to a second connector to obtain a second connection product, wherein the second connector is complementary to the enzyme cleavage end of the first enzyme cleavage product containing the first connector, and the second connection product contains a recognition sequence of a second restriction endonuclease.

[0043] As an embodiment, the nucleotide sequences of the first linker and the second linker of the present invention are different. As an embodiment, the connection can be a double-stranded DNA connection or a single-stranded DNA connection.

[0044] After obtaining the second ligation product, the present invention performs a second enzyme digestion on the second ligation product with the second restriction endonuclease to obtain a second enzyme digestion product, and the second restriction endonuclease is sensitive to 5mC.

[0045] As an embodiment, the second restriction endonuclease of the present invention can be but not limited to Pvu I or Hpa II.

[0046] After obtaining the second enzyme cleavage product, the present invention performs PCR amplification on the second enzyme cleavage product with a primer pair to obtain the high-throughput sequencing library enriched in methylated DNA; the upstream primer and the downstream primer of the primer pair respectively contain a connector; the connector includes a first connector and / or a second connector.

[0047] As an embodiment, the first connector and the second connector of the present invention match the high-throughput sequencing platform used subsequently, that is, the first connector and the second connector are associated with the high-throughput sequencing platform used subsequently.

[0048] Unless otherwise specified, the present invention has no particular limitations on the systems and procedures in the connection, enzyme digestion and PCR amplification processes in the construction method, and they can be adjusted according to the reagents or kits used.

[0049] The construction method of the present invention adds two restriction enzyme digestion steps on the basis of the existing high-throughput sequencing library construction method, and combines with specially designed linkers to achieve selective library construction for methylated CpG. The construction principle is as follows: Figure 1 The figure shows different DNA fragments in the process of constructing a high-throughput sequencing library enriched for methylated DNA. The left figure shows a DNA fragment without the first restriction endonuclease cleavage site, the middle figure shows a DNA fragment with the first restriction endonuclease cleavage site and CpG methylated therein, and the right figure shows a DNA fragment with the first restriction endonuclease cleavage site but CpG sites not methylated therein. Figure 1 It can be concluded that only DNA fragments containing the first restriction endonuclease cleavage site and in which CpG is methylated can achieve selective library construction for methylated CpG, thereby obtaining a high-throughput sequencing library enriched in methylated DNA.

[0050] Based on the above advantages, the present invention also provides the application of the construction method described in the above technical solution in DNA methylation detection.

[0051] Specifically, the present invention also provides a method for detecting DNA methylation, comprising:

[0052] Construct a high-throughput sequencing library enriched for methylated DNA using the sample to be tested according to the construction method described in the above technical solution;

[0053] Performing high-throughput sequencing based on the high-throughput sequencing library enriched for methylated DNA to obtain high-throughput sequencing data;

[0054] The high-throughput sequencing data is subjected to bioinformatics analysis to obtain DNA methylation detection results.

[0055] As an embodiment, the first adapter and the second adapter used in the process of constructing the high-throughput sequencing library enriched for methylated DNA match the sequencing platform of the high-throughput sequencing. The present invention does not specifically limit the strategy of the high-throughput sequencing, and it can be adjusted according to the routine of the detection items. The present invention does not specifically limit the method of the biological analysis, and the conventional sequence alignment and coverage depth analysis methods in the art can be used.

[0056] The present invention also provides the use of the construction method described in the above technical solution or the detection method described in the above technical solution in the preparation of a product having one or more functions of screening, diagnosis and adjuvant treatment of DNA methylation-related diseases. As an embodiment, the product of the present invention can be a reagent and / or a kit. As an embodiment, the DNA methylation-related disease of the present invention can be but is not limited to cancer.

[0057] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0058] The application of DNA methylation as a molecular marker in the field of early cancer screening is one of its most concerned application scenarios. Methylation signal detection based on high-throughput sequencing can analyze a large number of CpG sites at the same time, which greatly improves the sensitivity and reliability of early screening compared with single-site detection technology based on quantitative PCR. Extracellular free DNA (cfDNA) extracted from samples such as plasma and urine is often used as the object of methylation signal detection. Such samples often pose challenges to experimental techniques in terms of total DNA volume and fragment integrity. Conventional detection processes including bisulfite conversion and enzyme conversion methods include DNA conversion steps, which cause a large amount of sample loss, increase experimental difficulty, and reduce the upper limit of sensitivity. In addition, in order to reduce sequencing costs and information analysis costs, specific targets need to be enriched, which further makes the experimental process cumbersome and reduces stability.

[0059] Taking plasma cfDNA as an example, the methylation-specific library construction method and bisulfite conversion method of the present invention are used to prepare a high-throughput sequencing library in a single-stranded library construction mode.

[0060] Example 1

[0061] A method for constructing a high-throughput sequencing library enriched for methylated DNA and a DNA detection method, the steps are as follows:

[0062] 1. Sample: 10ng plasma cfDNA sample;

[0063] 2. Library preparation steps are as follows:

[0064] 1 Take 10 ng of DNA from step 1, add water to 15 μL, denature at 95°C for 2 min, and quickly transfer to ice to obtain the denatured product.

[0065] 2. Adoption The single-strand library construction kit (for Illumina) was prepared on ice according to the first ligation reaction system shown in Table 1. The P7 Buffer, P7 adapter, and P7 Enzyme Mix in Table 1 were thawed on ice in advance and then mixed. P7 Adapter is a form of the "first adapter".

[0066] Table 1 The first ligation reaction system

[0067] substance volume Denatured product from step 1 15μL P7Buffer 2.2μL P7 Connector 1μL P7EnzymeMix 1.8μL total 20μL

[0068] 3. Use a pipette to gently pipette the ligation reaction system mixed in step 2 to mix evenly, and centrifuge it instantaneously to place all the reaction liquid at the bottom of the PCR tube. Put the PCR tube into the PCR instrument and start the program in Table 2. When the temperature stabilizes to 37°C, put the reaction tube into the PCR instrument.

[0069] Table 2 First ligation reaction procedure

[0070] temperature time 37℃ 15min 95℃ 2min 4℃ Keep

[0071] 4. Prepare the double-stranded DNA synthesis reaction system according to Table 3:

[0072] Table 3 Double-stranded DNA synthesis reaction system

[0073] substance volume Step 3 Reaction Product 20μL 2ndStrandMasterMix 21μL 2ndStrandPrimer 1μL total 42μL

[0074] Use a pipette to gently pipette the double-stranded DNA synthesis reaction system to mix evenly, and centrifuge briefly to place all the reaction solution at the bottom of the PCR tube. Place the PCR tube in a PCR instrument and start the program in Table 4.

[0075] Table 4 Double-stranded DNA synthesis reaction program

[0076] temperature time 98℃ 1min 60℃ 2min 72℃ 5min 4℃ Keep

[0077] 5. Use The product obtained in step 4 was purified using SP Beads, eluted with 17 μL.

[0078] 6. After preparing the first restriction endonuclease reaction system according to Table 5, digest at 37° C. for 10 min, wherein FastDigestMspI in Table 5 is a form of the first restriction endonuclease in the present invention.

[0079] Table 5 The first enzyme digestion reaction system

[0080] substance volume Step 5: Purification of product 17μL FastDigestMspI(ThermoScientific) 1μL 10×FastDigestBuffer 2μL total 20μL

[0081] 7. Use SP Beads purification of step 6 product, 23 μL elution.

[0082] 8. Prepare the second ligation reaction system according to Table 6. P5-2 linker is a form of the "second linker", which is formed by annealing two single-stranded oligonucleotides, and its sense strand sequence is 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTCGAT-3' (SEQ ID NO: 1), and its antisense strand sequence is 5'-CGATCGAGATCGGAAGAGCGTCGTGA-3' (SEQ ID NO: 2), both without 5' modification.

[0083] Table 6 Second ligation reaction system

[0084] substance volume Step 7: Purification of product 23μL P5Buffer 13μL P5-2 connector 2μL P5Ligase 2μL total 40μL

[0085] Start the second ligation reaction program in Table 7, and when the temperature stabilizes to 20°C, place the reaction tube into the PCR instrument for ligation reaction.

[0086] Table 7 Second ligation reaction procedure

[0087] 20℃ 15min 4℃ Keep

[0088] 9. Use SP Beads purification of step 8 product, 17 μL elution.

[0089] 10. After preparing the second restriction endonuclease reaction system according to Table 8, digest at 37° C. for 10 minutes, wherein FastDigestPvu I in Table 8 is a form of the second restriction endonuclease in the present invention.

[0090] Table 8 Second enzyme digestion reaction system

[0091] Step 9: Purification of product 17μL FastDigestPvuI(ThermoScientific) 1μL 10×FastDigestBuffer 2μL Total 20μL

[0092] 11. Use SP Beads purification of step 10 product, 20 μL elution.

[0093] 12. Prepare the PCR amplification reaction system according to Table 9.

[0094] Table 9 PCR amplification reaction system

[0095]

[0096]

[0097] PCR amplification was performed according to the amplification reaction system prepared in Table 9. The PCR amplification program is shown in Table 10.

[0098] Table 10 PCR amplification program

[0099]

[0100] 13. Use The PCR amplification product of step 12 was purified using SP Beads, and 20 μL of the eluted product was used to obtain the library to be sequenced.

[0101] 3. Sequence the library obtained in step 2 on the Illumina Novasek 6000 platform in PE150 mode.

[0102] Comparative Example 1

[0103] The steps of the methylation library construction method and DNA methylation detection method based on bisulfite conversion are as follows:

[0104] 1. Sample: 10 ng of cfDNA sample from the same plasma source as in Example 1.

[0105] 2. Methylation library construction method based on bisulfite conversion, the steps are as follows:

[0106] Bisulfite conversion-based methylation library preparation Methylation library construction kit (Nanoda) with DNA methylation bisulfite conversion module (Naonda) was used for the hybridization capture. 60k methylation demo panel (Naonda) and Hybrid CaptureReagents (Naonda). All steps were performed according to the instructions.

[0107] 3. Sequence the library obtained in step 2 on the Illumina Novasek 6000 platform in PE150 mode.

[0108] The sequencing data in Example 1 and Comparative Example 1 were both taken as 10 Gb, and the coverage depth of the CpG double bases in the Msp I restriction site was calculated using the samtools software. Specifically, the aligned bam file and the bed file of the Msp I site were input into the samtools software, and the samtools depth tool was used for calculation. The results are as follows: Figure 2 As shown. Figure 2 WGBS and this method represent the library construction methods of Comparative Example 1 and Example 1, respectively.

[0109] Depend on Figure 2 It can be concluded that the average coverage depth of the Chinese library sequencing in Comparative Example 1 is about 3×, which cannot accurately reflect the methylation level of a single site. The average coverage depth of the Chinese library in Example 1 is about 4 times that of Comparative Example 1. Since the fragments will be cut off many times when the Msp I restriction site is close, it is difficult to retain, and the coverage depth of such sites is extremely low. Therefore, if only the sites with a front and rear spacing greater than 200bp are calculated, the sequencing depth of the library construction method in Example 1 is about 11 times that of Comparative Example 1, which can more accurately reflect the methylation level of a single site. There are about more than one million Msp I restriction sites that meet the conditions on the human genome. Direct sequencing of the library obtained in Example 1 can obtain a large number of sites that can accurately analyze the methylation level.

[0110] In order to obtain methylation sequencing data with high coverage depth for calculating the methylation level of a single CpG site, the library construction method in Comparative Example 1 needs to be combined with targeted capture. The demo panel of the methylation library construction kit #1002501 can be provided free of charge to researchers who test the effect of Example 1.) After capture, the sequencing data was 1Gb, and the average coverage depth of the targeted capture area was about 10000×, and 350× after deduplication. After calculating the CpG methylation level using MethylDackel software, the CpG that was also the Msp I restriction site was compared with the library construction method of Example 1.

[0111] The CpG methylation level of the library construction method in Example 1 is reflected by the number of reads at the Msp I site. Figures 3-4 As shown, in Figures 3-4 The present method and the traditional method respectively represent the results obtained by the library construction and sequencing method in Example 1 and the library construction method in Comparative Example 1 combined with targeted capture. Figure 3 The comparison results of all Msp I sites in the target region of the bisulfite conversion library hybridization capture in Comparative Example 1 are as follows: Figure 4 This is the comparison result of 573 sites in the hybridization capture target region of the bisulfite conversion library in Comparative Example 1 after excluding the adjacent Msp I site.

[0112] according to Figures 3-4 It can be concluded that the CpG methylation level of the library construction and sequencing method in Example 1 is highly consistent with the CpG methylation level of the library construction method combined with targeted capture in Comparative Example 1. 2 is 0.763, while at 573 sites with a distance of more than 200 bp adjacent to the Msp I site, R 2 Reached 0.9.

[0113] In the following, lambda DNA and pUC19 plasmid DNA are used as examples to prepare a methylation-specific high-throughput sequencing library in a double-stranded library construction mode using the library construction method of the present invention.

[0114] Example 2

[0115] A method for constructing a high-throughput sequencing library enriched for methylated DNA and a DNA detection method, the steps are as follows:

[0116] 10 ng of Lambda DNA (0% methylated, from Promega, D1521) and 10 ng of puc19 plasmid (100% methylated, from Zymo Research, D5017) were mixed and divided into 2 parts, each containing 10 ng.

[0117] use Rapid DNA digestion library construction kit v2 (Naonda), after fragmentation, end repair & A addition.

[0118] The connector connection steps will Rapid DNA digestion library construction kit v2 (Naonda) The Universal Stubby Adapter was replaced with an adapter having a positive strand of 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO: 3) and a negative strand of 5'-AACAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 4), ie, a form of the first adapter.

[0119] After the ligation product is purified, proceed according to steps 6-9 in Example 1.

[0120] After completing step 9, one of the copies continued to build the library according to steps 10-13 in Example 1 to generate libA;

[0121] The other one skipped steps 10-11 in Example 1 and directly performed steps 12-13 according to the steps in Example 1, that is, no second enzyme digestion treatment was performed to produce libB.

[0122] Both libraries were sequenced on Illumina Novaseq 6000 in PE150 mode.

[0123] The data after libA and libB sequencing were analyzed. Specifically, bwa was used to align the sequenced data to the reference genome containing the human genome, LambdaDNA sequence, and puc19 sequence to obtain the bam file. Then samtools was used to count the reads. The results are as follows: Figure 5 shown.

[0124] Depend on Figure 5 It can be concluded that in the library libB without the second enzyme digestion treatment, the reads of LambdaDNA and puc19 plasmid accounted for 65% and 35% respectively. Although they were mixed in a 1:1 ratio, the number of enzyme digestion sites was different, so it was not 50% and 50%. After the second enzyme digestion treatment, the proportion of Lambda DNA was reduced to less than 1%, demonstrating the characteristics of the method of the present invention in building a library specifically for methylated fragments.

[0125] It can be concluded from the above examples that the construction method of the present invention can realize the selective library construction of methylated CpG, and the constructed library can be directly subjected to high-throughput sequencing without the need for cumbersome base conversion and targeted enrichment steps.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a high-throughput sequencing library enriched for methylated DNA, characterized in that: The steps include: 1) Connecting a first adapter to one end of the DNA to be constructed to obtain a first connection product; 2) performing a first enzyme digestion on the first ligation product with a first restriction endonuclease to obtain a first enzyme digestion product containing a first linker, wherein the first restriction endonuclease has a CpG site in its cleavage site and is insensitive to 5mC; 3) connecting the first enzyme cleavage product containing the first linker to a second linker to obtain a second ligation product, wherein the second linker is complementary to the cleavage end of the first enzyme cleavage product containing the first linker, and the second ligation product contains a recognition sequence for a second restriction endonuclease; 4) performing a second digestion on the second ligation product with the second restriction endonuclease to obtain a second digestion product, wherein the second restriction endonuclease is sensitive to 5mC; 5) performing PCR amplification on the second enzyme digestion product with a primer pair to obtain the high-throughput sequencing library enriched in methylated DNA; the upstream primer and the downstream primer of the primer pair respectively contain a linker; the linker includes a first linker and / or a second linker.

2. The construction method according to claim 1, characterized in that: The nucleotide sequences of the first linker and the second linker are different.

3. The construction method according to claim 1, characterized in that: The DNA to be library constructed includes extracellular free DNA and / or fragmented DNA.

4. The construction method according to claim 1, characterized in that: The ligation in step 1) and step 3) includes double-stranded DNA ligation or single-stranded DNA ligation.

5. The construction method according to claim 1, characterized in that: The first restriction endonuclease includes Msp I 、 One or more of Taq I and Sfu I.

6. The construction method according to claim 1 or 5, characterized in that: The second restriction endonuclease comprises Pvu I and / or Hpa II.

7. Use of the construction method according to any one of claims 1 to 6 in DNA methylation detection.

8. A method for detecting DNA methylation, characterized in that: include: Constructing a high-throughput sequencing library enriched for methylated DNA from the sample to be tested according to the construction method described in any one of claims 1 to 6; Performing high-throughput sequencing based on the high-throughput sequencing library enriched for methylated DNA to obtain high-throughput sequencing data; The high-throughput sequencing data is subjected to bioinformatics analysis to obtain DNA methylation detection results.

9. The detection method according to claim 8, characterized in that: The first adapter and the second adapter used in the process of constructing the high-throughput sequencing library enriched with methylated DNA match the sequencing platform of the high-throughput sequencing.

10. Use of the construction method according to any one of claims 1 to 6 or the detection method according to claim 8 or 9 in the preparation of a product having one or more functions of screening, diagnosis and auxiliary treatment of DNA methylation-related diseases.

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