Method for constructing high-throughput sequencing library for enriched methylated DNA and use thereof

Abstract

The present invention belongs to the technical fields of DNA methylation library construction and DNA methylation detection, and specifically relates to a method for constructing a high-throughput sequencing library for enriched methylated DNA and use thereof. The construction method, on the basis of existing high-throughput sequencing library construction methods, incorporates two steps of restriction endonuclease digestion, and, combined with a specially designed linker, can achieve selective library construction for methylated CpG. Compared with existing conventional high-throughput sequencing library construction methods, the construction method incorporates only a small number of steps, making the overall procedure relatively simple. Moreover, high-throughput sequencing is directly performed on the prepared library, so that methylation states in widely distributed intervals on the whole genome can be obtained, thereby eliminating cumbersome steps of base conversion and targeted enrichment.

Description

A method for constructing high-throughput sequencing libraries enriched with methylated DNA and its applications

[0001] This application claims priority to Chinese Patent Application No. 202411712817.1, filed on November 27, 2024, entitled "A method for constructing a high-throughput sequencing library enriched with methylated DNA and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of DNA methylation library construction and DNA methylation detection technology, specifically involving a method for constructing a high-throughput sequencing library enriched with methylated DNA and its application. Background Technology

[0003] DNA methylation is a crucial epigenetic mechanism closely related to cell function, differentiation, embryonic development, and tumorigenesis. In mammals, DNA methylation primarily occurs at position 5 (5mC) of the C base in CpG dinucleotides. CpG methylation, as a molecular marker, possesses advantages of high abundance and high specificity, thus attracting increasing attention from researchers. For example, in early cancer screening, using methylation signals as tumor markers allows for the detection of tumor-specific methylation signals from a small amount of free nucleic acid with extremely high sensitivity. This is because, in terms of marker abundance, abnormal methylation in tumors affects a large number of genomic regions, while commonly used sequence variation markers are relatively scarce; and in terms of marker specificity, methylation usually occurs consecutively, meaning adjacent CpGs have the same methylation state. Multiple CpGs can be used together to determine whether a fragment is methylated, making it less susceptible to sequencing noise compared to point mutations, which have only a single positional variation.

[0004] There are several methods for detecting 5mC, the most common being the bisulfite conversion method. The basic principle is that bisulfite (usually sodium bisulfite) catalyzes the deamination of cytosine to uracil. In subsequent PCR and Sanger sequencing, uracil is identified as thymine, but 5mC remains unaffected and is still detected as cytosine, thus distinguishing unmethylated cytosine from 5mC. Bisulfite conversion can be combined with PCR for detecting single or small numbers of CpG sites, or with NGS for detecting a large number of CpG sites simultaneously. The converted library can be directly sequenced to analyze methylation status across the entire genome, or combined with library enrichment techniques, such as hybridization capture to capture target regions of interest. Additionally, 5mC detection can also utilize methylation-sensitive endonucleases to cleave unmethylated fragments while preserving methylated fragments. Methylation-sensitive endonucleases are often combined with PCR. By utilizing the principle that unmethylated fragments are broken after enzyme digestion and cannot be amplified, the methylation status of the site can be distinguished without transformation.

[0005] In some applications, current technologies still have various shortcomings. For example, in early cancer screening using methylation, the detection of methylation in low-volume cell-free DNA samples is often involved. The significant loss of template DNA during bisulfite conversion is a serious problem. Although enzymatic conversion techniques exist as alternatives to bisulfite conversion, they have not yet surpassed the gold standard in terms of conversion efficiency and consistency. Furthermore, when there are no clearly defined target sites and the goal is to analyze a broad range of genomic regions simultaneously, the cost of current targeted enrichment techniques increases with the size of the target region, creating significant cost pressures. Therefore, there is an urgent need in this field for a DNA methylation library construction method that does not rely on complex base conversion steps, is adaptable to low-quality samples, and can simultaneously detect the methylation status of a wide range of genomic regions. Summary of the Invention

[0006] The purpose of this application is to provide a method for constructing a high-throughput sequencing library enriched with methylated DNA and its application. The construction method can selectively construct libraries for methylated CpG, and the constructed libraries can be directly sequenced in high-throughput without the need for cumbersome base conversion and targeted enrichment steps.

[0007] This application provides a method for constructing a high-throughput sequencing library enriched with methylated DNA, comprising the following steps:

[0008] 1) The first adapter is ligated to one end of the DNA to be used to construct the library, and the first ligation product is obtained;

[0009] 2) The first ligation product is digested with the first restriction endonuclease to obtain a first digestion product containing the first adapter. The restriction endonuclease has a CpG site in its cleavage site and is not sensitive to 5mC.

[0010] 3) The first enzyme digestion product containing the first adapter is ligated to the second adapter to obtain the second ligation product. The second adapter is complementary to the enzyme digestion end of the first enzyme digestion product containing the first adapter, and the second ligation product contains the recognition sequence of the second restriction endonuclease.

[0011] 4) The second ligation product is digested with the second restriction endonuclease to obtain the second digested product. The second restriction endonuclease is sensitive to 5 mC.

[0012] 5) Perform PCR amplification on the second enzyme digestion product using the primer pair to obtain the high-throughput sequencing library enriched with methylated DNA; the upstream and downstream primers of the primer pair each contain adapters; the adapters include a first adapter and / or a second adapter.

[0013] Preferably, the nucleotide sequences of the first adapter and the second adapter are different.

[0014] Preferably, the DNA to be used for library construction includes extracellular free DNA and / or fragmented DNA.

[0015] Preferably, the ligation in steps 1) and 3) includes double-stranded DNA ligation or single-stranded DNA ligation.

[0016] Preferably, the first restriction endonuclease comprises MspI. 、 One or more of Taq I and Sfu I.

[0017] Preferably, the second restriction endonuclease includes Pvu I and / or Hpa II.

[0018] This application also provides the application of the construction method described in the above technical solution in DNA methylation detection.

[0019] This application also provides a method for detecting DNA methylation, including:

[0020] A high-throughput sequencing library enriched with methylated DNA was constructed from the sample to be tested according to the construction method described in the above technical solution.

[0021] High-throughput sequencing was performed based on the high-throughput sequencing library enriched with methylated DNA to obtain high-throughput sequencing data.

[0022] Bioinformatics analysis was performed on the high-throughput sequencing data to obtain DNA methylation detection results.

[0023] Preferably, the first and second adapters used in constructing the high-throughput sequencing library enriched with methylated DNA are matched with the high-throughput sequencing platform.

[0024] This application also provides the application of the construction method or detection method described in the above technical solutions in the preparation of products with one or more functions in screening, diagnosis and adjuvant treatment of DNA methylation-related diseases. Beneficial effects:

[0025] This application provides a method for constructing a high-throughput sequencing library enriched with methylated DNA. This method adds two restriction endonuclease digestion steps to existing high-throughput sequencing library construction methods, combined with specially designed adapters, enabling selective library construction of methylated CpGs. Compared to existing conventional high-throughput sequencing library construction methods, it adds only a few steps, making the overall process simpler. Furthermore, the prepared library can be directly sequenced using high-throughput sequencing to obtain the methylation status of widely distributed regions across the entire genome, eliminating the need for cumbersome base conversion and targeted enrichment steps. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0027] Figure 1 is a schematic diagram of the principle of constructing a high-throughput sequencing library enriched with methylated DNA in this application;

[0028] Figure 2 is a comparison of the coverage depth of the Msp I site by 10Gb sequencing data of the Chinese library in Example 1 and Comparative Example 1.

[0029] Figures 3 and 4 show the comparison of the methylation level detection results between the Chinese library of Example 1 and the Chinese library of Comparative Example 1.

[0030] Figure 5 shows the data proportions of Lambda DNA and puc19 plasmid under different enzyme digestion treatments in Example 2. Detailed Implementation

[0031] This application provides a method for constructing a high-throughput sequencing library enriched with methylated DNA, comprising the following steps:

[0032] 1) The first adapter is ligated to one end of the DNA to be used to construct the library, and the first ligation product is obtained;

[0033] 2) The first ligation product is digested with the first restriction endonuclease to obtain a first digestion product containing the first adapter. The restriction endonuclease has a CpG site in its cleavage site and is not sensitive to 5mC.

[0034] 3) The first enzyme digestion product containing the first adapter is ligated to the second adapter to obtain the second ligation product. The second adapter is complementary to the enzyme digestion end of the first enzyme digestion product containing the first adapter, and the second ligation product contains the recognition sequence of the second restriction endonuclease.

[0035] 4) The second ligation product is digested with the second restriction endonuclease to obtain the second digested product. The second restriction endonuclease is sensitive to 5 mC.

[0036] 5) Perform PCR amplification on the second enzyme digestion product using the primer pair to obtain the high-throughput sequencing library enriched with methylated DNA; the upstream and downstream primers of the primer pair each contain adapters; the adapters include a first adapter or a second adapter.

[0037] This application involves ligating a first adapter to one end of the DNA to be used as a library, resulting in a first ligation product. As one embodiment, the DNA to be used as a library in this application includes one or more of extracellular free DNA, fragmented FFPE sample DNA, and fragmented DNA. This application does not have a specific limitation on the length of the fragmented DNA; any conventional sequencing platform in the art will suffice.

[0038] In one embodiment, the first adapter of this application can be an adapter suitable for high-throughput sequencing platforms in the art; in one specific embodiment, the first adapter can be a P7 adapter; in another specific embodiment, the first adapter includes a positive strand and a negative strand, wherein the nucleotide sequence of the positive strand is: 5'-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3' (SEQ ID NO:3), and the nucleotide sequence of the negative strand is: 5'-AACAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO:4).

[0039] As one implementation method, the connection method described in this application can be double-stranded DNA ligation or single-stranded DNA ligation.

[0040] After obtaining the first ligation product, this application performs a first restriction endonuclease digestion on the first ligation product to obtain a first digestion product containing a first adapter. The first restriction endonuclease has a CpG site in its digestion site and is not sensitive to 5mC.

[0041] As one implementation method, the first restriction endonuclease in this application may be, but is not limited to, MspI. 、 Taq I and Sfu I.

[0042] After obtaining the first enzyme digestion product containing the first adapter, this application ligates the first enzyme digestion product containing the first adapter to a second adapter to obtain a second ligation product. The second adapter is complementary to the enzyme digestion end of the first enzyme digestion product containing the first adapter, and the second ligation product contains a recognition sequence of a second restriction endonuclease.

[0043] In one embodiment, the nucleotide sequences of the first and second adapters in this application are different. In another embodiment, the ligation can be a double-stranded DNA ligation or a single-stranded DNA ligation.

[0044] After obtaining the second ligation product, this application performs a second digestion of the second ligation product with the second restriction endonuclease to obtain a second digestion product. The second restriction endonuclease is sensitive to 5mC.

[0045] In one implementation, the second restriction endonuclease in this application may be, but is not limited to, Pvu I or Hpa II.

[0046] After obtaining the second enzyme digestion product, this application performs PCR amplification on the second enzyme digestion product using primer pairs to obtain the high-throughput sequencing library enriched with methylated DNA; the upstream and downstream primers of the primer pairs each contain adapters; the adapters include a first adapter and / or a second adapter.

[0047] In one implementation, the first and second adapters of this application are matched with the high-throughput sequencing platform used subsequently, that is, the first and second adapters are associated with the high-throughput sequencing platform used subsequently.

[0048] Unless otherwise specified, this application does not impose any special limitations on the system and procedure for ligation, enzyme digestion and PCR amplification in the construction method described herein, and adjustments can be made according to the reagents or kits used.

[0049] The construction method described in this application adds two restriction endonuclease digestion steps to the existing high-throughput sequencing library construction method. Combined with a specially designed adapter, it enables selective library construction for methylated CpG. The construction principle is illustrated in Figure 1, showing the different DNA fragments during the construction of a high-throughput sequencing library enriched with methylated DNA. The left figure shows a DNA fragment without a first restriction endonuclease cleavage site; the middle figure shows a DNA fragment containing a first restriction endonuclease cleavage site where CpG is methylated; and the right figure shows a DNA fragment containing a first restriction endonuclease cleavage site but where the CpG site is not methylated. Based on Figure 1, it can be concluded that only DNA fragments containing a first restriction endonuclease cleavage site where CpG is methylated can achieve selective library construction for methylated CpG, resulting in a high-throughput sequencing library enriched with methylated DNA.

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

[0051] Specifically, this application also provides a method for detecting DNA methylation, including:

[0052] A high-throughput sequencing library enriched with methylated DNA was constructed from the sample to be tested according to the construction method described in the above technical solution.

[0053] High-throughput sequencing was performed based on the high-throughput sequencing library enriched with methylated DNA to obtain high-throughput sequencing data.

[0054] Bioinformatics analysis was performed on the high-throughput sequencing data to obtain DNA methylation detection results.

[0055] In one implementation, the first and second adapters used in constructing the high-throughput sequencing library enriched with methylated DNA are matched to the high-throughput sequencing platform. This application does not specifically limit the high-throughput sequencing strategy; adjustments can be made routinely according to the detection requirements. This application also does not specifically limit the method of biological analysis; conventional sequence alignment and coverage depth analysis methods in the art can be used.

[0056] This application also provides the application of the construction method or detection method described in the above-described technical solutions in the preparation of products with one or more functions in the screening, diagnosis, and adjuvant treatment of DNA methylation-related diseases. As one embodiment, the product described in this application can be a reagent and / or a kit. As one embodiment, the DNA methylation-related diseases described in this application can be, but are not limited to, cancer.

[0057] To further illustrate this application, the technical solutions provided by this application will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of this application.

[0058] The application of DNA methylation as a molecular marker in early cancer screening is one of its most promising applications. High-throughput sequencing-based methylation signal detection can simultaneously analyze a large number of CpG sites, significantly improving the sensitivity and reliability of early screening compared to single-site detection techniques based on quantitative PCR. Cell-free extracellular DNA (cfDNA) extracted from samples such as plasma and urine is often used for methylation signal detection. These samples often present challenges to experimental techniques due to their varying DNA volume and fragment integrity. Conventional detection procedures, including bisulfite conversion and enzymatic conversion methods, involve DNA conversion steps, resulting in significant sample loss, increased experimental difficulty, and a lower sensitivity limit. Furthermore, to reduce sequencing and information analysis costs, specific target enrichment is required, further complicating the experimental process and reducing stability.

[0059] The following uses plasma cfDNA as an example, employing the methylation-specific library preparation method and bisulfite conversion method of this application to prepare high-throughput sequencing libraries in single-strand library preparation mode.

[0060] Example 1

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

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

[0063] II. The library preparation steps are as follows:

[0064] 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. Adopt The single-stranded library preparation kit (for Illumina) was used to prepare the first ligation reaction system on ice according to the system shown in Table 1. The P7 Buffer, P7 adapter, and P7 Enzyme Mix in Table 1 were thawed on ice and mixed beforehand. The P7 Adapter is one form of the "first adapter".

[0066] Table 1 First Connection Reaction System

[0067] 3. Using a pipette, gently pipette the ligation reaction mixture from step 2 to mix thoroughly. Briefly centrifuge to ensure all reaction solution is at the bottom of the PCR tube. Place the PCR tube into the PCR instrument and start the program in Table 2. Once the temperature has stabilized at 37°C, place the reaction tube into the PCR instrument.

[0068] Table 2 First Connection Reaction Procedure

[0069] 4. Prepare the double-stranded DNA synthesis reaction system according to Table 3:

[0070] Table 3. Double-stranded DNA Synthesis Reaction System

[0071] Use a pipette to gently mix the combined double-stranded DNA synthesis reaction mixture thoroughly, and then briefly centrifuge to ensure all reaction solution is at the bottom of the PCR tube. Place the PCR tube into the PCR instrument and start the program in Table 4.

[0072] Table 4. Procedure for Double-Stranded DNA Synthesis

[0073] 5. Use The product obtained in step 4 of SP Beads purification was eluted with 17 μL.

[0074] 6. After preparing the first enzyme digestion reaction system according to Table 5, digest at 37°C for 10 min, wherein FastDigest Msp I in Table 5 is a form of the first restriction endonuclease in this application.

[0075] Table 5. First Enzyme Digestion Reaction System

[0076] 7. Use SP Beads purification step 6 product, eluted with 23 μL.

[0077] 8. Prepare the second ligation reaction system according to Table 6. The P5-2 linker is a form of the "second linker" described above, which is derived from the annealing of two oligonucleotide single strands. 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), neither of which has a 5' modification.

[0078] Table 6 Second Connection Reaction System

[0079] Start the second ligation reaction program in Table 7, and when the temperature stabilizes at 20°C, place the reaction tube into the PCR instrument to perform the ligation reaction.

[0080] Table 7 Second Connection Reaction Procedure

[0081] 9. Use SP Beads purification step 8 product, eluted with 17 μL.

[0082] 10. After preparing the second restriction enzyme digestion 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 this application.

[0083] Table 8 Second Enzyme Digestion Reaction System

[0084] 11. Use SP Beads purification step 10 product, eluted with 20 μL.

[0085] 12. Prepare the PCR amplification reaction system according to Table 9.

[0086] Table 9 PCR amplification reaction system

[0087] PCR amplification will be performed according to the amplification reaction system prepared in Table 9. The PCR amplification procedure is shown in Table 10.

[0088] Table 10 PCR Amplification Procedure

[0089] 13. Use The PCR amplification product from step 12 was purified using SP Beads, and 20 μL was eluted to obtain the library ready for sequencing.

[0090] 3. Sequencing the library obtained in step 2 on the Illumina Novaseq 6000 platform in PE150 mode.

[0091] Comparative Example 1

[0092] The methylation library preparation method and DNA methylation detection method based on bisulfite conversion are as follows:

[0093] 1. Sample: 10 ng of cfDNA sample from the same plasma source as in Example 1.

[0094] II. A methylation library preparation method based on bisulfite conversion, the steps of which are as follows:

[0095] The preparation of methylated libraries based on bisulfite conversion was carried out using... Methylation Library Construction Kit (Nanoda) DNA methylation-bisulfite conversion module (Naonda) was used. Hybridization capture was performed using a 60k methylation demo panel (Naonda) and Hybrid Capture Reagents (Naonda). All steps were performed according to the instruction manual.

[0096] 3. Sequencing the library obtained in step 2 on the Illumina Novaseq 6000 platform in PE150 mode.

[0097] The sequencing data from Example 1 and Comparative Example 1 were both taken as 10Gb. The coverage depth of the CpG double bases in the Msp I restriction site was calculated using samtools software. Specifically, the aligned bam file and the BED file of the Msp I site were input into samtools software, and the depth was calculated using the samtools depth tool. The results are shown in Figure 2. In Figure 2, WGBS and the method described in this figure represent the library construction methods of Comparative Example 1 and Example 1, respectively.

[0098] Figure 2 shows that the average coverage depth of the Chinese library in Comparative Example 1 is approximately 3×, which cannot accurately reflect the methylation level of individual sites. The average coverage depth of the Chinese library in Example 1 is approximately 4 times that of Comparative Example 1. Because fragments are repeatedly cleaved when Msp I restriction sites are close together, they are difficult to preserve, resulting in extremely low coverage depth. Therefore, if only sites with a spacing greater than 200 bp are calculated, the sequencing depth of the library construction method in Example 1 is approximately 11 times that of Comparative Example 1, which can more accurately reflect the methylation level of individual sites. There are over one million Msp I restriction sites on the human genome that meet the criteria; direct sequencing of the library obtained in Example 1 can yield a large number of sites whose methylation levels can be accurately analyzed.

[0099] To obtain high-coverage methylation sequencing data for calculating single CpG site methylation levels, the library preparation method in Comparative Example 1 needs to be combined with targeted capture. Using a 60kb panel (TM500), (The demo panel accompanying the methylation library construction kit #1002501 is provided free of charge to researchers testing the effectiveness of Example 1.) After capture, 1 Gb of sequencing data was taken, with an average coverage depth of approximately 10000× for the targeted capture region, which was 350× after deduplication. The CpG methylation level was calculated using MethylDackel software, and the CpG that also served as Msp I restriction sites was compared with the library construction method in Example 1.

[0100] In Example 1, the CpG methylation level of the library construction method was reflected by the number of reads at Msp I sites. As shown in Figures 3 and 4, the results obtained by combining the library construction and sequencing method in Example 1 with the conventional method and the library construction method in Comparative Example 1 with targeted capture are respectively presented in Figures 3 and 4. Figure 3 shows the comparison results of all Msp I sites within the hybridization capture target region of the bisulfite-transformed library in Comparative Example 1, and Figure 4 shows the comparison results of 573 sites within the hybridization capture target region of the bisulfite-transformed library in Comparative Example 1 after excluding nearby Msp I sites.

[0101] Based on Figures 3 and 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. At 776 sites, R... 2 The value was 0.763, while at 573 sites with an interval of more than 200 bp adjacent to the Msp I site, R... 2 It reached 0.9.

[0102] The following uses lambda DNA and pUC19 plasmid DNA as examples to demonstrate the preparation of methylation-specific high-throughput sequencing libraries in double-stranded library construction mode using the library construction method of this application.

[0103] Example 2

[0104] A method for constructing a high-throughput sequencing library enriched with methylated DNA and a DNA detection method are provided, with the following steps:

[0105] Mix 10 ng of Lambda DNA (0% methylated, from Promega, D1521) and 10 ng of puc19 plasmid (100% methylated, from Zymo Research, D5017), and divide into two portions of 10 ng each.

[0106] use Rapid DNA restriction enzyme digestion library construction kit v2 (Nanoda), after fragmentation, end repair & A addition.

[0107] The connector connection steps will Rapid DNA Enzyme Digestion Library Construction Kit v2 (Nanoda) The Universal Stubby Adapter is replaced with a connector with a positive chain of 5'-P-GATCGGAAGAGCACAC GTCTGAACTCCAGTCAC-3' (SEQ ID NO:3) and a negative chain of 5'-AACAGACGTGTG CTCTTCCGATCT-3' (SEQ ID NO:4), which is one form of the first connector.

[0108] After purification of the ligation product, proceed according to steps 6-9 in Example 1.

[0109] After completing step 9, one of the copies continues to build the library according to steps 10-13 in Example 1, generating libA;

[0110] Another sample skipped steps 10-11 in Example 1 and directly followed steps 12-13 in Example 1, i.e., did not perform the second enzyme digestion treatment, and produced libB.

[0111] Both libraries were sequenced on an Illumina Novaseq 6000 in PE150 mode.

[0112] The data from libA and libB sequencing were analyzed as follows: bwa was used to align the sequencing data to a reference genome containing the human genome, Lambda DNA sequence, and puc19 sequence to obtain a bam file. Then, samtools was used to count the reads. The results are shown in Figure 5.

[0113] As shown in Figure 5, in the libB library without the second enzyme digestion, the reads of Lambda DNA and puc19 plasmid accounted for 65% and 35%, respectively. Although they were mixed in a 1:1 ratio, the number of restriction sites differed, so the ratios were not exactly 50% and 50%. After the second enzyme digestion, the proportion of Lambda DNA decreased to less than 1%, demonstrating the specificity of the proposed method for methylated fragment library construction.

[0114] From the above embodiments, it can be concluded that the construction method described in this application can achieve selective library construction of methylated CpG, and the constructed library can be directly sequenced in high-throughput without the need for cumbersome base conversion and targeted enrichment steps.

[0115] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.

Claims

1. A method for constructing a high-throughput sequencing library enriched for methylated DNA, characterized in that, The method comprises the following steps: 1) connecting a first adaptor to one end of DNA to be constructed, to obtain a first ligation product; 2) performing first enzyme digestion on the first ligation product with a first restriction endonuclease, to obtain a first enzyme digestion product containing the first adaptor, wherein the enzyme digestion site of the first restriction endonuclease contains a CpG site and the first restriction endonuclease is not sensitive to 5mC; 3) connecting a second adaptor to the first enzyme digestion product containing the first adaptor, to obtain a second ligation product, wherein the second adaptor is complementary to the enzyme digestion end of the first enzyme digestion product containing the first adaptor, and the second ligation product contains a recognition sequence of a second restriction endonuclease; 4) performing second enzyme digestion on the second ligation product with the second restriction endonuclease, to obtain a second enzyme 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, wherein the upstream primer and the downstream primer of the primer pair respectively contain an adaptor; and the adaptor comprises the first adaptor and / or the second adaptor.

2. The construction method of claim 1, wherein, The nucleotide sequences of the first adaptor and the second adaptor are different.

3. The construction method of claim 1, wherein, The DNA to be constructed comprises extracellular free DNA and / or fragmented DNA.

4. The construction method of claim 1, wherein, The ligation in steps 1) and 3) comprises double-stranded DNA ligation or single-stranded DNA ligation.

5. The construction method of claim 1, wherein, The first restriction endonuclease comprises one or more of Msp I, 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 by, The method comprises: constructing a high-throughput sequencing library enriched in methylated DNA from a sample to be detected according to the construction method of any one of claims 1 to 6; performing high-throughput sequencing based on the high-throughput sequencing library enriched in methylated DNA, to obtain high-throughput sequencing data; performing bioinformatics analysis on the high-throughput sequencing data, to obtain a DNA methylation detection result.

9. The detection method according to claim 8, characterized in that, The adaptors used in the process of constructing the high-throughput sequencing library enriched in methylated DNA are matched with the sequencing platform of the high-throughput sequencing.

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

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