Construction method and application of methylation target region capture sequencing library based on modified Tn5

Abstract

The invention discloses a construction method and application of a methylation target area capture sequencing library based on modified Tn5, the construction method is based on transposon characteristics of Tn5 transposons, modified sequencing joints with double-end barcodes are directly embedded into Tn5 transposase, and a batch of Tn5 transposons with different sequence tags are formed; different Tn5 transposons are used for fragmenting a plurality of samples, so that different samples can carry different sequence tags, the purpose of distinguishing the samples is achieved, mixing of a plurality of low-initial-quantity samples is achieved, and a sequencing result of a capture sequence is finally obtained by processing the mixed library. Compared with a traditional methylation target area capturing method, the method has the advantages that a large number of samples can be detected in batches without any complex and precise instrument, and the limitation of a conventional methylation capturing and sequencing method in the aspect is overcome. In addition, according to the method, a low-initial-quantity sample can be converted into a high-quality to-be-sequenced sample only in one day.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and in particular to a method for constructing a methylation target region capture sequencing library based on modified Tn5 and an application thereof. Background Art

[0002] DNA methylation is an important epigenetic modification that selectively adds methyl groups to specific bases through DNA methyltransferases to form 5-methylcytosine (5-mC) and other small amounts of methylated structures. This chemical modification can change genetic expression without changing the DNA sequence, and plays an important role in maintaining normal cell function, chromatin structure modification, embryonic development, and human tumorigenesis. There are certain differences in DNA methylation levels in different cell types and tissues, and this difference can affect gene expression and function. Therefore, researchers integrate DNA methylation with other omics content in order to reveal the molecular mechanisms of disease occurrence and discover potential early screening markers.

[0003] In recent years, various types of cancer have become increasingly threatening to human health. Compared with normal cells, the overall genomic methylation level of cancer cells is relatively low, but the promoter region of their genes shows a high methylation trend. Human genome research has shown that the high methylation level of the promoter region of tumor suppressor genes can inhibit the expression of tumor suppressor genes by changing the chromatin structure, DNA conformation and DNA-protein interaction. After losing the tumor suppressor function, cells will re-enter the growth cycle, eventually leading to the formation of tumors.

[0004] At present, people often prevent the occurrence of tumors by detecting changes in characteristic methylation structures in certain cancer cells or tissues. The main methods for DNA methylation research include bisulfite sequencing, restriction endonuclease sequencing, and affinity enrichment. Among them, bisulfite sequencing is currently recognized as the "gold standard" for DNA methylation sequencing. The unmethylated C on the genome of DNA treated with bisulfite will be converted into thymine (T), while the methylated C remains unchanged. After PCR amplification, the product is sequenced and the methylation sites in the DNA sequence can be determined by comparing the sequencing results with the untreated sequence.

[0005] Although whole-genome bisulfite sequencing can achieve high-resolution and comprehensive methylation pattern detection, it is not suitable for some samples due to the high input DNA amount during library construction.

[0006] Therefore, it is necessary to design a method for constructing a methylation capture sequencing library that is suitable for sequencing low starting amounts of DNA and meets the requirements of methylation capture. Summary of the invention

[0007] The present invention provides a method for constructing a methylation target region capture sequencing library based on modified Tn5 and its application, so as to be applicable to the sequencing of low starting amount of DNA and meet the requirements of methylation capture.

[0008] In view of this, the scheme of the present invention is:

[0009] The first aspect of the present invention is to provide a method for constructing a methylation capture sequencing library, the steps comprising:

[0010] The sequencing adapter with double-end tags is embedded in the Tn5 transposase to construct the Tn5 transposon;

[0011] The assembled Tn5 transposon is used to fragment the genomic DNA of several samples respectively, to obtain fragmentation products of each sample containing different tags;

[0012] Perform end-filling and repair on the fragmentation products of each sample;

[0013] The repaired products were mixed in equal amounts, and blocking primers were added for hybridization;

[0014] The product is subjected to a conversion treatment to convert the unmethylated cytosine into uracil;

[0015] The converted DNA was amplified by PCR to obtain a methylation-captured sequencing library.

[0016] Furthermore, the sequencing adapter includes a first adapter with a nucleotide sequence as shown in SEQ ID NO: 1, a second adapter with a nucleotide sequence as shown in SEQ ID NO: 2, and a third adapter with a nucleotide sequence as shown in SEQ ID NO: 3; the base C in the second adapter and the third adapter are both methylated.

[0017] Preferably, it is characterized in that the Tn5 transposon assembly process uses a first primer and a second primer, and all base Cs in the primers are methylated; the first primer is selected from any one of the nucleotide sequences shown in SEQ ID NOs: 4-6, and the second primer is selected from any one of the primers with nucleotide sequences shown in SEQ ID NOs: 7-14.

[0018] Furthermore, the sample is fragmented and then terminated using SDS, followed by the addition of a neutralizing reagent to eliminate SDS.

[0019] Furthermore, the fragmented product repair process uses Klenow enzyme, T4 polymerase and four bases to fill in the ends, and then uses Klenow enzyme without 3'-5' exo-cutting activity to repair.

[0020] Furthermore, the hybridization process is to concentrate the mixed repair product into dry powder, add blocking primers, probes and hybridization buffer, and then perform hybridization. After the hybridization is completed, streptavidin magnetic beads are used for adsorption and washed with an elution reagent to remove excess fragments and redundant impurities.

[0021] Furthermore, during the PCR amplification, primers with nucleotide sequences as shown in SEQ ID NOs: 15-16 are added.

[0022] The second aspect of the present invention is to provide a methylation capture sequencing library obtained by the construction method.

[0023] The third aspect of the present invention is to propose the use of the methylation capture sequencing library described in the second aspect in the detection of methylation of non-diagnostic target genes.

[0024] A fourth aspect of the present invention is to provide a sequencing method, comprising the following steps:

[0025] A methylation capture sequencing library is prepared according to the construction method described in the first aspect;

[0026] Sequencing the methylation capture sequencing library to obtain sequencing data;

[0027] The sequencing data is compared with the reference genome data, and the methylation sites of the genomic DNA of each sample are obtained based on the comparison information and the labels of each library.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The library construction method of the present invention is based on the transposition characteristics of Tn5 transposon, and directly embeds the modified sequencing adapter with double-end barcode into Tn5 transposase to form a batch of Tn5 transposons with different sequence tags; using different Tn5 transposons to fragment multiple samples can make different samples carry different sequence tags, so as to achieve the purpose of distinguishing samples, realize mixed sequencing of multiple low-starting amount samples, and greatly improve the experimental throughput; compared with the traditional whole genome methylation sequencing method, the advantage of the present invention is that it does not require any complex and precise instruments, and conventional experimental equipment can complete all operations, and it only takes 1 day to convert low-starting amount samples into high-quality samples to be sequenced.

[0030] The present invention can process samples with a starting DNA amount of 500ng or even 400ng, which has significant advantages for experimental samples with a small total DNA amount. In addition, the method requires a low starting amount of DNA, with an average capture specificity of more than 90% and a call rate of 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure is a flowchart of library construction for Tn5-based whole-genome methylation capture sequencing of the present invention.

[0032] Figure 2 This is a diagram of the structure of a single sample library of the present invention.

[0033] Figure 3 This is the electrophoresis diagram after 500 ng gDNA was digested with different enzyme amounts and electrophoresed on 1% agarose gel after the digestion was terminated.

[0034] Figure 4 This is a peak graph of DNA after enzyme shearing, end repair and sorting using Angilent 5300 quality inspection in an example of the present invention.

[0035] Figure 5 In one embodiment of the present invention, the mixed library after PCR enrichment and purification is subjected to a peak graph after quality inspection using Q-seq. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In order to make the experimental methods, technical solutions and advantages and disadvantages in the embodiments of the present invention clearer, the specific experimental steps in the embodiments of the present invention are described in detail and completely below. If the manufacturer of the reagent or instrument is not indicated in the embodiments, conventional products can be purchased from the market. If the specific conditions are not indicated, they can be carried out according to conventional conditions or conditions recommended by the manufacturer.

[0038] Related reagent list:

[0039]

[0040]

[0041] In one embodiment, a method for constructing a DNA methylation capture sequencing library is provided, such as Figure 1 As shown, specifically including:

[0042] 1. Transposome preparation:

[0043] The present invention uses the independently developed Tn5 enzyme, embeds the sequencing adapter with barcode into the transposase to form a transposome. Each sample corresponds to a pair of different double-end barcodes. In specific implementation, the same number of TN5s can be constructed and used according to the actual sample demand. The transposon adapter sequence is shown in Table 1.

[0044] Table 1:

[0045]

[0046] Among them, NNNNNNNN represents an 8-base barcode, the degenerate base N represents A / T / C / G, and the C bases in P5 and P7 need to be methylated.

[0047] 2. DNA fragmentation: Use Tn5 transposon with double-end barcode to fragment DNA, end-repair and connect adapters;

[0048] 3. Enzyme digestion termination and neutralization: Use SDS to terminate the previous step of the disruption and linker reaction, and then add the neutralization reaction reagent to eliminate the effect of SDS on the protein;

[0049] 4. End repair: fill the gap and extend to a complete double-stranded DNA structure;

[0050] 5. Product sorting: Use magnetic beads for sorting and purification;

[0051] 6. Mixing, hybridization and elution: Mix equal amounts of up to 48 libraries, add blocking primers and hybridize overnight, and use elution reagent to remove excess fragments and impurities;

[0052] 7. Bisulfite conversion: convert unmethylated C to U;

[0053] 8. PCR enrichment: Use amplification primers to enrich and convert uracil U in DNA into thymine T;

[0054] 9. PCR product purification: Magnetic beads are used for purification and library extraction. The structure diagram of a single sample library is shown in the figure below. Figure 2 shown.

[0055] Example

[0056] In this example, methylation capture sequencing was performed on 24 DNA samples, and all steps from transposome preparation to sample mixing were performed in a 96-well plate.

[0057] The specific steps of the second-generation sequencing method in this example are as follows:

[0058] 1.24 Preparation of transposons

[0059] (1) It includes embedding the sequencing adapter with double-end barcode into Tn5 transposase to form Tn5 transposon. The specific reaction system is: 40ul of 1.5ug / ul transposase stock solution, 20ul of 100uM annealed adapter (P5\P7 equal mixture), totaling 60ul.

[0060] (2) The specific linker sequences are shown in Table 2.

[0061] Table 2: Linker sequences

[0062]

[0063]

[0064] Among them, the eight bases in the underlined part are barcodes, and the base C in all sequences is methylated.

[0065] (3) The positions in the 96-well plate are shown in Table 3.

[0066] Table 3:

[0067] Hole pattern P5-P501 P5-P502 P5-P503 P7-P701 A1 A2 A3 P7-P702 B1 B2 B3 P7-P703 C1 C2 C3 P7-P704 D1 D2 D3 P7-P705 E1 E2 E3 P7-P706 F1 F2 F3 P7-P707 G1 G2 G3 P7-P708 H1 H2 H3

[0068] (4) After the above reaction system is fully mixed, the temperature is kept at 25°C for 1 hour to obtain 24 Tn5 transposons with different double-end barcodes, which are then stored at -20°C for future use.

[0069] 2. Fragmentation

[0070] That is, the DNA sample processing step includes using the prepared Tn5 transposon to process the DNA sample to be tested. Only through one-step processing, the fragmented, end-repaired and adapter-connected fragmented DNA samples can be obtained, wherein Lambda-DNA does not contain methylated C, which is used for the subsequent evaluation of the sample conversion rate. The specific reaction system is shown in Table 4.

[0071] Table 4:

[0072]

[0073]

[0074] After the above reaction system is fully mixed, it is placed on a PCR instrument and the program shown in Table 5 is run.

[0075] Table 5:

[0076]

[0077] 3. End of enzyme digestion

[0078] That is, the Tn5 enzymatic reaction termination step includes adding the termination reaction solution to the fragmented reaction product to terminate the Tn5 enzymatic reaction. Specifically, SDS is added to the reaction system after being kept at 55°C for 10 minutes in "2. Fragmentation", and the reaction is terminated after being fully mixed and placed at room temperature for 10 minutes.

[0079] 4. Neutralization

[0080] Add tritomx to the reaction solution in step 3, gently invert to mix, then centrifuge, let stand for 10 minutes before proceeding to the next step.

[0081] Different enzyme amounts of 50pmol, 60pmol, 70pmol, 80pmol, and 90pmol were used to cut 500ng gDNA. After the enzyme cutting was terminated, the electrophoresis was performed on 1% agarose. The electrophoresis diagram is as follows: Figure 3 shown.

[0082] 5. End repair

[0083] That is, the Klenow (exo) enzyme with strand displacement function is used to fill the 9bp gap after the restriction digestion reaction and extend to the complete DNA double strand. Among the four bases used for repair, the C base is a methylated C base to ensure that the subsequent sulfite treatment will not affect the adapter sequence. The specific reaction system is shown in Table 6.

[0084] Table 6:

[0085]

[0086]

[0087] After the above reaction system is fully mixed, it is placed on a PCR instrument and the program described in Table 7 is run.

[0088] Table 7:

[0089]

[0090] Fragment sorting was performed immediately after the program was completed.

[0091] 6. Product sorting

[0092] 1) Take out the kapa beads from the 4℃ refrigerator in advance, let them stand to equilibrate to room temperature, and vortex or shake to mix before use.

[0093] 2) After the PCR program is completed, add 45ul of kapa beads directly into the 96-well plate and mix well by pipetting.

[0094] 3) After standing at room temperature for 10 minutes, transfer the 96-well plate to a magnetic rack, absorb the magnet for 10 minutes, and then transfer the supernatant to a new 96-well plate.

[0095] 4) Add 55ul of kapa beads to the 96-well plate in step 6.3 and mix well by pipetting.

[0096] 5) After standing at room temperature for 10 minutes, transfer the 96-well plate to a magnetic rack and magnetize for 10 minutes before discarding the supernatant.

[0097] 6) Keep the 96-well plate on the magnetic stand, add 200ul 80% ethanol, let stand for 30s, and discard all supernatant.

[0098] 7) Repeat step 6.6 once.

[0099] 8) Keep the 96-well plate on the magnetic stand and use a 10ul pipette to remove the ethanol remaining at the bottom of the tube.

[0100] 9) Dry the magnetic beads for 2-3 minutes. After the alcohol evaporates completely (the color of the magnetic beads changes from bright brown to matte brown), add 22ul DEPC water and mix by pipetting.

[0101] 10) Let stand at room temperature for 5 minutes and place on a magnetic rack for 5 minutes until the solution becomes clear. Carefully pipette 20 μL of the supernatant into another 96-well plate for later use.

[0102] Figure 4 This is the peak graph after DNA is digested, end-repaired and sorted, and is quality-checked using Angilent 5300.

[0103] 7. Target area capture

[0104] That is, a probe with biotin is used to capture the functional sites of the sample to be tested, and then streptavidin magnetic beads are used to adsorb the target DNA. It mainly includes four steps: library mixing, hybridization, magnetic bead binding and elution.

[0105] 1) Library mixing

[0106] Mix the prepared 24 libraries in equal amounts of 50 ng per sample into a new PCR tube to form a mixed library. Add cot DNA and Universal Hybridization Blockers library blocking mixture to the PCR tube according to Table 8, vortex or shake to mix, centrifuge briefly to collect the liquid to the bottom of the tube, place in a concentrator to concentrate into dry powder, and save for later use.

[0107] Table 8:

[0108]

[0109] 2) Library hybridization

[0110] Take out the Hybridization Buffer and place it at room temperature to thaw. At the same time, take out the probe and place it on ice to thaw. Use a pipette tip to mix well, centrifuge briefly, and place it on ice for later use.

[0111] In step 1), 15ul of Hybridization Buffer and 2ul of Wild type were added to the PCR tube in sequence. The mixture was mixed by pipetting with a pipette tip and then centrifuged. The liquid was collected at the bottom of the tube and incubated at room temperature for 5-10 minutes. The tube was then placed on a PCR instrument and the program shown in Table 9 was run to complete hybridization capture.

[0112] Table 9:

[0113]

[0114]

[0115] 3) Magnetic bead binding

[0116] Take out the streptavidin magnetic beads and equilibrate them at room temperature for at least 30 minutes.

[0117] Take out the Hybridization Buffer and 1× Capture Beads Wash Buffer and place them at room temperature to dissolve until no crystals appear.

[0118] Vortex the streptavidin beads to mix well and aliquot 20ul of M270 beads into a new PCR tube for later use.

[0119] Add 100ul 1×Capture Beads Wash Buffer, mix by pipetting, and centrifuge briefly to collect the liquid at the bottom of the tube. Place the tube on a magnetic rack for 2-3 minutes until the solution is clear, discard the supernatant, and repeat this step three times.

[0120] Take out the PCR tube, add 15ul Hybridization Buffer and 2ul DEPC water, and use a pipette to gently mix the magnetic beads to avoid generating bubbles.

[0121] After the 16-18 h hybridization procedure was completed, the elution procedure shown in Table 9 was set on another PCR instrument.

[0122] Table 10:

[0123]

[0124] Preheat the resuspended M-270 magnetic beads in the above PCR instrument for 5 minutes.

[0125] Quickly transfer all the hybridization reaction solution from step 2) to the preheated M-270 magnetic beads, mix gently 10-15 times with a pipette, and continue incubating on the PCR instrument for 45 minutes. Mix 10 times with a pipette every 15 minutes to ensure sufficient contact between the magnetic beads and the DNA.

[0126] 4) Library elution

[0127] Take out Wash buffer S, Wash buffer Ⅰ, Wash buffer Ⅱ, and Wash buffer Ⅲ and place them at room temperature to melt until no crystals appear. The main components of Wash buffer Ⅰ, Wash buffer Ⅱ, and Wash buffer Ⅲ are SDS, NaCl, and sodium citrate.

[0128] Gently vortex the buffer to mix thoroughly and minimize the generation of bubbles.

[0129] Before hot elution, preheat the above-prepared Wash buffer S and Wash buffer I at 68°C and 65°C in a PCR instrument for at least 15 minutes, respectively.

[0130] Step 3) After the 45-minute incubation, add 100 ul of 65°C preheated Wash buffer I to the PCR tube and mix gently using a pipette.

[0131] Place the PCR tube on a magnetic stand for 1-2 minutes, wait for the solution to clarify, and discard the supernatant.

[0132] Take out the PCR tube, add 150ul of Wash buffer S preheated at 68℃, mix gently with a pipette, put it in the PCR instrument and incubate at 68℃ for 5 minutes. After incubation, place it on the magnetic stand for 1-2 minutes, wait for the solution to clarify, discard the supernatant, and then repeat this step once.

[0133] Take out the PCR tube, add 150ul of room temperature Wash buffer I, mix gently with a pipette, incubate at room temperature for 2 minutes, and vortex to mix every 30 seconds.

[0134] After a brief centrifugation, place the PCR tube on a magnetic rack for 1 minute, wait for the solution to clarify, and discard the supernatant.

[0135] Take out the PCR tube, add 150ul of room temperature Wash buffer II, mix gently with a pipette, incubate at room temperature for 2 minutes, and vortex mix every 30 seconds.

[0136] After a brief centrifugation, place the PCR tube on a magnetic rack for 1 minute, wait for the solution to clarify, and discard the supernatant.

[0137] Take out the PCR tube, add 150ul of room temperature Wash buffer III, mix gently with a pipette, incubate at room temperature for 2 minutes, and vortex mix every 30 seconds.

[0138] After a brief centrifugation, place the PCR tube on a magnetic stand for 1 minute. Wait for the solution to become clear, discard the supernatant, and use a small pipette tip to remove a small amount of residual liquid.

[0139] Take out the PCR tube, add 15ul 0.1M NaOH to the tube, mix gently with a pipette, and incubate at room temperature for 15min.

[0140] After incubation, place the PCR tube on a magnetic stand for 5-10 minutes, transfer the supernatant to a new 200ul PCR tube, and add 2.5ul of diluted concentrated hydrochloric acid for neutralization. Then add water to 20ul and proceed to the next step. The main purpose of this step is to dissociate DNA from streptavidin with a strong base and neutralize with hydrochloric acid to avoid adverse effects on bisulfite conversion due to the pH value of the solution.

[0141] 8. Bisulfite Conversion

[0142] That is, under the action of bisulfite, cytosine (cytosine C) in DNA is converted into uracil, while 5-methylcytosine (5mC) remains unchanged. The present invention uses EpiArt DNA MethylationBisulfite Kit (Vazyme, EM101-02) for bisulfite conversion.

[0143] Add 130 μl of freshly prepared CT Conversion Mix to the PCR tube in step 7, mix thoroughly by pipetting, and place on a PCR instrument to run the program shown in Table 11.

[0144] Table 11:

[0145]

[0146] After the program is completed, use EpiArt DNA Methylation Bisulfite Kit (Vazyme, EM101-02) for deamination, and finally add 21ul DEPC water to the EpiArt DNA Column for remelting. The output obtained is the mixed library after transformation.

[0147] 9. PCR Enrichment

[0148] Transfer all the mixed libraries obtained in the previous step to a new 200ul PCR tube, add 25ul 2×HIFIMIX and 5ul 4uM PPM-1 and PPM-2 mixture, the sequence is as follows:

[0149] PPM-1: 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 15)

[0150] PPM-2: 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 16)

[0151] Mix well with a pipette tip, centrifuge briefly, and place in a PCR instrument to run the program shown in Table 12.

[0152] Table 12:

[0153]

[0154] Here, the number of PCR enrichment cycles is related to the hybridization input amount and the number of probes. In actual operation, the number of cycles can be adjusted accordingly.

[0155] 10. PCR product purification

[0156] Take out the kapa beads from the 4℃ refrigerator in advance, let them stand for 30 minutes to equilibrate to room temperature, and vortex or shake to mix before use.

[0157] After the PCR program is completed, add 60ul of kapa beads directly into the PCR tube, mix well by pipetting, and let stand at room temperature for 10 minutes.

[0158] Place the PCR tube on a magnetic rack and discard the supernatant after magnetic absorption for 10 minutes.

[0159] Keep the PCR tube on the magnetic rack, add 200ul 80% ethanol, let it stand for 30s, and discard all the supernatant.

[0160] Repeat the above steps once.

[0161] Keep the PCR tube on the magnetic stand and use a 10ul pipette to remove the ethanol remaining at the bottom of the tube.

[0162] Dry the magnetic beads for 2-3 minutes. After the alcohol evaporates completely (the color of the magnetic beads changes from bright brown to matte brown), remove the PCR tube and add 15ul DEPC water, pipette to mix, and let stand at room temperature for 5 minutes.

[0163] Place the PCR tube on the magnetic rack for 5 minutes until the solution becomes clear, and carefully pipette the supernatant into another new 1.5 ml tube.

[0164] Take 1ul of library and use Qubit dsDNA HS Assay Kit for quantification and record the library concentration.

[0165] Take 1ul sample and dilute to 2ng / ul. Use Agilent 5300 Bioanalyzer system (High Sensitivity DNA Kit) or Q-seq to measure the length of library fragments. The length of the library is about 330bp. Figure 5 shown.

[0166] 11. After quality inspection, the DNA was sequenced using the Illumina sequencing platform.

[0167] 12. The sequencing results were analyzed on the machine. The results are shown in Table 13.

[0168] Table 13:

[0169]

[0170]

[0171] (1) Methyl conversion rate (BST)

[0172] As can be seen from Table 13, the methyl conversion rates of samples T1 to T24 are all between 96% and 98%. Such data indicate that 96% to 98% of the C bases that were not methylated at the CA, CT, and CC positions were converted from C to U after sulfite treatment, and the corresponding bases read by sequencing were all T. The BST calculation method is to count the total number of C bases at the CA, CT, and CC positions after sulfite conversion and the total number of C bases at the CA, CT, and CC positions that were not changed after sulfite treatment in the sequencing results. The sum of the methylated C bases divided by the total C bases is the methylation rate, and the methylation rate minus 1 is the BS treatment conversion rate.

[0173] (2) Hybridization capture rate

[0174] The data in Table 13 show that the hybridization capture rates of samples T1 to T24 are all above 90%, which shows that the method of hybridization followed by transformation adopted in the present invention is completely feasible, and the fragment breakage and fragment loss caused by bisulfite have little effect on the hybridization library.

[0175] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a methylation capture sequencing library, characterized in that the steps include: The sequencing adapter with double-end tags is embedded in the Tn5 transposase to construct the Tn5 transposon; The assembled Tn5 transposon is used to fragment the genomic DNA of several samples respectively, to obtain fragmentation products of each sample containing different tags; Perform end-filling and repair on the fragmentation products of each sample; The repaired products were mixed in equal amounts, and blocking primers were added for hybridization; The product is subjected to a conversion treatment to convert the unmethylated cytosine into uracil; The converted DNA was amplified by PCR to obtain a methylation-captured sequencing library.

2. The construction method according to claim 1, characterized in that: The sequencing adapter includes a first adapter with a nucleotide sequence as shown in SEQ ID NO: 1, a second adapter with a nucleotide sequence as shown in SEQ ID NO: 2, and a third adapter with a nucleotide sequence as shown in SEQ ID NO: 3; the base C in the second adapter and the third adapter are both methylated.

3. The construction method according to claim 2, characterized in that: The Tn5 transposon assembly process uses a first primer and a second primer, and all base Cs in the primers are methylated; the first primer is selected from any one of the nucleotide sequences shown in SEQ ID NOs: 4-6, and the second primer is selected from any one of the primers with nucleotide sequences shown in SEQ ID NOs: 7-14.

4. The construction method according to claim 1, characterized in that: After the sample fragmentation, SDS was used to terminate the fragmentation, and then a neutralizing reagent was added to eliminate SDS.

5. The construction method according to claim 1, characterized in that: The fragment product repair process uses Klenow enzyme, T4 polymerase and four bases to fill in the ends, and then uses Klenow enzyme without 3'-5' exonuclease activity to repair.

6. The construction method according to claim 1, characterized in that: The hybridization process is to concentrate the mixed repair products into dry powder, add blocking primers, probes and hybridization buffer, and then perform hybridization. After the hybridization is completed, streptavidin magnetic beads are used for adsorption and washed with an elution reagent to remove excess fragments and redundant impurities.

7. The construction method according to claim 1, characterized in that: During the PCR amplification, primers with nucleotide sequences as shown in SEQ ID NO: 15-16 are added.

8. The methylation capture sequencing library obtained by the construction method according to any one of claims 1 to 7.

9. Use of the methylation capture sequencing library according to claim 8 in non-diagnostic target gene methylation detection.

10. A sequencing method, characterized in that: The following steps are involved: A methylation capture sequencing library is prepared according to the construction method according to any one of claims 1 to 7; Sequencing the methylation capture sequencing library to obtain sequencing data; The sequencing data is compared with the reference genome data, and the methylation sites of the genomic DNA of each sample are obtained based on the comparison information and the labels of each library.

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