CN113550013B - Method for rapidly constructing RRBS sequencing library by using formalin-fixed paraffin embedded sample - Google Patents

Abstract

The invention discloses a method for quickly constructing an RRBS sequencing library by using a formalin-fixed paraffin embedded sample, belonging to the field of molecular biology and comprising the following steps: taking a paraffin-embedded tissue sample to obtain genome DNA; repair with end modifying enzyme plus ddATP; digesting the CpG island of the genome DNA by using restriction endonuclease; thirdly, carrying out end repair on the genome DNA by using end modification enzyme and adding A-Tailing at the 3' end; step four, carrying out methylation joint connection by using DNA ligase, and mixing the samples; converting the unmethylated cytosine by using bisulfite; sixthly, enriching the methylation library by PCR; seventhly, cutting and recycling the rubber; the method solves the problem of high failure Rate of RRBS (RRBS) for constructing the FFPE DNA library caused by formalin soaking in the prior art, and has wide application prospect and market value.

Description

Method for rapidly constructing RRBS sequencing library by using formalin-fixed paraffin embedded sample

Technical Field

The invention relates to the field of molecular biology, in particular to a method for quickly constructing an RRBS sequencing library by using formalin-fixed paraffin-embedded samples.

Background

DNA methylation is a chemical modification of DNA in eukaryotic organisms that can alter genetic behavior without altering the DNA sequence. DNA methylation usually occurs at CpG dinucleotides by the addition of a methyl group at the 5' carbon of cytosine by the action of methyltransferases. Methylation may be inherited by daughter cells from parent cells or may be synthesized de novo in the cells. The methylation degree is used as a reversible DNA modification mode and is involved in the regulation of gene expression, and abnormal hypermethylation or hypomethylation can cause various diseases, so that the methylation degree can be used as a detection standard for the health of cells and organisms.

Methylation of the genome can be sequenced by combining bisulfite conversion techniques with high throughput sequencing techniques of genomic DNA. Simplified representative methylation sequencing by enriching CpG island regions with methylation insensitive enzymes, followed by bisulfite conversion, sequencing, high resolution for single base detection and high utilization of sequencing data can be achieved.

Based on the characteristics of high sensitivity, multiple loci, tissue specificity and the like of a methylation signal, the gene can be widely applied to the fields of early screening of cancers, monitoring of curative effects, tissue tracing and the like in clinical practice. In clinical practice, paraffin embedding is a common means for preserving tissue samples, can preserve morphological characteristics of tissue cells for a long time, and is convenient for pathological diagnosis. However, in the formalin soaking process, the damage to the genomic DNA in the cell nucleus is large, so that the extracted genomic DNA has a large number of broken small fragments, the broken genomic DNA is connected with a linker and enters a library, a large amount of data waste is caused, and the enrichment effect of enzyme digestion on CpG islands is diluted. Therefore, the failure rate of constructing the FFPE DNA library by using the traditional RRBS is high, and the obstruction is brought to the clinical development and popularization of the methylation sequencing technology. The market needs a technology which can hinder the connection of genome DNA and a methylation joint, enlarge the effect of enzyme digestion enrichment CpG island region, simultaneously use the same buffer solution, simplify the operation process, reduce the cost of reagents and personnel, reduce the DNA loss caused by repeated purification, and easily convert the DNA loss into an automatic library construction process, and the invention solves the problems.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a method for quickly constructing an RRBS sequencing library by using a formalin-fixed paraffin-embedded sample.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for rapidly constructing an RRBS sequencing library by using a formalin-fixed paraffin-embedded sample comprises the following steps:

taking a paraffin-embedded tissue sample to obtain genome DNA; performing addatp repair on the end of the genomic DNA with an end modifying enzyme;

digesting the genome DNA by using restriction endonuclease;

step three, carrying out end repair on the genome DNA by using end modifying enzyme, adding A-Tailing at the 3 'end, and doping dATP into the 3' end of the blunt-ended DNA fragment;

step four, carrying out methylation joint connection on the DNA by using DNA ligase, and mixing samples marked by all the label sequences;

converting the unmethylated cytosine by using bisulfite, and immediately carrying out polymerase chain reaction amplification on the converted DNA or storing the DNA at-80 ℃;

sixthly, enriching the methylation library by PCR;

and seventhly, performing gel cutting recovery on the DNA fragment of 170-400bp of each mixed sample through gel electrophoresis, performing quality control and mixing on the library subjected to gel cutting recovery, and finally performing Illumina platform sequencing, wherein the sequencing sample loading quantity is subjected to cluster generation according to 60% density of the conventional Illumina library.

Further, the paraffin-embedded tissue sample in the first step is a tissue sample which is embedded in paraffin for less than 18 months.

Further, the terminal-modifying enzyme includes: a combination of one or more of Klenow Exo-enzyme, DNA polymerase.

Further, the restriction enzyme in the second step is one or more of MspI and HaeIII.

Further, the methylation joint in the fourth step is a Y-shaped double-stranded structure formed by putting two single-stranded complementary nucleic acids into a water bath at 98 ℃ for 5 minutes, closing the water bath, naturally cooling to room temperature of 25 ℃ and finishing annealing, wherein all cytosine nucleotides in the two single-stranded nucleic acids are methylated, and the 5' end of the nucleic acid at the bottom of the Y-shaped joint is subjected to phosphorylation treatment; the 3' end comprises a tag sequence consisting of 6 nucleotides.

Further, the methylated linker comprises: RRBS-1, RRBS-2, RRBS-3, RRBS-4, RRBS-5, RRBS-6, RRBS-7, RRBS-8, RRBS-9, RRBS-10, RRBS-11, RRBS-12; the upstream sequence of RRBS-1 is shown as SEQ01, and the downstream sequence is shown as SEQ13; the upstream sequence of RRBS-2 is shown as SEQ02, and the downstream sequence is shown as SEQ14; the upstream sequence of RRBS-3 is shown as SEQ03, and the downstream sequence is shown as SEQ15; the upstream sequence of RRBS-4 is shown as SEQ04, and the downstream sequence is shown as SEQ16; the upstream sequence of RRBS-5 is shown as SEQ05, and the downstream sequence is shown as SEQ17; the upstream sequence of RRBS-6 is shown as SEQ06, and the downstream sequence is shown as SEQ18; the upstream sequence of RRBS-7 is shown as SEQ07, and the downstream sequence is shown as SEQ19; the upstream sequence of RRBS-8 is shown as SEQ08, and the downstream sequence is shown as SEQ20; the upstream sequence of RRBS-9 is shown as SEQ09, and the downstream sequence is shown as SEQ21; the upstream sequence of RRBS-9 is shown as SEQ10, and the downstream sequence is shown as SEQ22; the upstream sequence of RRBS-10 is shown as SEQ11, and the downstream sequence is shown as SEQ23; the upstream sequence of RRBS-11 is shown as SEQ12, and the downstream sequence is shown as SEQ24.

Further, the DNA ligase in the fourth step is T4 DNA ligase.

Further, the specific method for PCR enrichment of methylation library in the sixth step comprises: before enriching the library, taking part of the library after bisulfite conversion to carry out PCR cycle number optimization; distributing the obtained PCR reaction mixture into each PCR hole, centrifuging the PCR hole, performing subsequent PCR amplification, and sequentially increasing 2 cycles from 10 PCR cycles to determine the optimal cycle number for obtaining a reliable library; and amplifying and purifying the residual bisulfite-converted library according to the optimal cycle number, and eluting the DNA of the library.

Further, the amplification primers for PCR enrichment of the methylation library in the sixth step comprise: universal Primer SEQ25, RRBS-R701 SEQ26, RRBS-R702 SEQ27, RRBS-R703 SEQ28, RRBS-R704 SEQ29, RRBS-R705 SEQ30 and RRBS-R706 SEQ31.

After the technical scheme is adopted, the invention has the advantages that:

the method can be used for constructing the methylation library by utilizing the FFPE sample in a large scale, the whole process of generating 96 FFPE DNA simplified representative methylation libraries only needs 3 days, and the construction time of the methylation library is greatly saved;

according to the method, a sample set can be mixed with 24 genomic DNA methylation libraries and a method for removing the adaptor dimer by using VAHTS DNA Clean Beads magnetic Beads, so that the complexity of a single-cell methylation library and the loss caused by electrophoretic selection are eliminated, large-scale library generation is allowed, and the automatic setting is easy, so that the total cost of each sample is reduced;

according to the method, ddATP repair is carried out on the tail end of the genome DNA by using the tail end modification enzyme, so that a large number of short segments in FFPE DNA are prevented from being connected with joints, the efficiency of combining the joints with enzyme cutting sites is improved, the coverage of CpG islands is improved, the effect of enzyme cutting enriched CpG island regions is amplified, and methylation information is provided in a more economic and efficient mode;

the method can use FFPE DNA with low content and a plurality of short segments as a starting material for research, can use the residual sample after pathological research, and does not need repeated sampling;

the method can retrospectively research the tumor methylation condition of the previous case, thereby obtaining more tumor methylation data.

Drawings

FIG. 1 is a schematic view of the process flow of the present invention.

FIG. 2 is a schematic diagram of the experimental flow of FFPE DNA library construction of the present invention.

FIG. 3 is a diagram of the quality control of RRBS library in the experiment of the present invention, using Agilent bioanalyzer high sensitivity DNA analysis kit.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

As shown in fig. 1 and 2, a method for rapidly constructing an RRBS sequencing library by using formalin-fixed paraffin-embedded samples comprises the following steps:

taking a tissue sample fixed by paraffin-embedded FFPE, scraping the tissue sample by using a clean blade, putting the tissue sample into cell lysate for digestion, and releasing DNA by digested cells to obtain genome DNA; performing addatp repair on the end of the genomic DNA with an end-modifying enzyme; preferably, the paraffin-embedded tissue sample is a tissue sample embedded in paraffin for more than 18 months, the sample has no specific embedding time limit, but generally, the longer the embedding time is, the greater the damage to the tissue is, the adverse effect on the library quality after library construction is caused, and therefore, the sample embedding limit is recommended to be optimal within a certain period of time. .

Digesting the CpG island of the genome DNA by using restriction endonuclease; as an example, the restriction enzyme is a combination of one or more of MspI and HaeIII.

Step three, carrying out terminal repair on the genome DNA by using terminal modification enzyme, adding A-Tailing at the 3 'end, and doping dATP into the 3' end of the blunt-ended DNA fragment; end-modifying enzymes include: a combination of one or more of Klenow Exo-enzyme and DNA polymerase, both of which are realized from the enzyme function perspective; preferably, the end-modifying enzyme is Klenow Exo-enzyme; the Klenow Exo-enzyme lacks 3'-5' exonuclease activity and, if the enzyme has 3'-5' exonuclease activity, the end repair is followed by a blunt-ended library that is difficult to ligate to downstream linker sequences.

Step four, carrying out methylation joint connection on the DNA by using DNA ligase, and mixing samples marked by all the label sequences;

preferably, the DNA ligase is T4 DNA ligase.

The methylated linker is two single-stranded complementary nucleic acids, and is diluted to 0.15 μ M before use and stored at-80 deg.C; the conditions for annealing the methylated joint to form the Y-shaped double-stranded structure are as follows: water bath is carried out at the temperature of 98 ℃ for 5 minutes, the water bath kettle is closed, the temperature is naturally cooled to 25 ℃ at room temperature, a Y-shaped double-stranded structure is formed, all cytosine nucleotides in two single-stranded nucleic acids are methylated, and phosphorylation treatment is carried out on the 5' end of the bottom nucleic acid of the Y-shaped joint; the 3' end comprises a tag sequence consisting of 6 nucleotides.

Converting the unmethylated cytosine by using bisulfite, and immediately performing polymerase chain reaction amplification on the converted DNA or storing the DNA at-80 ℃;

as a preference, the bisulfite conversion kit used is the QiaGen EpiTect fast bisulfate conversion kit, which, after the temperature has dropped to 20 ℃ after the conversion, immediately enters the purification step of the bisulfite converted DNA library.

Sixthly, enriching the methylation library by PCR;

as an example, a specific method for PCR enrichment of methylation libraries comprises:

the reaction system and PCR procedure are as follows in tables 1 and 2:

reaction systems Table 1

It should be noted that: PCR positive and negative primers for library amplification are diluted to 25 mu M by TE buffer solution, and can be stored for 1 year at-20 ℃.

PCR procedure Table 2

Then purifying the library after PCR enrichment; as a preference, 1.3 times VAHTS DNA Clean Beads are used for purification.

As a preference, the specific method for PCR enrichment of methylation library comprises: before enriching the library, taking part of the library after bisulfite conversion for PCR cycle number optimization, thus ensuring sufficient generation of library DNA and avoiding introducing higher duplicate by over-amplification; distributing the obtained PCR reaction mixture into each PCR hole, centrifuging the PCR hole, performing subsequent PCR amplification, and sequentially increasing 2 cycles from 10 PCR cycles to determine the optimal cycle number for obtaining a reliable library; and amplifying the residual bisulfite-converted library according to the optimal cycle number, wherein the specific amplification process is shown in experiment I, purifying and eluting the DNA of the library. Purification as a preference, after a linker with a different tag sequence is added to the sample, the sample is mixed into the same tube for magnetic bead purification. Purification is preferably performed using 1.8 × VAHTS DNA Clean Beads.

And seventhly, performing gel cutting recovery on each mixed sample through gel electrophoresis on the DNA fragment with the band length of 170-400bp, performing quality control and mixing on the library subjected to gel cutting recovery, and finally performing Illumina platform sequencing, wherein the sequencing sample loading amount is subjected to cluster generation according to 60% density of the conventional Illumina library.

The specific process is as follows:

1) Gel electrophoresis using 4% agarose TAE-gel for each mixed sample, the band was excised from the 170-400bp DNA fragment and recovered using the QiaGen MinElute gel purification kit;

2) Performing quality control and mixing on the library recovered by the photoresist, wherein the quality control is to perform quality control on the size and the concentration of fragments of the library by using an Agilent high-sensitivity chip, and the mixing is to perform equal proportion molar weight mixing on the library with different tag sequences according to the distribution of data quantity;

3) Sequencing loads should be clustered at 60% density of conventional Illumina libraries, and as a preference, mixed sequencing libraries should incorporate 30% -50% phiX balanced libraries to ensure library diversity.

The technical effect of the invention is verified by the following specific implementation process:

the library construction specific process comprises the following steps:

with FFPE in different embedding times as the material, genomic DNA was obtained, and the sample information is as follows in table 3:

TABLE 3

Step one, DNA end modification

1. 15ng FFPE DNA was taken from each sample, and then filled up with water to 17. Mu.l, and added to a 96-well plate. The DNA end modification reaction was carried out according to the following reaction system of Table 4:

TABLE 4

Note: klenow Exo-, ddATP (1 mM), and 10 × CutSmart buffer were mixed in advance according to the number of samples to avoid sample-to-sample errors caused by sample addition. The following restriction enzyme digestion, end repair and linker ligation reactions were performed using similar methods to mix the reagents in advance.

2. 3 μ l of DNA end-modifying reaction mixture was added to each sample well.

3. The reaction was mixed by gentle shaking and centrifuged briefly.

4. The samples were placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reactions were performed according to the following procedure in table 5:

TABLE 5

5. After the incubation was completed, centrifugation was performed for 30 seconds.

Step two, restriction enzyme digestion

1. The restriction enzyme digestion reaction was carried out according to the reaction system of the following Table 6:

TABLE 6

2. Add 1 μ l of digestion mixture to each sample well.

3. The reaction was mixed by gentle shaking and centrifuged briefly.

4. The samples were placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reactions were performed according to the following schedule table 7:

TABLE 7

5. After the incubation was completed, centrifugation was performed for 30 seconds.

Step three, end repair

1. The end-point repair reaction was carried out according to the reaction system of the following Table 8:

TABLE 8

2. 2 μ l of end repair mixture was added to each sample well.

3. The reaction was mixed by gentle shaking and centrifuged briefly.

4. The samples were placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reactions were performed according to the following schedule 9:

TABLE 9

5. After the incubation, centrifugation was carried out for 30 seconds.

Step four, joint connection

1. The linker ligation reaction was carried out according to the following reaction system 10:

watch 10

After the methylation joint is purchased and synthesized, dissolving to 100 mu M by using low TE and storing as mother liquor, and then taking part of the mother liquor to dilute to 0.15 mu M of working solution.

TABLE 11 linker sequence information

0.5 μ l linker ligation mixture and 2.5 μ l linker were added to each sample.

2. The reaction was mixed by gentle shaking and centrifuged briefly.

3. The samples were placed in a thermal cycler with the thermal lid set at 25 ℃ and the incubation reactions were performed according to the following schedule 12:

TABLE 12

4. After the incubation, centrifugation was carried out for 30 seconds.

5. The 24 samples labeled with different tag sequences were all transferred to a 1.5ml centrifuge tube for mixing, and then each sample well was washed with 30 μ l of low TE and finally mixed with the sample.

6. To each pooled sample library was added 1.8 times VAHTS DNA Clean Beads and the tube was rotated at room temperature for 30 minutes to facilitate binding of the Beads to the library DNA.

7. The sample tube was centrifuged briefly, the tube was placed on a DynaMagTM-2 (Life Technologies) magnetic rack to separate the beads, separated at room temperature for 10 minutes, and the supernatant solution was carefully removed, taking care not to touch the beads.

8. The beads were washed twice with 1 ml of 80% freshly prepared ethanol.

9. After the ethanol was completely evaporated, the library DNA was eluted with 40. Mu.l of low TE buffer.

Step five, bisulfite conversion

1. Methylation processing was performed according to the product instructions of the QIAGEN epitec rapid bisulfite conversion kit with some modifications using a two cycle bisulfite conversion protocol as shown in table 13 below:

watch 13

When the temperature of the thermal cycler is lowered to 20 ℃, the purification steps of the kit should be performed as soon as possible to reduce DNA loss.

2. The DNA after completion of the transformation was eluted with 30. Mu.l of TE buffer.

The key steps are as follows: the bisulfite converted DNA should be immediately amplified by polymerase chain reaction or stored at-80 ℃. If left at room temperature or 4 ℃, bisulfite converted DNA will degrade rapidly, causing failure of the library.

Step six, library enrichment

1. (optional) if multiple similar pooled libraries are available, it is recommended that one of the pooled libraries be used to validate the PCR cycle and the final enrichment of the DNA library be performed with the lowest number of cycles (from which sufficient pool DNA can be generated) and the PCR reaction system is as follows:

TABLE 14

TABLE 15PCR amplification primer information

2. Mu.l of each of the above 50. Mu.l PCR reaction mixtures was dispensed into 4 different PCR wells. The PCR plate wells were simply centrifuged for subsequent PCR amplification with the amplification parameters as follows:

TABLE 16

Usually 10 cycles are started, each time with 2 cycles to find the best number of cycles to enrich the library, and no more than 20 cycles are recommended, otherwise large amounts of PCR by-products tend to occur.

3. The PCR amplification system was prepared as follows:

TABLE 17

4. After brief centrifugation, PCR amplification was performed with the amplification parameters as in table 18 below:

watch 18

5. To each enriched library, 1.3 times of VAHTS DNA Clean Beads were added and the tube was rotated at room temperature for 30 minutes to facilitate binding of the Beads to the library DNA.

6. The sample tube was briefly centrifuged, the sample tube was placed on a DynaMagTM-2 (Life Technologies) magnetic rack to separate the magnetic beads, separated for 10 minutes at room temperature, and the supernatant solution was carefully removed, taking care not to touch the beads.

7. The beads were washed twice with 1 ml of 80% freshly prepared ethanol.

8. After complete ethanol evaporation, the library DNA was eluted with 20. Mu.l of low TE buffer.

Step seven, fragment recovery

1. The FFPE-RRBS library quality inspection results are shown in FIG. 3, the target library fragments are all between 180-1000bp, and a large amount of PCR primer dimers are removed, by using the quality control maps of 4 library fragments generated by an Agilent Bioanalyzer (Agilent Technologies, agilent Bioanalyzer 2100) and a high-sensitivity DNA analysis kit (Agilent Technologies, cat No. 5067-4626).

2. Preparation 4% agarose TAE-gel agarose gel.

3. The DNA library was electrophoresed on an agarose gel at 4% agarose TAE-gel for 2 hours at 1: staining was done for 1 hour in 10000 diluted SYBR Green I.

4. The 170-400bp DNA fragment was recovered by cutting the DNA ladder gel. The 170-400bp DNA fragment was purified using QIAGEN MinElute gel purification kit to remove non-target sequences (including PCR primer sequences, primer dimers, and large fragment sequences).

5. The library DNA was eluted with 20. Mu.l of low TE buffer.

6. Library DNA QC the mass concentration of the library and the DNA fragment size were determined using the Qubit dsDNA HS Assay Kit and Agilent HS high sensitive chip from Invitrogen, respectively.

7. Different libraries of the same band size were mixed in equal molar amounts depending on the amount of data required.

8. The mixed library was subjected to 100bp double-ended sequencing on the Illumina sequencing platform. As with other bisulfite sequencing libraries, single cell reduced representative methylation libraries should also be loaded onto sequencing chips (flow cells) at relatively low cluster densities (-60% of the density of conventional Illumina sequencing libraries). Meanwhile, 30-50% of phiX DNA equilibrium library is added to increase the base diversity of the sequencing library and improve the sequencing quality.

According to the method, ddATP repair is carried out on the tail end of the genome DNA by using the tail end modification enzyme, the problem of high failure rate of RRBS (rapid reverse transcriptase polymerase chain reaction) for constructing an FFPE (fringe field programmable peptide) DNA library caused by formalin soaking in the prior art is solved, the efficiency of combining a linker and an enzyme cutting site is improved, the coverage of a CpG island is improved, the effect of enzyme cutting on enriching the CpG island region is amplified, methylation information is provided in a more economical and efficient mode, and the method has wide application prospects and market values.

Other embodiments of the present invention than the preferred embodiments described above, and those skilled in the art can make various changes and modifications according to the present invention without departing from the spirit of the present invention, should fall within the scope of the present invention defined in the claims.

Sequence listing

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Claims (1)

1. A method for rapidly constructing an RRBS sequencing library by using a formalin-fixed paraffin-embedded sample is characterized by comprising the following steps:

taking a paraffin-embedded intestinal cancer or lung cancer tissue sample to obtain genome DNA; add dATP repair to the ends of genomic DNA with end modifying enzymes:

1) Taking 15ng of FFPE DNA from each sample, filling the FFPE DNA into 17 mu l of the FFPE DNA with water, adding the FFPE DNA into a 96-well plate, and carrying out DNA end modification reaction according to the following reaction system: 17. Mu.l of DNA, klenow Exo-0.5. Mu.l, 0.5. Mu.l of 1mM ddATP, 2. Mu.l of 10X CutSmart buffer;

2) Adding 3 mul of DNA end modification reaction mixture into each sample well;

3) Mixing the reaction system by gentle shaking and centrifuging for a short time;

4) The sample was placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reaction was performed according to the following procedure: procedure 1 ℃ 10 min; procedure 210 min at 37 ℃; program 3 ℃ 5 min; procedure 4 ℃ hold;

5) After the incubation is finished, centrifuging for 30 seconds;

and step two, digesting the genome DNA by using restriction endonuclease:

1) The restriction enzyme digestion reaction was carried out according to the following reaction system: the product obtained in the previous step is 20 mul, mspI 0.5 mul, 10X CutSmart 0.1 mul and water 0.4 mul;

2) Adding 1 μ l of digestion mixture to each sample well;

3) Mixing the reaction system by gentle shaking and centrifuging for a short time;

4) The sample was placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reaction was performed according to the following procedure: procedure 1 ℃ 30 min; procedure 2 ℃ 10 min; procedure 3 hold at 4 ℃;

5) After the incubation is finished, centrifuging for 30 seconds;

and step three, performing end repair on the genomic DNA by using an end modifying enzyme and adding A-Tailing at the 3 'end to incorporate dATP into the 3' end of the blunt-ended DNA fragment:

1) The end repair reaction was carried out according to the following reaction system:

21 mul of the product obtained in the previous step, klenowExo-0.5 mul, 0.4 mul of dNTP, 0.2 mul of 10X CutSmart and 0.9 mul of water; wherein the dNTPs consist of 10mM dATP,1mM dCTP,1mM dGTP;

2) Adding 2 mul of end repairing mixture into each sample hole;

3) Mixing the reaction system by gentle shaking and centrifuging for a short time;

4) The sample was placed in a thermal cycler with the thermal lid set at 85 ℃ and the incubation reaction was performed according to the following procedure: procedure 1 ℃ 10 min; procedure 210 min at 37 ℃; program 3 ℃ 5 min, program 4 ℃ hold;

5) Centrifuging for 30 seconds after the incubation is finished;

step four, carrying out methylation joint connection on the DNA by using DNA ligase, and mixing samples marked by all the label sequences:

1) The linker ligation reaction was carried out according to the following reaction system: 23 mul of the product obtained in the previous step, 0.1 mul of 100mM ATP, 0.2 mul of T4 ligase, 0.3 mul of 10X CutSmart buffer and 2.4 mul of 0.15 mu M methylated linker;

after the methylation joint is purchased and synthesized, dissolving to 100 mu M by using low TE buffer solution to be used as mother liquor for storage, and then taking part of the mother liquor to dilute to 0.15 mu M working solution;

2) Mixing the reaction system by gentle shaking and centrifuging for a short time;

3) The samples were placed in a thermal cycler with the thermal lid set at 25 ℃ and the incubation reactions were performed according to the following procedure: procedure 1 temperature 16 ℃ 1-3 hours; procedure 2 ℃ 10 min; procedure 3 hold at 4 ℃;

4) After the incubation is finished, centrifuging for 30 seconds;

5) Transferring all the 24 samples marked by different label sequences into a 1.5ml centrifuge tube for mixing, washing each sample well with 30 microliters of lowTE buffer solution, and finally mixing with the samples;

6) Adding 1.8 times of VAHTS DNA Clean Beads to each mixed sample library, and rotating the test tube at room temperature for 30 minutes to promote the combination of the Beads and the library DNA;

7) Centrifuging the sample tube for a short time, placing the sample tube on a DynaMagTM-2 magnetic frame to separate magnetic beads, separating for 10 minutes at room temperature, and carefully taking out a supernatant solution, wherein the supernatant solution is not touched with the magnetic beads;

8) Washing the magnetic beads twice with 1 ml of 80% freshly prepared ethanol;

after the ethanol is completely volatilized, eluting the library DNA by using 40 mu l of lowTE buffer solution;

converting the unmethylated cytosine by using bisulfite, immediately performing polymerase chain reaction amplification on the converted DNA or storing the DNA at-80 ℃:

1) Methylation processing was performed according to the product instructions of the qiagen epistec rapid bisulfite conversion kit using a two cycle bisulfite conversion protocol, the procedure being as follows: procedure 1 ℃ 5 min at 98 ℃; procedure 2 20 min at 60 ℃; program 3 ℃ 5 min, program 4 ℃ 20 min, program 5 ℃ hold;

when the temperature of the thermal cycler is reduced to 20 ℃, the purification step of the kit is started as soon as possible to reduce the DNA loss;

2) Eluting the DNA after the conversion by using 30 mul of TE buffer solution; the DNA after bisulfite conversion should be immediately amplified by polymerase chain reaction or stored at-80 ℃;

sixthly, enriching the methylation library by PCR;

performing gel cutting recovery on the DNA fragment of 170-400bp of the band in each mixed sample through gel electrophoresis, performing quality control and mixing on the library subjected to gel cutting recovery, and finally performing Illumina platform sequencing, wherein the sequencing sample loading amount is subjected to cluster generation according to 60% density of the conventional Illumina library; wherein the DNA library was electrophoresed on a 4% agarose gel for 2 hours at 1: staining in 10000 diluted SYBR Green I for 1 hour; cutting according to the DNA ladder to recover a DNA fragment of 170-400 bp; purifying the 170-400bp DNA fragment by using a QIAGEN MinElute gel purification kit, and removing non-target sequences comprising a PCR primer sequence, a primer dimer and a large fragment sequence;

the methylated linker in step four comprises: RRBS-1, RRBS-2, RRBS-3, RRBS-4, RRBS-5, RRBS-6, RRBS-7, RRBS-8, RRBS-9, RRBS-10, RRBS-11, RRBS-12; the upstream sequence of RRBS-1 is shown as SEQ01, and the downstream sequence is shown as SEQ13; the upstream sequence of RRBS-2 is shown as SEQ02, and the downstream sequence is shown as SEQ14; the upstream sequence of RRBS-3 is shown as SEQ03, and the downstream sequence is shown as SEQ15; the upstream sequence of RRBS-4 is shown as SEQ04, and the downstream sequence is shown as SEQ16; the upstream sequence of RRBS-5 is shown as SEQ05, and the downstream sequence is shown as SEQ17; the upstream sequence of RRBS-6 is shown as SEQ06, and the downstream sequence is shown as SEQ18; the upstream sequence of RRBS-7 is shown as SEQ07, and the downstream sequence is shown as SEQ19; the upstream sequence of RRBS-8 is shown as SEQ08, and the downstream sequence is shown as SEQ20; the upstream sequence of RRBS-9 is shown as SEQ09, and the downstream sequence is shown as SEQ21; the upstream sequence of RRBS-9 is shown as SEQ10, and the downstream sequence is shown as SEQ22; the upstream sequence of RRBS-10 is shown as SEQ11, and the downstream sequence is shown as SEQ23; the upstream sequence of RRBS-11 is shown as SEQ12, and the downstream sequence is shown as SEQ24;

the paraffin-embedded tissue sample in the step one is a tissue sample embedded in paraffin within 18 months;

the specific method for enriching the methylation library in the sixth step by PCR comprises the following steps: before enriching the library, taking part of the library after bisulfite conversion for PCR cycle number optimization; distributing the obtained PCR reaction mixture into each PCR hole, centrifuging the PCR hole, performing subsequent PCR amplification, and sequentially increasing 2 cycles from 10 PCR cycles to determine the optimal cycle number for obtaining a reliable library; amplifying and purifying the residual bisulfite-converted library according to the optimal cycle number, and eluting the DNA of the library;

the amplification primers for PCR enrichment of the methylation library in the sixth step comprise: universal Primer SEQ25, RRBS-R701 SEQ26, RRBS-R702 SEQ27, RRBS-R703 SEQ28, RRBS-R704 SEQ29, RRBS-R705 SEQ30 and RRBS-R706 SEQ31.

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