阅读说明：本技术 一种利用血液循环肿瘤dna快速构建rrbs测序文库的方法 (Method for rapidly constructing RRBS sequencing library by using blood circulation tumor DNA ) 是由 谷红仓 王云飞 车仙荣 陶宛琪 钱飞箭 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种利用血液循环肿瘤DNA快速构建RRBS测序文库的方法,属于分子生物学领域,构建方法包括：步骤一,以血液样本获得ctDNA；用末端修饰酶对ctDNA的末端进行加ddATP修复；步骤二,采用限制性内切酶消化基因组DNA的CpG岛；步骤三,用末端修饰酶对基因组DNA进行末端修复并在3’端加A-Tailing；步骤四,用DNA连接酶对DNA进行甲基化接头连接；步骤五,采用亚硫酸氢盐转化非甲基化胞嘧啶；步骤六,甲基化文库PCR富集；步骤七,切胶回收；本方法解决了现有技术利用血液进行RRBS构建ctDNA文库失败率高的问题,提高接头和酶切位点结合的效率,推进了液体活检技术的发展。(The invention discloses a method for quickly constructing an RRBS sequencing library by utilizing blood circulation tumor DNA, which belongs to the field of molecular biology and comprises the following steps: step one, obtaining ctDNA from a blood sample; performing addatp repair on the end of ctDNA with an end modifying enzyme; 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 on the DNA by using DNA ligase; converting non-methylated 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 constructing the ctDNA library by RRBS (reverse transcription-reverse transcription) by using blood in the prior art, improves the combination efficiency of the joint and the enzyme cutting site, and promotes the development of the liquid biopsy technology.)

1. A method for rapidly constructing an RRBS sequencing library by using blood circulation tumor DNA is characterized by comprising the following steps:

step one, taking a freshly collected blood sample which is not subjected to freeze thawing as a material to obtain ctDNA; performing addatp repair on the end of ctDNA 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 performing 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 with the band of 170-400bp in the DNA library by gel electrophoresis, purifying and eluting, performing quality control and mixing on the library subjected to gel cutting recovery, finally performing Illumina platform sequencing, performing cluster generation on sequencing sample loading according to 60% density of the conventional Illumina library, and doping 30-50% of phiX equilibrium library into the mixed sequencing library.

2. The method for rapidly constructing RRBS sequencing library by using blood circulation tumor DNA, according to claim 1, wherein the terminal modifying enzyme comprises: a combination of one or more of Klenow Exo-enzyme, DNA polymerase.

3. The method for rapidly constructing RRBS sequencing library according to claim 1, wherein the restriction enzyme in step two is a combination of one or more of MspI and HaeIII.

4. The method for rapidly constructing the sequencing library of RRBS by using the blood circulating tumor DNA, according to claim 1, wherein the methylation joint in the fourth step is a Y-shaped double-stranded structure formed by two single-stranded complementary nucleic acids in a water bath at 98 ℃ for 5 minutes, closing the water bath kettle, naturally cooling to room temperature of 25 ℃ and finishing annealing, all cytosine nucleotides in the two single-stranded nucleic acids are methylated, and the 5' end of the bottom nucleic acid of the Y-shaped joint is subjected to phosphorylation treatment; the 3' end comprises a tag sequence consisting of 6 nucleotides.

5. The method for rapidly constructing RRBS sequencing library according to claim 4, wherein the methylation 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 SEQ 13; the upstream sequence of RRBS-2 is shown as SEQ02, and the downstream sequence is shown as SEQ 14; the upstream sequence of RRBS-3 is shown as SEQ03, and the downstream sequence is shown as SEQ 15; the upstream sequence of RRBS-4 is shown as SEQ04, and the downstream sequence is shown as SEQ 16; the upstream sequence of RRBS-5 is shown as SEQ05, and the downstream sequence is shown as SEQ 17; the upstream sequence of RRBS-6 is shown as SEQ06, and the downstream sequence is shown as SEQ 18; the upstream sequence of RRBS-7 is shown as SEQ07, and the downstream sequence is shown as SEQ 19; the upstream sequence of RRBS-8 is shown as SEQ08, and the downstream sequence is shown as SEQ 20; the upstream sequence of RRBS-9 is shown as SEQ09, and the downstream sequence is shown as SEQ 21; the upstream sequence of RRBS-10 is shown as SEQ10, and the downstream sequence is shown as SEQ 22; the upstream sequence of RRBS-11 is shown as SEQ11, the downstream sequence is shown as SEQ23, the upstream sequence of RRBS-12 is shown as SEQ12, and the downstream sequence is shown as SEQ 24.

6. The method for rapidly constructing RRBS sequencing library according to claim 1, wherein the DNA ligase in the fourth step is T4 DNA ligase.

7. The method for rapidly constructing the sequencing library of RRBS by using the circulating tumor DNA as claimed in claim 1, wherein the specific method for PCR enrichment of the methylation library in the sixth step comprises: 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; and amplifying and purifying the residual bisulfite-converted library according to the optimal cycle number, and eluting the DNA of the library.

8. The method for rapidly constructing the sequencing library of RRBS by using the circulating tumor DNA as claimed in claim 1, wherein the amplification primers enriched by the PCR 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 SEQ 31.

9. The method as claimed in claim 1, wherein the specific method for performing gel cutting recovery on the DNA fragment with band of 170-400bp in the DNA library by gel electrophoresis in the seventh step is as follows: after preparation of 4% agarose TAE-gel agarose gel, the DNA library was electrophoresed on 4% agarose TAE-gel agarose gel for 2 hours at 1: 10000 diluted SYBR Green I staining for 1 hours, according to the DNA ladder gel recovery 170 and 400bp DNA fragment.

Technical Field

The invention relates to the field of molecular biology, in particular to a method for quickly constructing an RRBS sequencing library by utilizing blood circulation tumor DNA.

Background

DNA methylation is a chemical modification of DNA in eukaryotes 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 degree of methylation is a reversible mode of DNA modification, and is involved in the regulation of gene expression, and abnormal hypermethylation or hypomethylation can be closely related to many diseases including cancers.

Methylation of the genome can be sequenced by combining bisulfite conversion techniques with high throughput sequencing techniques of genomic DNA. Simplified DNA methylation sequencing can achieve resolution of single base detection and high utilization of sequencing data by enriching regions of high CpG content using a methylation insensitive restriction enzyme, followed by bisulfite conversion, PCR amplification and Illumina sequencing. Based on the characteristics of high sensitivity, multiple loci, tissue specificity and the like of a methylation signal, the kit can be widely applied to the fields of early screening of cancers, monitoring of curative effects, cancer tracing and the like in clinical practice.

As a noninvasive detection method, the liquid biopsy can be used for obtaining samples more quickly and simply in clinic, can also reduce the harm to the body of a patient to the maximum extent, is more easily accepted, and can be used for dynamic monitoring by collecting the samples for multiple times. DNA released into the blood by necrotic or apoptotic cells is called circulating free DNA (cfdna); whereas in the blood of tumor patients a part of cfDNA is derived from dead tumor cells, this part of DNA is defined as circulating tumor DNA (ctdna). ctDNA and cfDNA are very close in size and cannot be separated by routine experimental means at present. Even in tumor patients, ctDNA accounts for only a very small fraction of DNA isolated from blood, and therefore, naturally-broken cfDNA joins adaptors and enters libraries, resulting in a large amount of data waste and diluting the enrichment of CpG islands by enzymatic digestion. Therefore, the failure rate of constructing the ctDNA library by using the traditional RRBS is high, and the development and popularization of the liquid biopsy technology are hindered. The market needs a new method which can block the connection of ctDNA and a methylated linker and amplify the effect of enzyme digestion on enriched CpG island regions. Meanwhile, the method has the advantages of simple operation process, reduced reagent and personnel cost, reduced DNA loss caused by repeated purification and easy conversion into the RRBS sequencing library of the automatic library building process by using the same buffer solution, and 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 utilizing blood circulating tumor DNA, solves the problem of high failure rate of constructing a ctDNA library by utilizing blood RRBS in the prior art, and promotes the development of a liquid biopsy technology.

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

a method for rapidly constructing an RRBS sequencing library by utilizing blood circulation tumor DNA comprises the following steps:

step one, taking a freshly collected blood sample which is not subjected to freeze thawing as a material to obtain ctDNA; performing addatp repair on the end of ctDNA 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 performing 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 with the band of 170-400bp in the DNA library by gel electrophoresis, purifying and eluting, performing quality control and mixing on the library subjected to gel cutting recovery, finally performing Illumina platform sequencing, performing cluster generation on sequencing sample loading according to 60% density of the conventional Illumina library, and doping 30-50% of phiX equilibrium library into the mixed sequencing library.

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 SEQ 13; the upstream sequence of RRBS-2 is shown as SEQ02, and the downstream sequence is shown as SEQ 14; the upstream sequence of RRBS-3 is shown as SEQ03, and the downstream sequence is shown as SEQ 15; the upstream sequence of RRBS-4 is shown as SEQ04, and the downstream sequence is shown as SEQ 16; the upstream sequence of RRBS-5 is shown as SEQ05, and the downstream sequence is shown as SEQ 17; the upstream sequence of RRBS-6 is shown as SEQ06, and the downstream sequence is shown as SEQ 18; the upstream sequence of RRBS-7 is shown as SEQ07, and the downstream sequence is shown as SEQ 19; the upstream sequence of RRBS-8 is shown as SEQ08, and the downstream sequence is shown as SEQ 20; the upstream sequence of RRBS-9 is shown as SEQ09, and the downstream sequence is shown as SEQ 21; the upstream sequence of RRBS-10 is shown as SEQ10, and the downstream sequence is shown as SEQ 22; the upstream sequence of RRBS-11 is shown as SEQ11, the downstream sequence is shown as SEQ23, the upstream sequence of RRBS-12 is shown as SEQ12, and the downstream sequence is shown as SEQ 24.

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

Further, the specific method for PCR enrichment of the methylation library in the sixth step 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; 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 SEQ 31.

Further, the specific method for performing gel cutting recovery on the DNA fragment with the band of 170-400bp in the DNA library by gel electrophoresis in the step seven comprises the following steps: after preparation of 4% agarose TAE-gel agarose gel, the DNA library was electrophoresed on 4% agarose TAE-gel agarose gel for 2 hours at 1: 10000 diluted SYBR Green I staining for 1 hours, according to the DNA ladder gel recovery 170 and 400bp DNA fragment.

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

according to the method, phosphate groups are digested, and ddATP repair is carried out on the tail end of ctDNA by using a tail end modifying enzyme, so that a large number of short fragments in the ctDNA are prevented from being connected with joints, the efficiency of combining the joints with enzyme cutting sites is improved, and the coverage of CpG islands is improved, so that methylation information is provided in a more economic and efficient manner, the problem of high failure rate of construction of a ctDNA library by using blood RRBS in the prior art is solved, and the development of a liquid biopsy technology is promoted;

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

by adopting the method, 24 ctDNA methylation libraries and VAHTS DNA Clean Beads can be mixed in one sample set to remove the adaptor dimer, the tedious property of a single traditional methylation library and the loss caused by electrophoresis selection are eliminated, the large-scale library generation is allowed, and the automatic setting is easy, so the total cost of each sample is reduced.

The method can take the tumor DNA with ultra-low content in blood as a starting material for research, obtains the sample by a non-invasive method, and is suitable for researching the DNA methylation condition of cancer patients who are not suitable for obtaining the sample by an operation means.

The method obtains the sample through a non-invasive means, can better understand the characteristics of the cancer tumor in clinic, and can dynamically monitor the development and treatment condition of the cancer in real time by collecting the sample for multiple times.

Drawings

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

FIG. 2 is a schematic diagram of the experimental procedure for constructing the ctDNA library of the present invention.

Detailed Description

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

As shown in FIG. 1, a method for rapidly constructing an RRBS sequencing library by using blood circulation tumor DNA comprises the following steps:

step one, taking a freshly collected blood sample which is not subjected to freeze thawing as a material to obtain ctDNA; performing addatp repair on the end of ctDNA with an end modifying enzyme;

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 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; end-modifying enzymes include: a combination of one or more of Klenow Exo-enzyme, DNA polymerase; from an enzyme function point of view, Klenow Exo-enzyme, DNA polymerase are both available; 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 bisulphite conversion kit used is the QiaGen EpiTect fast bisulfate conversion kit, which, after a temperature drop to 20 ℃ after conversion, immediately enters the purification step of the bisulphite converted DNA library.

Sixthly, enriching the methylation library by PCR;

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

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

reaction System 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, 1.3X VAHTS DNA Clean Beads are used for purification.

As a preference, the specific method for enriching the methylation library by PCR comprises the following steps: 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 adapters with different tag sequences are 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 the DNA fragment with the band of 170-400bp in the DNA library by gel electrophoresis, purifying and eluting, performing quality control and mixing on the library subjected to gel cutting recovery, finally performing Illumina platform sequencing, performing cluster generation on sequencing sample loading according to 60% density of the conventional Illumina library, and doping 30-50% of phiX equilibrium library into the mixed sequencing library.

The specific method for performing gel cutting recovery on the DNA fragment with the band of 170-400bp in the DNA library by gel electrophoresis comprises the following steps: after preparation of 4% agarose TAE-gel agarose gel, the DNA library was electrophoresed on 4% agarose TAE-gel agarose gel for 2 hours at 1: 10000 diluted SYBR Green I staining for 1 hours, according to the DNA ladder gel recovery 170-.

Preferably, the DNA fragment of 170-400bp is purified by using QIAGEN MinElute gel purification kit, and PCR primer dimer is removed; elution library DNA was eluted with 20. mu.l of low TE buffer; quality control library DNA QC is adopted, and an Agilent HS high sensitive chip is used for determining the size and the molar concentration of the library DNA fragments. The mixing method comprises the following steps: different libraries of the same band size were mixed in equal molar amounts depending on the amount of data required. It should be noted that: the above are preferable, and any reagent or method capable of achieving the same function can be applied to the present invention.

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

as shown in fig. 2, the library construction is a specific process:

the method comprises the steps of taking freshly collected 8 cases of peripheral blood samples of lung cancer as detection objects, carrying out secondary centrifugation on the peripheral blood of a patient to separate plasma, and then separating ctDNA or small-fragment cell-free DNA (cfDNA) from the plasma, wherein all samples are from Shore-eff hospitals affiliated to Zhejiang university medical colleges. Wherein samples 1, 2, 3 and 4 were tested according to the contents of steps one through seven, and samples 5, 6, 7 and 8 were tested according to the contents of steps two through seven.

Step one, DNA end modification

1. Samples 1, 2, 3 and 4 were filled to 17 μ l with 15ng ctDNA and added to a 96-well plate. The DNA end-modification reactions were performed according to the reaction system of table 3 below, and samples 5, 6, 7 and 8 were run directly from step two:

TABLE 3

Note: klenow Exo-, ddATP (1mM), 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. 1 μ l of the 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 4:

TABLE 4

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

Step two, restriction enzyme digestion

1. Samples 1, 2, 3 and 4 were subjected to restriction enzyme digestion according to the reaction system of Table 5, and samples 5, 6, 7 and 8 were subjected to restriction enzyme digestion according to the reaction system of Table 6:

TABLE 5

TABLE 6

2. 1 μ l and 3 μ l of digestion mixture were added to wells of samples 1, 2, 3 and 4 and samples 5, 6, 7 and 8, respectively.

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 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 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:

TABLE 9

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

Step four, joint connection

1. Linker ligation was performed according to the following reaction system 10, with the methylated linker sequence shown in Table 11:

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

2. 0.5 μ l linker ligation mixture and 2.5 μ l linker were added to each sample.

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 25 ℃ and the incubation reactions were performed according to the following schedule 12:

TABLE 12

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

6. 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.

7. 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.

8. 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.

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

10. 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 reduced to 20 ℃, the purification step of the kit should be performed as soon as possible to reduce the 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 a failure to pool.

Step six, library enrichment

1. (this step is preferred and can be skipped.) if multiple similar pooled libraries are available, it is recommended that one of the pooled libraries be used to validate the PCR cycles and the final enrichment of the DNA library be performed with the lowest number of cycles (from which sufficient library DNA can be generated) in the PCR reaction system of Table 14 below:

TABLE 14

TABLE 15 PCR 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 from 10 cycles, each time increased by 2 cycles, to find the library enrichment of the best cycle number, recommended no more than 20 cycles, otherwise easily occurs in a large amount of by-products.

3. The following PCR amplification System Table 17 was prepared:

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 the ethanol was completely evaporated, the library DNA was eluted with 20. mu.l of low TE buffer.

Step seven, fragment recovery

1. A4% agarose TAE-gel was prepared.

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

3. A DNA fragment of 170-400bp was recovered from the DNA ladder gel cut. The 170-400bp DNA fragment was purified using QIAGEN MinElute gel purification kit to remove PCR primer dimers.

4. The library DNA was eluted with 20. mu.l of low TE buffer.

5. Library DNA QC for samples 1-8 the mass concentration of the library and the DNA fragment size were determined using the Qubit dsDNA HS Assay Kit and Agilent HS high sensitivity chip from Invitrogen, respectively.

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

7. 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.

8. Finally, the ctDNA library of 8 blood samples was sequenced, resulting in the following results, as shown in table 19:

watch 19

And (4) analyzing results: the coverage detected by the ctDNA-RRBS library is in CpG numbers above 10x, and CpG island numbers above 10x and 50 x. In the modified 4 ctDNA samples, all samples can detect >60 ten thousand cpgs with coverage of 10x or more; in all 4 samples >7000 CpG islands with a coverage of more than 50x could be detected. In 4 samples without DNA fragment modification, the CpG with the coverage rate of more than 10X is less than 40 ten thousand, the CpG islands with the coverage rate of more than 50X are less than 4000, and the effectiveness of data can be remarkably improved after the original template is modified.

From the above experiments, it can be known that ddATP repair is performed on the tail end of ctDNA by using a tail end modifying enzyme, a large number of short segments in the ctDNA are prevented from connecting joints, the efficiency of combining the joints and enzyme cutting sites is improved, and the coverage of CpG islands is improved, so that methylation information is provided in a more economic and efficient manner, the problem of high failure rate of construction of a ctDNA library by using blood in the prior art is solved, and the development of a liquid biopsy technology is promoted.

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.

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<212> DNA

<213> Artificial Sequence

<400> 5

ctacacgacg ctcttccgat ctcggtagt 29

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 6

ctacacgacg ctcttccgat ctctattgt 29

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 7

ctacacgacg ctcttccgat ctgcattct 29

<210> 8

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 8

ctacacgacg ctcttccgat ctgttgagt 29

<210> 9

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 9

ctacacgacg ctcttccgat cttctctgt 29

<210> 10

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 10

ctacacgacg ctcttccgat cttgctgct 29

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 11

ctacacgacg ctcttccgat ctagaaggt 29

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 12

ctacacgacg ctcttccgat ctgaagtct 29

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 13

ggttgtagat cggaagagca cacgtctgaa c 31

<210> 14

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 14

gtgagtagat cggaagagca cacgtctgaa c 31

<210> 15

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 15

catcctagat cggaagagca cacgtctgaa c 31

<210> 16

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 16

gtcgatagat cggaagagca cacgtctgaa c 31

<210> 17

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 17

ctaccgagat cggaagagca cacgtctgaa c 31

<210> 18

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 18

caatagagat cggaagagca cacgtctgaa c 31

<210> 19

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 19

gaatgcagat cggaagagca cacgtctgaa c 31

<210> 20

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 20

ctcaacagat cggaagagca cacgtctgaa c 31

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 21

cagagaagat cggaagagca cacgtctgaa c 31

<210> 22

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 22

gcagcaagat cggaagagca cacgtctgaa c 31

<210> 23

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 23

ccttctagat cggaagagca cacgtctgaa c 31

<210> 24

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 24

gacttcagat cggaagagca cacgtctgaa c 31

<210> 25

<211> 58

<212> DNA

<213> Artificial Sequence

<400> 25

aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58

<210> 26

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 26

caagcagaag acggcatacg agattcgcct tagtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 27

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 27

caagcagaag acggcatacg agatctagta cggtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 28

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 28

caagcagaag acggcatacg agatttctgc ctgtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 29

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 29

caagcagaag acggcatacg agatgctcag gagtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 30

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 30

caagcagaag acggcatacg agataggagt ccgtgactgg agttcagacg tgtgctcttc 60

cgatct 66

<210> 31

<211> 66

<212> DNA

<213> Artificial Sequence

<400> 31

caagcagaag acggcatacg agatcatgcc tagtgactgg agttcagacg tgtgctcttc 60

cgatct 66