WO2020232621A1 - 微量dna甲基化高通量测序方法 - Google Patents

微量dna甲基化高通量测序方法 Download PDF

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WO2020232621A1
WO2020232621A1 PCT/CN2019/087743 CN2019087743W WO2020232621A1 WO 2020232621 A1 WO2020232621 A1 WO 2020232621A1 CN 2019087743 W CN2019087743 W CN 2019087743W WO 2020232621 A1 WO2020232621 A1 WO 2020232621A1
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stranded
double
strand
component
stranded dna
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French (fr)
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杨林
张艳艳
陈恬
杨贵芳
陈芳
蒋慧
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深圳华大智造科技有限公司
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Priority to PCT/CN2019/087743 priority Critical patent/WO2020232621A1/zh
Priority to CN201980092829.XA priority patent/CN113490750B/zh
Publication of WO2020232621A1 publication Critical patent/WO2020232621A1/zh

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the invention belongs to the field of biotechnology, and specifically relates to a high-throughput sequencing method for trace DNA methylation.
  • DNA methylation is an apparent regulatory modification, which participates in the regulation of protein synthesis without changing the base sequence.
  • DNA methylation is a very wonderful chemical modification. The care of relatives, the body's aging, smoking, alcoholism and even obesity will be truthfully recorded on the genome by methylation. The genome is like a diary, and methylation is used as text to record the experience of the human body.
  • DNA methylation is an important epigenetic marker information. Obtaining the methylation level data of all C sites in the whole genome is of great significance for the study of epigenetic spatio-temporal specificity.
  • mapping the DNA methylation level of the whole genome, and analyzing the high-precision methylation modification patterns of specific species will surely have a milestone significance in epigenomics research.
  • Whole Genome Methylation Sequencing WGBS Whole Genome Bisulfite Sequencing
  • 1Bisulfite treatment will single-strand DNA and cause serious damage
  • 2Unmethylated C bases after bisulfite treatment will be converted into U Base
  • the GC content of the entire genome has extreme changes, resulting in great preference for subsequent amplification
  • 3The library requires microgram-level starting DNA, and it is difficult to have a very effective method of library building for trace DNA.
  • WGBS is roughly divided into two categories, one is Pre-BS after Bisulfite treatment, and the other is Post-BS after Bisulfite treatment.
  • the library will cause 90% DNA fragmentation under the action of sulfite, resulting in a very low library conversion rate.
  • the library is built after Bisulfite treatment, and the library is prepared for the treated single-stranded DNA after sulfite treatment. It is divided into single-strand ligation method (low ligation efficiency and complicated steps) and random primer extension method (which has serious effects on template strands). Selection preference, complicated operation), too many experimental steps also aggravated the loss of DNA. Therefore, inventing a simple and efficient method for library construction is of great significance for methylation research.
  • the invention provides a high-throughput sequencing method for trace DNA methylation.
  • the present invention provides a method for preparing a methylation sequencing library, which sequentially includes the following steps:
  • Joint 1 is composed of two single-stranded DNA molecules, named single-strand 1-1 and single-strand 1-2; in the ligation product, the double-stranded DNA fragment The 3'end of each chain is connected to the 5'end of the single-stranded 1-1 (directly connected), and the 5'end of each chain of the double-stranded DNA fragment is adjacent to but not connected to the 3'end of the single-stranded 1-2 (Form a nick, nike);
  • Universal primer 1 is used for DNA replication; the 3'end segment of universal primer 1 is reverse complementary to single-stranded 1-1; the polymerase used for DNA replication meets the following conditions: it has the ability to amplify U;
  • the ligation product of step (1) consists of the following segments in sequence: linker 1, double-stranded DNA fragment, and linker 1.
  • the ligation product of step (1) is a double-stranded DNA molecule with nicks.
  • the nicks are caused by the fact that the 5'end of each strand of the double-stranded DNA fragment is adjacent to the 3'end of the single-strand 1-2 but not connected.
  • step (1) the preparation method of the double-stranded DNA fragment is specifically as follows: blunting the double-stranded DNA molecule and adding A to the 3'end.
  • the realization of the blunt end includes but is not limited to end repair.
  • the double-stranded DNA molecule may specifically be as follows (a) or (b):
  • the amount of the double-stranded DNA molecule can be 1-3000 ng, more specifically 200-400 ng.
  • the length of the double-stranded DNA molecule can be 100-600 bp, more specifically 150-300 bp.
  • the double-stranded DNA fragment is a double-stranded DNA with sticky ends, and each strand of the double-stranded DNA has a sticky end A at the 3'end.
  • step (1) the 5'end of each strand in the double-stranded DNA fragment is blocked and modified.
  • step (1) the single-stranded 1-1 and the single-stranded 1-2 are reversely complementary.
  • step (1) the single-stranded 1-1 and the single-stranded 1-2 are partially complementary in reverse.
  • step (1) the 3'end of the single strand 1-2 is blocked and the other parts are reversely complementary to the single strand 1-1.
  • the purpose of the blocking modification is to block the connection between the single-stranded 1-2 and the double-stranded DNA fragment. Any blocking modification in the prior art can be used, such as amino modification.
  • the blocking modification is a dideoxynucleotide.
  • the blocking modification is a dideoxynucleotide.
  • the blocking modification is a dideoxynucleotide ddT.
  • linker 1 is a methylated tag linker.
  • step (1) the 5'end of the single strand 1-1 is phosphorylated.
  • step (1) C in the single-stranded 1-1 is 5-methylcytosine deoxyribonucleotide.
  • the single-stranded 1-1 is specifically shown in Sequence 1 of the Sequence Listing.
  • C in the linker 1-1 all represent 5-methylcytosine deoxyribonucleotides.
  • the 5'end of the linker 1-1 is phosphorylated.
  • the single strand 1-2 is specifically shown in Sequence 2 of the Sequence Listing.
  • the sulfite treatment includes but is not limited to bisulfite treatment or bisulfite treatment.
  • the sulfite treatment can also be other sulfite treatments known to those skilled in the art.
  • step (3) the polymerase also satisfies the following conditions: it has the activity of adding A to the 3'end (used to add A to the 3'end of the double-stranded DNA product).
  • step (3) the polymerase also meets the following conditions: it has displacement activity (used to open the high GC structure).
  • the 3'end segment of the universal primer 1 refers to 10-30 nucleotides upstream from the second nucleotide of the 3'end.
  • the universal primer 1 may or may not contain the sample tag sequence.
  • step (3) the nucleotide at the 3'end of the universal primer 1 is T.
  • the general primer 1 is specifically shown in sequence 3 of the sequence listing.
  • connection in the 3'end connection sequencing adapter can be direct connection or indirect connection (that is, connection through a spacer sequence).
  • the sequencing adapter When directly connected, the sequencing adapter may be adapter 2.
  • Joint 2 consists of two single-stranded DNA molecules, named single-stranded 2-1 and single-stranded 2-2.
  • the nucleotide at the 3'end of the single strand 2-1 is T, and the other part is reverse complementary to the single strand 2-2.
  • the adapter 2 may or may not contain the sample tag sequence.
  • the linker 2 may be single-stranded or double-stranded.
  • the 5'end of the single strand 2-2 is phosphorylated.
  • the single strand 2-1 is specifically shown in Sequence 4 of the Sequence Listing.
  • the single strand 2-2 is specifically shown in sequence 5 of the sequence listing.
  • a universal primer can be used for amplification first, and then a primer complementary to the universal primer with a sequencing adapter can be connected.
  • step (4) can be used for subsequent multi-sample mixed parallel sequencing, and can also be used for PCR-free library construction.
  • the method may also include the following step (5): after step (4) is completed, PCR amplification is performed with universal primers. Step (5) is used to enrich DNA.
  • the sample tag can be added by universal primers.
  • the universal primer may specifically be universal primer 2 and universal primer 3.
  • the general primer 2 is specifically shown in sequence 6 of the sequence listing.
  • the general primer 3 is specifically shown in sequence 7 of the sequence listing.
  • the present invention also protects a kit for preparing a methylated sequencing library, which includes component 1, component 2, component 3 and component 4;
  • the function of the component 1 is: to connect the double-stranded DNA fragment with the adapter 1 to obtain the ligation product; the component 1 includes the component 1-1 and the component 1-2; the component 1-1 is the adapter 1; the component 1-2 is used The reagent or reagent set for realizing the connection; the adapter 1 is composed of two single-stranded DNA molecules, named single-stranded 1-1 and single-stranded 1-2; in the connection product, 3 of each strand of the double-stranded DNA fragment The'end is connected to the 5'end of the single-stranded 1-1, and the 5'end of each strand of the double-stranded DNA fragment is adjacent to but not connected to the 3'end of the single-stranded 1-2;
  • the function of the component 2 is: sulfite treatment of DNA;
  • the function of the component 3 is: DNA replication; the component 3 includes the component 3-1 and the component 3-2; the component 3-1 is the universal primer 1; the component 3-2 is the reagent or Reagent set; the 3'end segment of universal primer 1 is reversely complementary to single-stranded 1-1; component 3-2 includes DNA polymerase; the DNA polymerase meets the following conditions: it has the ability to amplify U;
  • the function of the component 4 is to connect a sequencing adapter to the 3'end of the double-stranded DNA.
  • the double-stranded DNA fragment is a double-stranded DNA with sticky ends, and each strand of the double-stranded DNA has a sticky end A at the 3'end.
  • the ligation product In the function of component 1, the ligation product consists of the following segments in sequence: linker 1, double-stranded DNA fragment, and linker 1.
  • the ligation product In the function of component 1, the ligation product is a double-stranded DNA molecule with nicks. The nicks are caused by the fact that the 5'end of each strand of the double-stranded DNA fragment is adjacent to the 3'end of the single-strand 1-2 but not connected. of.
  • the kit also includes a component 5; the function of the component 5 is: blunt-end double-stranded DNA molecules and add A to the 3'end.
  • the realization of the blunt end includes but is not limited to end repair.
  • the double-stranded DNA molecule may specifically be as follows (a) or (b):
  • the length of the double-stranded DNA molecule can be 100-600 bp, more specifically 150-300 bp.
  • the 5'end of each strand in the double-stranded DNA fragment is modified to block.
  • Single strand 1-1 and single strand 1-2 are reverse complementary.
  • the single strand 1-1 and the single strand 1-2 are partially complementary in reverse.
  • the 3'end of the single strand 1-2 is blocked and the other part is reverse complementary to the single strand 1-1.
  • the purpose of the blocking modification is to block the connection between the single-stranded 1-2 and the double-stranded DNA fragment. Any blocking modification in the prior art can be used, such as amino modification.
  • the blocking modification is a dideoxynucleotide.
  • the blocking modification is a dideoxynucleotide.
  • the blocking modification is a dideoxynucleotide ddT.
  • Linker 1 is a methylated tag linker.
  • the 5'end of the single strand 1-1 is phosphorylated.
  • the C in single-stranded 1-1 is all 5-methylcytosine deoxyribonucleotide.
  • the single-stranded 1-1 is specifically shown in Sequence 1 of the Sequence Listing.
  • C in the linker 1-1 all represent 5-methylcytosine deoxyribonucleotides.
  • the 5'end of the linker 1-1 is phosphorylated.
  • the single strand 1-2 is specifically shown in Sequence 2 of the Sequence Listing.
  • Sulfite treatment includes, but is not limited to, bisulfite treatment or bisulfite treatment.
  • the sulfite treatment may also be other sulfite treatments known to those skilled in the art.
  • DNA polymerase also satisfies the following conditions: it has the activity of adding A to the 3'end (used to add A to the 3'end of the double-stranded DNA product).
  • DNA polymerase also meets the following conditions: it has displacement activity (used to open a high GC structure).
  • the 3'end segment of the universal primer 1 refers to 10-30 nucleotides upstream from the 2nd nucleotide of the 3'end.
  • the universal primer 1 may or may not contain the sample tag sequence.
  • the nucleotide at the 3'end of the universal primer 1 is T.
  • the general primer 1 is specifically shown in sequence 3 of the sequence listing.
  • connecting the 3'end of the double-stranded DNA with the sequencing adapter is realized by direct connection or indirect connection (that is, connection through a spacer sequence).
  • Joint 2 consists of two single-stranded DNA molecules, named single-stranded 2-1 and single-stranded 2-2.
  • the nucleotide at the 3'end of the single strand 2-1 is T, and the other part is reverse complementary to the single strand 2-2.
  • the adapter 2 may or may not contain the sample tag sequence.
  • the linker 2 may be single-stranded or double-stranded.
  • the 5'end of the single strand 2-2 is phosphorylated.
  • the single strand 2-1 is specifically shown in Sequence 4 of the Sequence Listing.
  • the single strand 2-2 is specifically shown in sequence 5 of the sequence listing.
  • the component 4 includes a universal primer and a linker primer.
  • Universal primers are used for DNA amplification.
  • the adaptor primer has a sequencing adaptor and has a segment complementary to the universal primer.
  • the kit also includes component 6.
  • the function of the component 6 is: PCR amplification.
  • the component 6 includes universal primers for PCR amplification.
  • the universal primer may or may not have a sample label.
  • the universal primer may specifically be universal primer 2 and universal primer 3.
  • the general primer 2 is specifically shown in sequence 6 of the sequence listing.
  • the general primer 3 is specifically shown in sequence 7 of the sequence listing.
  • methylation sequencing libraries includes, but is not limited to, whole genome methylation sequencing libraries.
  • small fragment DNA small fragment DNA or cfDNA obtained after large fragment DNA is broken
  • undergo end repair and add A then add single-stranded sequencing adaptor 1 by way of double-stranded linker, and then perform sub- Sulfate treatment, after treatment, use universal primers to use the single-stranded sequencing sequence as an anchor to replicate the sulfite-treated DNA, and add a linker 2 to the end of the newly generated double-stranded DNA in a double-stranded manner.
  • the obtained product After the obtained product is purified, it can be directly subjected to high-throughput sequencing, or it can be subjected to subsequent PCR on the template with the linker sequence added by the universal primer to complete the construction of the high-throughput library.
  • Figure 1 is a schematic diagram of the database construction process.
  • Figure 2 is a schematic diagram of the joint connection.
  • Figure 3 shows the comparison of library yields.
  • Figure 4 shows the comparison of the repetition rate of the offline data.
  • Figure 5 shows the distribution of methylation rates of CpG sites on different chromosomes.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.
  • A represents adenine deoxyribonucleotide
  • T represents thymine deoxyribonucleotide
  • C represents cytosine deoxyribonucleotide
  • G represents bird Purine deoxyribonucleotides.
  • ⁇ -DNA 200-400ng
  • ⁇ -DNA as an exogenous reference, Thermo Fisher Scientific, USA, catalog number SD0011
  • use the covaris-S2 interrupter to fragment fragments, and then recover DNA fragments between 150-300 bp. Re-dissolve it in 56 ⁇ l TE buffer to form a DNA fragment solution.
  • step 1 Take the DNA fragment solution obtained in step 1, and prepare the reaction system in a 1.5 mL centrifuge tube according to Table 1. Place the reaction system on a PCR machine and perform the reaction (reaction conditions: 20°C, 30 minutes, then 65°C, 30 minutes). After the reaction is completed, use AMPureXP magnetic beads for purification, and then dissolve the purified product in 34 ⁇ l TE buffer, which is the product solution.
  • Ultra TM End Repair/dA-Tailing Module US NEB company, article number E7442S; product website: https://www.neb.com/products/e7442-nebnext-ultra-end-repair-da-tai ling-module.
  • Connect connector 1 (connector 1 is methylated label connector)
  • step 2 Take the product solution obtained in step 2 and prepare the reaction system according to Table 2.
  • the reaction system was placed on a Thermomixer (Eppendorf) at 20°C and reacted for 15 minutes. After the reaction is completed, use AMPure magnetic beads for purification, and then dissolve the purified product in 22 ⁇ l TE buffer, which is the product solution.
  • the product solution obtained in step 2 18 ⁇ L 2 ⁇ Rapid connection buffer 25 ⁇ L Connector 1 solution 4 ⁇ L T4 DNA Ligase 3 ⁇ L total capacity 50 ⁇ L
  • the active ingredient provided by the joint 1 solution is joint 1.
  • the concentration of the connector 1 is 10 ⁇ M.
  • the joint 1 is formed of a joint 1-1 and a joint 1-2.
  • Linker 1-1 5'-AGTCGGAGGCCAAGCG-3';
  • Linker 1-2 5'-CGCTTGGCCTCCGACTT-3'.
  • Both linker 1-1 and linker 1-2 are single-stranded DNA molecules.
  • linker 1-1 all represent 5-methylcytosine deoxyribonucleotides.
  • the 5'end of the linker 1-1 is phosphorylated.
  • the last nucleotide T (underlined) at the 3'end is the dideoxy nucleotide ddT (the purpose is to block modification to prevent ligation with the target fragment).
  • T4 DNA Ligase is T4 DNA Ligase (Rapid). 2 ⁇ Rapid connection buffer, which is 2X Rapid Ligation Buffer (B1010). T4 DNA Ligase (Rapid): Specification 600,000U/ml. T4 DNA Ligase (Rapid) and 2X Rapid Ligation Buffer (B1010): enzymatic company, article number L6030-HC-L; product website http://www.enzymatics.com/products/t4-dna-ligase-rapid/.
  • step 3 Take all the product solutions obtained in step 3, use EZ DNA Methylation-Gold Kit TM (ZYMO, catalog number D5042) and operate according to the kit instructions (the purpose is to subject the DNA to bisulfite treatment) to obtain 18 ⁇ l of product solution.
  • kit instructions the purpose is to subject the DNA to bisulfite treatment
  • step 4 Take the product solution obtained in step 4 and prepare the reaction system according to Table 3. First, react at 95°C for 5 minutes, then reduce to 60°C and add 1 ⁇ l BST enzyme, then react at 60°C for 15 minutes, and then react at 75°C for 15 minutes to obtain a product solution.
  • the effective ingredient provided by the universal primer 1 solution is universal primer 1.
  • the concentration of universal primer 1 is 10 ⁇ M.
  • Universal primer 1 is as follows:
  • the underlined mark is the distinguishing mark (refers to the mark that distinguishes different samples), and N is any of A, T, C, and G.
  • Universal primer 1 is a single-stranded DNA molecule.
  • BST enzyme is Bst 3.0 DNA Polymerase, the specification is 8000 units/ml, New England Biolabs, catalog number M0374S. 10 ⁇ Bst buffer is the supporting buffer for BST enzyme.
  • step 5 Take the product solution obtained in step 5, and prepare the reaction system according to Table 4.
  • the reaction system was placed on a Thermomixer (Eppendorf) at 20°C, reacted for 15 minutes, and then purified with AMPureXP magnetic beads. Finally, the purified product was dissolved in 22 ⁇ l TE buffer, which was the product solution.
  • the product solution obtained in step 5 20 ⁇ L 2 ⁇ Rapid connection buffer 25 ⁇ L Connector 2 solution 2 ⁇ L T4 DNA Ligase 3 ⁇ L total capacity 50 ⁇ L
  • the active ingredient provided by the joint 2 solution is joint 2.
  • the concentration of the connector 2 is 10 ⁇ M.
  • the joint 2 is formed of a joint 2-1 and a joint 2-2.
  • Connector 2-2 5'-AGTCGGATCGTAGCCATGTCGTTC-3'.
  • Both the linker 2-1 and the linker 2-2 are single-stranded DNA molecules.
  • the 5'end of the linker 2-2 was phosphorylated.
  • T4 DNA Ligase is T4 DNA Ligase (Rapid). 2 ⁇ Rapid connection buffer is 2X Rapid Ligation Buffer (B1010). T4 DNA Ligase (Rapid): Specification 600,000U/ml. T4 DNA Ligase (Rapid) and 2X Rapid Ligation Buffer (B1010): enzymatic company, article number L6030-HC-L; product website http://www.enzymatics.com/products/t4-dna-ligase-rapid/.
  • PCR reaction conditions 94°C for 1min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 12 cycles; 72°C for 5min; 12°C hold.
  • After the reaction use AMPure magnetic beads for purification, and finally dissolve the purified product in 22 ⁇ l TE buffer, which is the library solution.
  • the product solution obtained in step 6 20 ⁇ L 2 ⁇ kapa HIFI hot start master mix 25 ⁇ L Universal primer 2 solution 2.5 ⁇ L Universal primer 3 solution 2.5 ⁇ L total capacity 50 ⁇ L
  • the effective component provided by the universal primer 2 solution is universal primer 2.
  • the concentration of universal primer 2 is 10 ⁇ M.
  • Universal primer 2 is a single-stranded DNA molecule.
  • Universal primer 2 (same sequence as linker 2-1): 5'-GAACGACATGGCTACGATCCGACTT-3'.
  • the effective component provided by the universal primer 3 solution is universal primer 3.
  • the concentration of universal primer 3 is 10 ⁇ M.
  • Universal primer 3 is a single-stranded DNA molecule.
  • the library solution obtained in step 1 is subjected to quality control, and qualified for high-throughput sequencing.
  • Quality control Use Bioanalyzer analysis system (Agilent, Santa Clara, USA) to detect the size and content of the inserted fragments in the library.
  • High-throughput sequencing sequencing platform BGIseq-500, sequencing type PE100, sequencing depth 30X; after sequencing data after comparison, statistics on various basic parameters, including offline data, available data, data utilization, comparison rate, GC content And repetition rate etc.
  • Embodiment 2 Specific application examples of the method
  • Optimization method 2 425375773 397726348 91.5% 21.60% 3.4% 99.51% Optimization method 3 413005078 388224773 91.6% 21.90% 2.7% 99.48% Conventional method 1 432030698 401356518 89.6% 21.80% 17.6% 99.47% Conventional method 2 452180042 435689478 94.7% 21.00% 16.9% 99.51% Conventional method 3 422549120 399875641 90.3% 21.20% 21.3% 99.49%
  • the comparison of library yield is shown in Figure 3.
  • the library yield of the conventional method is 150ng.
  • the library yield of the method provided by the present invention is 1100 ng.
  • the method provided by the present invention can greatly increase the yield of the library and greatly improve the efficiency of DNA utilization.
  • the comparison of the repetition rate of the offline data is shown in Figure 4.
  • the data repetition rate of the conventional method is 18.6%.
  • the data repetition rate of the method provided by the present invention is 2.8%.
  • the method provided by the present invention can greatly improve data utilization efficiency.
  • Figure 5 shows the distribution of methylation rates of CpG sites on different chromosomes.
  • the technical effects of the present invention (1) The transformation efficiency of the library can be greatly improved, which is 10 times that of the traditional library construction; (2) Compared with the post-BS construction of the single-strand connection mode, the operation steps of the inventive method are simple ; Compared with the post-BS library construction of random primer extension method, the inventive method has very little preference; (3) PCR-free WGBS library preparation can be completed, and the library obtained by this method can be directly applied to high-throughput without PCR Sequencing is performed on the sequencing platform, while other library building methods need to convert U bases to T bases by PCR to perform subsequent DNB preparation (MGI platform) or bridge amplification (Illumina platform).
  • the beneficial effects brought by the technical solution of the present invention are: (1) The utilization efficiency of the template is greatly improved, and the conversion efficiency of the library is improved; (2) The simple operation steps can complete the PCR-free library preparation and reduce the sequencing process. Preference. Industrialization prospects: methylation library building kits, clinical methylation detection products: early tumor screening, tumor detection and diagnosis, etc.

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Abstract

本发明公开了一种微量DNA甲基化高通量测序方法。本发明提供的方法，依次包括如下步骤：（1）将双链DNA片段连接接头1，得到连接产物；接头1由两条单链DNA分子组成，命名为单链1-1和单链1-2；连接产物中，双链DNA片段的每条链的3'末端与单链1-1的5'末端连接，双链DNA片段的每条链的5'末端与单链1-2的3'末端相邻但不连接；（2）进行亚硫酸盐处理；（3）采用通用引物1进行DNA复制；（4）3'末端连接测序接头。

Description

[根据细则37.2由ISA制定的发明名称]　微量DNA甲基化高通量测序方法 技术领域
本发明属于生物技术领域，具体涉及一种微量DNA甲基化高通量测序方法。
背景技术
DNA甲基化是一种表观调控修饰，它在不改变碱基序列的情况下，参与调控蛋白质合成的多少。对人类来说，DNA甲基化是一种非常奇妙的化学修饰，亲人的关怀，机体的衰老、抽烟、酗酒甚至肥胖，都会被甲基化如实地记录到基因组上。基因组就像是一个日记本，甲基化作为文字，记录下人体的经历。DNA甲基化是重要的表观遗传学标记信息，获得全基因组范围内所有C位点的甲基化水平数据，对于表观遗传学的时空特异性研究具有重要意义。以新一代高通量测序平台为基础，进行全基因组DNA甲基化水平图谱绘制，特定物种的高精确度甲基化修饰模式的分析，必将在表观基因组学研究中具有里程碑式的意义，并为细胞分化、组织发育等基础机制研究，以及动植物育种、人类健康与疾病研究奠定基础。
全基因组甲基化测序WGBS(Whole Genome Bisulfite Sequencing)，即全基因组亚硫酸氢盐测序，是研究生物甲基化的最常用手段，它可以覆盖所有甲基化位点，能够获得更加全面的甲基化图谱。但其在高通量测序中遇到了很多挑战：①亚硫酸氢盐处理会对DNA单链化并造成严重的损伤；②亚硫酸氢盐处理后的未甲基化C碱基会转变成U碱基，整个基因组的GC含量发生极端变化，造成后续扩增产生极大的偏好性；③建库需要微克级别的起始DNA，对于微量DNA很难有很有效的建库方法。
现有的WGBS大致分为两类，一类是Bisulfite处理前建库(Pre-BS)，另一类是Bisulfite处理后建库(Post-BS)。Bisulfite处理前建库，在加完测序接头后进行亚硫酸盐处理，其文库在亚硫酸盐的作用下会造成90％的DNA断裂，最后导致文库转化率极低。Bisulfite处理后建库，在亚硫酸盐处理后对处理过后的单链DNA进行文库的制备，分为单链连接法(连接效率低，步骤复杂)和随机引物延伸法(对模板链具有严重的选择偏好 性，操作复杂)，过多的实验步骤也加重了DNA的丢失。因此发明一个简单、高效的建库方法对于甲基化研究具有重要意义。
发明公开
本发明提供了一种微量DNA甲基化高通量测序方法。
本发明提供了一种制备甲基化测序文库的方法，依次包括如下步骤：
(1)将双链DNA片段连接接头1，得到连接产物；接头1由两条单链DNA分子组成，命名为单链1-1和单链1-2；连接产物中，双链DNA片段的每条链的3’末端与单链1-1的5’末端连接(直接连接)，双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接(形成缺刻，nike)；
(2)进行亚硫酸盐处理；
(3)采用通用引物1进行DNA复制；通用引物1的3’端区段与单链1-1反向互补；DNA复制时采用的聚合酶满足如下条件：具有扩增U的能力；
(4)3’末端连接测序接头。
步骤(1)的连接产物依次由如下区段组成：接头1、双链DNA片段、接头1。
步骤(1)的连接产物为具有缺刻的双链DNA分子，缺刻是由于双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接引起的。
步骤(1)中，所述双链DNA片段的制备方法具体如下：将双链DNA分子进行平末端化以及3’末端加A。所述平末端化的实现方式包括但不限于末端修复。
所述双链DNA分子具体可为如下(a)或(b)：
(a)基因组DNA进行打断得到的DNA片段；
(b)cfDNA。
所述双链DNA分子的量可为1-3000ng，更具体可为200-400ng。
所述双链DNA分子的长度可为100-600bp，更具体可为150-300bp。
步骤(1)中，所述双链DNA片段为具有粘末端的双链DNA，该双链DNA的每条链的3’末端均具有一个粘末端A。
步骤(1)中，双链DNA片段中的每条链的5’末端进行阻断修饰。
步骤(1)中，单链1-1和单链1-2反向互补。
步骤(1)中，单链1-1和单链1-2部分反向互补。
步骤(1)中，单链1-2中3’末端进行阻断修饰、其他部分与单链1-1反向互补。
阻断修饰的目的是阻断单链1-2与所述双链DNA片段进行连接。可以采用任意现有技术中的阻断修饰，例如氨基修饰。所述阻断修饰为双脱氧核苷酸。所述阻断修饰为一个双脱氧核苷酸。所述阻断修饰为一个双脱氧核苷酸ddT。
步骤(1)中，接头1为甲基化标签接头。
步骤(1)中，单链1-1的5’末端进行磷酸化修饰。
步骤(1)中，单链1-1中的C均为5-甲基胞嘧啶脱氧核糖核苷酸。
单链1-1具体如序列表的序列1所示。接头1-1中的C均代表5-甲基胞嘧啶脱氧核糖核苷酸。接头1-1的5’末端进行磷酸化修饰。
单链1-2具体如序列表的序列2所示。
步骤(2)中,亚硫酸盐处理包括但不限于亚硫酸氢盐处理或者重亚硫酸盐处理。
步骤(2)中,亚硫酸盐处理也可以为本领域技术人员公知的其它亚硫酸盐处理。
步骤(3)中,聚合酶还满足如下条件：具有3’末端加A活性(用于在双链的DNA产物3’末端加上A)。
步骤(3)中,聚合酶还满足如下条件：具有置换活性(用于打开高GC结构)。
步骤(3)中,通用引物1的3’端区段指的是从3’末端第2位核苷酸开始的上游的10-30个核苷酸。
步骤(3)中，通用引物1可以包含样本标签序列，也可以不包含样本标签序列。
步骤(3)中，通用引物1的3’末端的核苷酸为T。
通用引物1具体如序列表的序列3所示。
步骤(4)中，3’末端连接测序接头中所述连接的实现可以为直接连接也可为间接连接(即通过间隔序列连接)。
直接连接时，所述测序接头可为接头2。接头2由两条单链DNA分子组成，命名为单链2-1和单链2-2。单链2-1的3’末端的核苷酸为T，其他部 分与单链2-2反向互补。接头2可以包含样本标签序列，也可以不包含样本标签序列。接头2可以进行单链连接，也可以进行双链连接。单链2-2的5’末端进行磷酸化修饰。单链2-1具体如序列表的序列4所示。单链2-2具体如序列表的序列5所示。
间接连接时，可先采用通用引物进行扩增再连接具有测序接头的与通用引物互补的引物。
步骤(4)的产物，可以用于后续的多样本混合平行测序，也可以用于PCR-free文库构建。
所述方法还可包括如下步骤(5)：完成步骤(4)后，采用通用引物进行PCR扩增。步骤(5)用于富集DNA。步骤(5)中，可以通过通用引物添加样本标签。通用引物具体可为通用引物2和通用引物3。通用引物2具体如序列表的序列6所示。通用引物3具体如序列表的序列7所示。
以上任一所述方法在甲基化测序中的应用也属于本发明的保护范围。
本发明还保护一种制备甲基化测序文库的试剂盒，包括组件1、组件2、组件3和组件4；
所述组件1的功能为：给双链DNA片段连接接头1，得到连接产物；所述组件1包括组件1-1和组件1-2；组件1-1为接头1；组件1-2为用于实现所述连接的试剂或试剂组；接头1由两条单链DNA分子组成，命名为单链1-1和单链1-2；连接产物中，双链DNA片段的每条链的3’末端与单链1-1的5’末端连接，双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接；
所述组件2的功能为：对DNA进行亚硫酸盐处理；
所述组件3的功能为：进行DNA复制；所述组件3包括组件3-1和组件3-2；组件3-1为通用引物1；组件3-2为用于实现所述复制的试剂或试剂组；通用引物1的3’端区段与单链1-1反向互补；组件3-2包括DNA聚合酶；所述DNA聚合酶满足如下条件：具有扩增U的能力；
所述组件4的功能为：给双链DNA的3’末端连接测序接头。
所述双链DNA片段为具有粘末端的双链DNA，该双链DNA的每条链的3’末端均具有一个粘末端A。
组件1的功能中，所述连接产物依次由如下区段组成：接头1、双链DNA片段、接头1。组件1的功能中，所述连接产物为具有缺刻的双链DNA 分子，缺刻是由于双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接引起的。
所述试剂盒还包括组件5；所述组件5的功能为：将双链DNA分子进行平末端化以及3’末端加A。所述平末端化的实现方式包括但不限于末端修复。
所述双链DNA分子具体可为如下(a)或(b)：
(a)基因组DNA进行打断得到的DNA片段；
(b)cfDNA。
所述双链DNA分子的长度可为100-600bp，更具体可为150-300bp。
双链DNA片段中的每条链的5’末端进行阻断修饰。
单链1-1和单链1-2反向互补。
单链1-1和单链1-2部分反向互补。
单链1-2中3’末端进行阻断修饰、其他部分与单链1-1反向互补。
阻断修饰的目的是阻断单链1-2与所述双链DNA片段进行连接。可以采用任意现有技术中的阻断修饰，例如氨基修饰。所述阻断修饰为双脱氧核苷酸。所述阻断修饰为一个双脱氧核苷酸。所述阻断修饰为一个双脱氧核苷酸ddT。
接头1为甲基化标签接头。
单链1-1的5’末端进行磷酸化修饰。
单链1-1中的C均为5-甲基胞嘧啶脱氧核糖核苷酸。
单链1-1具体如序列表的序列1所示。接头1-1中的C均代表5-甲基胞嘧啶脱氧核糖核苷酸。接头1-1的5’末端进行磷酸化修饰。
单链1-2具体如序列表的序列2所示。
亚硫酸盐处理包括但不限于亚硫酸氢盐处理或者重亚硫酸盐处理。
亚硫酸盐处理也可以为本领域技术人员公知的其它亚硫酸盐处理。
DNA聚合酶还满足如下条件：具有3’末端加A活性(用于在双链的DNA产物3’末端加上A)。
DNA聚合酶还满足如下条件：具有置换活性(用于打开高GC结构)。
通用引物1的3’端区段指的是从3’末端第2位核苷酸开始的上游的10-30个核苷酸。
通用引物1可以包含样本标签序列，也可以不包含样本标签序列。
通用引物1的3’末端的核苷酸为T。
通用引物1具体如序列表的序列3所示。
组件4的功能中，给双链DNA的3’末端连接测序接头中所述连接的实现为直接连接或间接连接(即通过间隔序列连接)。
直接连接时，所述组件4包括接头2。接头2由两条单链DNA分子组成，命名为单链2-1和单链2-2。单链2-1的3’末端的核苷酸为T，其他部分与单链2-2反向互补。接头2可以包含样本标签序列，也可以不包含样本标签序列。接头2可以进行单链连接，也可以进行双链连接。单链2-2的5’末端进行磷酸化修饰。单链2-1具体如序列表的序列4所示。单链2-2具体如序列表的序列5所示。
间接连接时，所述组件4包括通用引物和接头引物。通用引物用于DNA扩增。接头引物具有测序接头且具有与通用引物互补的区段。
所述试剂盒还包括组件6。所述组件6的功能为：PCR扩增。所述组件6中包括用于PCR扩增的通用引物。所述通用引物中可以具有样本标签，也可以不具有样本标签。通用引物具体可为通用引物2和通用引物3。通用引物2具体如序列表的序列6所示。通用引物3具体如序列表的序列7所示。
以上任一所述试剂盒在甲基化测序中的应用也属于本发明的保护范围。
以上任一所述甲基化测序文库包括但不限于全基因组甲基化测序文库。
本发明的技术方案：小片段DNA(大片段DNA打断后得到的小片段DNA或cfDNA)进行末端修复和加A，然后通过双链连接接头的方式加上单链测序接头1，然后进行亚硫酸盐处理，处理过后，用通用引物以单链测序序列为锚定位点对亚硫酸盐处理过后的DNA进行复制，在得到新生成双链DNA的末端以双链连接的方式加上接头2，得到的产物进行纯化后，可以直接进行高通量测序，也可以通过通用引物对加上接头序列的模板进行后续PCR，完成高通量文库的构建。
附图说明
图1为建库流程示意图。
图2为接头连接方式示意图。
图3为文库产量比较。
图4为下机数据重复率比较。
图5为在不同染色体上CpG位点的甲基化率分布。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。如无特殊说明，实施例中的DNA分子中，A均代表腺嘌呤脱氧核糖核苷酸，T均代表胸腺嘧啶脱氧核糖核苷酸,C均代表胞嘧啶脱氧核糖核苷酸,G均代表鸟嘌呤脱氧核糖核苷酸。
实施例1、全基因组甲基化测序(WGBS)方法的建立
建库流程见图1，接头连接方式见图2。
一、文库的制备
1、DNA片段化
取基因组DNA(200-400ng)，利用covaris-S2打断仪(美国Thermo Fisher Scientific公司，Covaris S2/S220 System，按照说明书操作)进行片段破碎，然后回收150-300bp之间的DNA片段，重新溶于56μl TE缓冲液中，即为DNA片段溶液。
取λ-DNA(200-400ng)(λ-DNA为外源参照，美国Thermo Fisher Scientific公司,货号SD0011)，利用covaris-S2打断仪进行片段破碎，然后回收150-300bp之间的DNA片段，重新溶于56μl TE缓冲液中，即为DNA片段溶液。
2、末端修复和加A
取步骤1得到的DNA片段溶液，按照表1在1.5mL的离心管中配制反应体系。将反应体系置于PCR仪上，进行反应(反应条件：20℃、30分钟，然后65℃、30分钟)。反应完成后，用AMPureXP磁珠进行纯化，然后将纯化产物溶于34μl TE缓冲液，即为产物溶液。
表1
步骤1得到的DNA片段溶液 55.5μL
末端修复缓冲液(10×) 6.5μL
末端修复酶体系 3.0μL
总体积 65μL
末端修复酶体系和末端修复缓冲液(10×)为“
Figure PCTCN2019087743-appb-000001
Ultra TM End Repair/dA-Tailing Module”的组件。
Figure PCTCN2019087743-appb-000002
Ultra TM End Repair/dA-Tailing Module：美国NEB公司，货号E7442S；产品网址：https://www.neb.com/products/e7442-nebnext-ultra-end-repair-da-tai ling-module。
3、连接接头1(接头1即甲基化标签接头)
取步骤2得到的产物溶液，按照表2配制反应体系。将反应体系置于20℃的Thermomixer(Eppendorf)上，反应15min。反应完成后，用AMPure磁珠进行纯化，然后将纯化产物溶于22μl TE缓冲液，即为产物溶液。
表2
步骤2得到的产物溶液 18μL
2×Rapid连接缓冲液 25μL
接头1溶液 4μL
T4 DNA连接酶 3μL
总体积 50μL
接头1溶液提供的有效成分为接头1。接头1溶液中，接头1的浓度为10μM。
接头1由接头1-1和接头1-2形成。
接头1-1：5’-AGTCGGAGGCCAAGCG-3’；
接头1-2：5’-CGCTTGGCCTCCGACTT-3’。
接头1-1和接头1-2均为单链DNA分子。
接头1-1中的C均代表5-甲基胞嘧啶脱氧核糖核苷酸。接头1-1的5’末端进行磷酸化修饰。
接头1-2中，3’末端的最后一个核苷酸T(下划线标注)为双脱氧核苷酸ddT(目的是作为阻断修饰防止和目标片段进行连接)。
T4 DNA连接酶，为T4 DNA Ligase(Rapid)。2×Rapid连接缓冲液，为 2X Rapid Ligation Buffer(B1010)。T4 DNA Ligase(Rapid)：规格600,000U/ml。T4 DNA Ligase(Rapid)和2X Rapid Ligation Buffer(B1010)：enzymatic公司，货号L6030-HC-L；产品网址http://www.enzymatics.com/products/t4-dna-ligase-rapid/。
4、亚硫酸盐处理
取步骤3得到的全部产物溶液，采用EZ DNA Methylation-Gold Kit  TM(ZYMO，货号D5042)并按试剂盒说明操作(目的是将DNA进行亚硫酸氢盐处理)，得到18μl产物溶液。
5、DNA复制
取步骤4得到的产物溶液，按照表3配制反应体系，先95℃反应5min，然后降至60℃并加入1μl BST酶，然后60℃反应15分钟，然后75℃反应15分钟，得到产物溶液。
表3
步骤4得到的产物溶液 16μL
10×Bst buffer 2μL
通用引物1溶液 1μL
总体积 19μL
通用引物1溶液提供的有效成分为通用引物1。通用引物1溶液中，通用引物1的浓度为10μM。
通用引物1如下：
5’-TGTGAGCCAAGGAGTTG NNNNNNNNNNTTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’。
通用引物1中，下划线标注的为区别标识(指的是区分不同样本的标识)，N为A、T、C、G任意。
通用引物1为单链DNA分子。
BST酶，为Bst 3.0 DNA Polymerase，规格为8000units/ml，New England Biolabs，货号M0374S。10×Bst buffer为BST酶的配套缓冲液。
6、连接接头2(接头2即测序接头)
取步骤5得到的产物溶液，按表4配制反应体系。反应体系置于20℃的Thermomixer(Eppendorf)上，反应15min，然后用AMPureXP磁珠进行纯化，最后将纯化产物溶于22μl TE缓冲液，即为产物溶液。
表4
步骤5得到的产物溶液 20μL
2×Rapid连接缓冲液 25μL
接头2溶液 2μL
T4 DNA连接酶 3μL
总体积 50μL
接头2溶液提供的有效成分为接头2。接头2溶液中，接头2的浓度为10μM。
接头2由接头2-1和接头2-2形成。
接头2-1：5’-GAACGACATGGCTACGATCCGACTT-3’；
接头2-2：5’-AGTCGGATCGTAGCCATGTCGTTC-3’。
接头2-1和接头2-2均为单链DNA分子。
接头2-2的5’末端进行磷酸化修饰。
T4 DNA连接酶，为T4 DNA Ligase(Rapid)。2×Rapid连接缓冲液，为2X Rapid Ligation Buffer(B1010)。T4 DNA Ligase(Rapid)：规格600,000U/ml。T4 DNA Ligase(Rapid)和2X Rapid Ligation Buffer(B1010)：enzymatic公司，货号L6030-HC-L；产品网址http://www.enzymatics.com/products/t4-dna-ligase-rapid/。
7、PCR扩增(可选步骤)
取步骤6得到的产物溶液，按照表5配制PCR反应体系。PCR反应条件：94℃1min；94℃30s、55℃30s、72℃30s，12个循环；72℃5min；12℃保持。反应结束后用AMPure磁珠进行纯化，最后将纯化产物溶于22μl TE缓冲液，即为文库溶液。
表5
步骤6得到的产物溶液 20μL
2×kapa HIFI hot start master mix 25μL
通用引物2溶液 2.5μL
通用引物3溶液 2.5μL
总体积 50μL
通用引物2溶液提供的有效成分为通用引物2。通用引物2溶液中，通用引物2的浓度为10μM。
通用引物2为单链DNA分子。
通用引物2(同接头2-1序列)：5’-GAACGACATGGCTACGATCCGACTT-3’。
通用引物3溶液提供的有效成分为通用引物3。通用引物3溶液中，通用引物3的浓度为10μM。
通用引物3为单链DNA分子。
通用引物3：5’-TGTGAGCCAAGGAGTTG-3’。
2×kapa HIFI hot start master mix：KAPA BIOSYSTEMS公司，货号KK2602。
二、测序和数据分析
步骤一得到的文库溶液，进行质控，合格的进行高通量测序。
质控：使用Bioanalyzer分析系统(Agilent,Santa Clara,USA)检测文库插入片段的大小及含量。
高通量测序：测序平台BGIseq-500，测序类型PE100,测序深度30X；测序后数据经过比对后统计各项基本参数，包括下机数据、可用数据、数据利用率、比对率、GC含量和重复率等。
三、以λ-DNA的测序结果为参照，获得基因组DNA的全基因组甲基化测序结果。
实施例2、方法的具体应用示例
采用human genomic gDNA(Promega公司，货号G1521)的基因组DNA。按照实施例1的方法进行全基因组甲基化测序，结果见表6。进行三次重复试验，在表6中分别用优化方法1、优化方法2和优化方法3表示。
采用human genomic gDNA基因组DNA。采用MGIEasy全基因组甲基化文库制备试剂盒(货号1000005251)(按说明书操作)进行文库的制备，然后按照实施例1的步骤二进行测序和数据分析，结果见表6。进行三次重复试验，在表6中分别用常规方法1、常规方法2和常规方法3表示。
通过lanmbdaDNA的结果推算出亚硫酸盐的转化效率。
表6
  原始数据 可用数据 比对率 GC含量 重复率 亚硫酸盐转化率
优化方法1 439209013 415052517 94.5％ 21.90％ 2.3％ 99.45％
优化方法2 425375773 397726348 91.5％ 21.60％ 3.4％ 99.51％
优化方法3 413005078 388224773 91.6％ 21.90％ 2.7％ 99.48％
常规方法1 432030698 401356518 89.6％ 21.80％ 17.6％ 99.47％
常规方法2 452180042 435689478 94.7％ 21.00％ 16.9％ 99.51％
常规方法3 422549120 399875641 90.3％ 21.20％ 21.3％ 99.49％
文库产量比较见图3。常规方法的文库产量为150ng。本发明提供的方法的文库产量为1100ng。相对于常规方法，本发明提供的方法能够大幅度提高文库的产量，极大地提高DNA利用效率。
下机数据重复率比较见图4。常规方法的数据重复率为18.6％。本发明提供的方法的数据重复率为2.8％。相对于常规方法，本发明提供的方法能够极大地提高数据利用效率。
在不同染色体上CpG位点的甲基化率分布见图5。
工业应用
本发明的技术效果：(1)可以极大地提高文库的转化效率，是传统文库建库效率的10倍；(2)相对于单链连接方式的post-BS建库，发明方法的操作步骤简单；相对于随机引物延伸方式的post-BS建库，发明方法偏好性极小；(3)可以完成PCR-free的WGBS的文库制备，该方法得到的文库不经过PCR可直接应用于高通量测序平台进行测序，而其他建库方法需要通过PCR将U碱基转变为T碱基才能进行后面的DNB制备(MGI平台)或者桥式扩增(Illumina平台)。本发明技术方案带来的有益效果：(1)极大地提高了模板的利用效率，提高了文库的转化效率；(2)简单的操作步骤，可以完成PCR-free的文库制备,减少测序过程中的偏好性。产业化前景：甲基化建库试剂盒，临床甲基化检测产品：肿瘤早筛，肿瘤检测诊断等。

Claims (32)

  1. 一种制备甲基化测序文库的方法，依次包括如下步骤：
    (1)将双链DNA片段连接接头1，得到连接产物；接头1由两条单链DNA分子组成，命名为单链1-1和单链1-2；连接产物中，双链DNA片段的每条链的3’末端与单链1-1的5’末端连接，双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接；
    (2)进行亚硫酸盐处理；
    (3)采用通用引物1进行DNA复制；通用引物1的3’端区段与单链1-1反向互补；DNA复制时采用的聚合酶满足如下条件：具有扩增U的能力；
    (4)3’末端连接测序接头。
  2. 如权利要求1所述的方法，其特征在于：步骤(1)中的所述双链DNA片段为具有粘末端的双链DNA，该双链DNA的每条链的3’末端均具有一个粘末端A。
  3. 如权利要求2所述的方法，其特征在于：步骤(1)中的所述双链DNA片段的制备方法如下：将双链DNA分子进行平末端化以及3’末端加A。
  4. 如权利要求3所述的方法，其特征在于：
    所述双链DNA分子为如下(a)或(b)：
    (a)基因组DNA进行打断得到的DNA片段；
    (b)cfDNA。
  5. 如权利要求1所述的方法，其特征在于：步骤(1)中，双链DNA片段中的每条链的5’末端进行阻断修饰。
  6. 如权利要求1所述的方法，其特征在于：步骤(1)中，单链1-2中3’末端进行阻断修饰、其他部分与单链1-1反向互补。
  7. 如权利要求5或6所述的方法，其特征在于：所述阻断修饰为双脱氧核苷酸。
  8. 如权利要求1所述的方法，其特征在于：步骤(1)中，单链1-1的5’末端进行磷酸化修饰。
  9. 如权利要求1所述的方法，其特征在于：步骤(1)中，单链1-1中的C均为5-甲基胞嘧啶脱氧核糖核苷酸。
  10. 如权利要求1所述的方法，其特征在于：步骤(2)中,亚硫酸盐处理为亚硫酸氢盐处理或者重亚硫酸盐处理。
  11. 如权利要求1所述的方法，其特征在于：步骤(3)中,聚合酶满足如下条件：具有3’末端加A活性。
  12. 如权利要求1所述的方法，其特征在于：步骤(3)中,聚合酶满足如下条件：具有置换活性。
  13. 如权利要求1所述的方法，其特征在于：步骤(4)中，3’末端连接测序接头中所述连接的实现为直接连接或间接连接。
  14. 如权利要求13所述的方法，其特征在于：步骤(4)中，所述连接的实现为直接连接时，所述测序接头为接头2；接头2由两条单链DNA分子组成，命名为单链2-1和单链2-2；单链2-1的3’末端的核苷酸为T，其他部分与单链2-2反向互补。
  15. 如权利要求14所述的方法，其特征在于：单链2-2的5’末端进行磷酸化修饰。
  16. 如权利要求1所述的方法，其特征在于：所述方法还包括如下步骤(5)：完成步骤(4)后，采用通用引物进行PCR扩增。
  17. 一种制备甲基化测序文库的试剂盒，包括组件1、组件2、组件3和组件4；
    所述组件1的功能为：给双链DNA片段连接接头1，得到连接产物；所述组件1包括组件1-1和组件1-2；组件1-1为接头1；组件1-2为用于实现所述连接的试剂或试剂组；接头1由两条单链DNA分子组成，命名为单链1-1和单链1-2；连接产物中，双链DNA片段的每条链的3’末端与单链1-1的5’末端连接，双链DNA片段的每条链的5’末端与单链1-2的3’末端相邻但不连接；
    所述组件2的功能为：对DNA进行亚硫酸盐处理；
    所述组件3的功能为：进行DNA复制；所述组件3包括组件3-1和组件3-2；组件3-1为通用引物1；组件3-2为用于实现所述复制的试剂或试剂组；通用引物1的3’端区段与单链1-1反向互补；组件3-2包括DNA聚合酶；所述DNA聚合酶满足如下条件：具有扩增U的能力；
    所述组件4的功能为：给双链DNA的3’末端连接测序接头。
  18. 如权利要求17所述的试剂盒，其特征在于：所述双链DNA片段为具有粘末端的双链DNA，该双链DNA的每条链的3’末端均具有一个粘末端A。
  19. 如权利要求18所述的试剂盒，其特征在于：所述试剂盒还包括组件5；所述组件5的功能为：将双链DNA分子进行平末端化以及3’末端加A。
  20. 如权利要求17所述的试剂盒，其特征在于：双链DNA片段中的每条链的5’末端进行阻断修饰。
  21. 如权利要求17所述的试剂盒，其特征在于：单链1-2中3’末端进行阻断修饰、其他部分与单链1-1反向互补。
  22. 如权利要求20或21所述的试剂盒，其特征在于：所述阻断修饰为双脱氧核苷酸。
  23. 如权利要求17所述的试剂盒，其特征在于：单链1-1的5’末端进行磷酸化修饰。
  24. 如权利要求17所述的试剂盒，其特征在于：单链1-1中的C均为5-甲基胞嘧啶脱氧核糖核苷酸。
  25. 如权利要求17所述的试剂盒，其特征在于：亚硫酸盐处理为亚硫酸氢盐处理或者重亚硫酸盐处理。
  26. 如权利要求17所述的试剂盒，其特征在于：DNA聚合酶满足如下条件：具有3’末端加A活性。
  27. 如权利要求17所述的试剂盒，其特征在于：DNA聚合酶满足如下条件：具有置换活性。
  28. 如权利要求17所述的试剂盒，其特征在于：给双链DNA的3’末端连接测序接头中所述连接的实现为直接连接或间接连接。
  29. 如权利要求28所述的试剂盒，其特征在于：其特征在于：所述连接的实现为直接连接时，所述组件4包括接头2；接头2由两条单链DNA分子组成，命名为单链2-1和单链2-2；单链2-1的3’末端的核苷酸为T，其他部分与单链2-2反向互补。
  30. 如权利要求29所述的试剂盒，其特征在于：单链2-2的5’末端进行磷酸化修饰。
  31. 权利要求1至16中任一所述方法在甲基化测序中的应用。
  32. 权利要求17至30中任一所述试剂盒在甲基化测序中的应用。
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