CN111074353B

CN111074353B - Single-chain library construction method of whole genome methylation library and obtained whole genome methylation library

Info

Publication number: CN111074353B
Application number: CN201811216020.7A
Authority: CN
Inventors: 李艳; 耿春雨; 杨心石; 蒋慧
Original assignee: MGI Tech Co Ltd
Current assignee: MGI Tech Co Ltd
Priority date: 2018-10-18
Filing date: 2018-10-18
Publication date: 2023-10-13
Anticipated expiration: 2038-10-18
Also published as: CN111074353A

Abstract

A whole genome methylation library single-chain library construction method and the obtained whole genome methylation library, the method comprises the following steps: (a) Performing bisulfite treatment on the interrupted or unbroken genome DNA to convert unmethylated C base into U base and form random AP site on the DNA; (b) 5 'end dephosphorylation treatment is carried out on the reaction product of the previous step by adopting dephosphorylase so as to remove the phosphate group at the 5' end; (c) Amplifying the denatured single strand of the reaction product of the previous step under the action of DNA polymerase and random primers to synthesize a second strand; and (d) removing the AP site using an endonuclease having an AP site removal function; wherein step (d) is performed at any stage after step (a). The method of the invention has the advantages of low initial quantity, high library quality, high data utilization rate and data reliability.

Description

Single-chain library construction method of whole genome methylation library and obtained whole genome methylation library

Technical Field

The invention relates to the technical field of genome methylation research, in particular to a single-chain library construction method of a whole genome methylation library and the whole genome methylation library obtained by the method.

Background

The variety of epigenetic modifications is rich and mainly comprises 5-methylcytosine (5 mC), 5-hydroxymethylcytosine (5 hmC) and N6-methyladenine (6 mA) modifications. Genome-wide methylation modification affects genome imprinting, gene expression, RNA alternative splicing, nucleosome localization and dynamic binding of transcription factors at different levels of precision, and is thus an important regulatory factor for biological traits. Whole genome methylation pool sequencing (WGBS) is the fundamental and important research tool in epigenetic histology. The methylation sites are detected at the whole genome level with single base resolution, so that not only can the change of the methylation level of common areas such as CpG islands be found with high precision, but also the difference of the methylation level of areas such as a genome body area and an intergenic area can be analyzed, the influence of methylation on the state of a chromosome and the change of a gene structure can be analyzed, and the biological problem can be solved from multidimensional analysis.

The current high throughput whole genome methylation detection technology mainly comprises: chip-based methylation profile analysis such as Illumina Human Methylation850 head Chip and flight mass spectrum detection of Sequenom Mass ARRAY platform by using Illumina TruSeq, NEB Next Ultra, swift Bioscience Accel-NGS, QIAGEN EpiTect and BGI MGIEasy as main flow technologies of whole-gene methylation library construction and second generation sequencing; a methylation three-generation sequencing technology developed by Pacific Biosciences company. Sequencing analysis methods other than mass-to-flight detection can distinguish between the methylation and unmethylation of the sample at each base site.

The second generation sequencing technology (Next Generation Sequencing) has high flux and low cost, and shows unique charm in the application of various fields such as DNA, RNA, appearance and the like. On the tenth International Association of genomics (ICG-10) at 24 th 10 months 2015 and the ICG-12 at 26 th 10 months 2017, the Huada gene successively released its novel desktop sequencing system BGISEQ-500, which was developed independently, and MGISEQ-2000, which was faster, more abundant, and of higher sequencing quality. In 7 months 2018, hua Dazhi issued a library product suitable for use in the large autonomous sequencers BGISEQ-500 and MGISEQ-2000: the MGIEasy whole genome methylation sequencing library is prepared into a kit V1.0, and the kit adopts a pre-BS-WGBS library building process, namely, sample DNA is subjected to joint adding, bisulfite (Bisulfite) treatment and amplification, and library building is completed.

The currently mainstream whole genome methylation pool-building sequencing technology comprises: pre-BS-WGBS library construction, conventional POST-BS-WGBS, and POST-BS-WGBS-SPLAT. The pre-BS-WGBS library construction method is characterized in that joints are added at two ends of broken double-stranded DNA, and then the double-stranded DNA is subjected to bisulfite conversion treatment and then amplified by PCR polymerase capable of amplifying uracil, so that the double-stranded DNA library construction method is adopted. The bisulfite conversion treatment causes DNA to be single-stranded and fragmented, and generates a plurality of AP sites (apurinic/apyrimidinic site, no base sites) on the DNA, wherein the AP sites are randomly generated from A, T, C, G bases, and the pre-BS-WGBS library construction method carries out the bisulfite conversion treatment after the addition of the linker, so that the effective template is reduced to about 1/10.

The conventional POST-BS-WGBS library construction method is that bisulfite is converted and then a joint is added, so that a single chain library construction method is adopted, and a new chain is usually synthesized and sequenced. The library construction method does not repair abasic sites generated by the bisulfite conversion treatment, which reduces the effectiveness of library construction. The single-strand library-building two-strand synthesis method generally selects a random primer, the base composition of the commonly used primer is N (A, G, C, T), the proportion of common C in DNA subjected to bisulfite conversion treatment is very low, and the base composition of the random primer is N, so that the random primer is not suitable for two-strand synthesis of most templates.

The POST-BS-WGBS-SPLAT method converts bisulfite and then adds a linker, which is a double-strand library building method, and each strand of the genome and the synthesized two strands are sequenced. The 3 '-end and the 5' -end of the library construction method are connected by adopting a joint of single-stranded DNA and double chains in sequence, and the efficiency is lower. The original strand and the newly synthesized complementary strand of the bisulfite conversion treatment are sequenced, which means that the same segment of DNA original template is sequenced twice, and the sequencing data is wasted by one time although the functions of mutual verification accuracy among certain data are achieved.

Disclosure of Invention

The invention provides a single-chain library construction method of a whole genome methylation library and the obtained whole genome methylation library, wherein the method carries out 5 '-end dephosphorylation treatment on genome DNA subjected to heavy sulfite treatment, so that a gap is formed between the 3' -end and the connector when the connector is added to an original chain, the gap cannot be effectively amplified, and the synthesized two chains can be effectively amplified, therefore, the library construction connector can adopt connectors without special modification such as methylation, and the PCR polymerase does not need to adopt special PCR enzyme capable of amplifying U bases, and the library construction cost is greatly reduced.

According to a first aspect, in one embodiment there is provided a single strand pooling method of whole genome methylation library, comprising the steps of:

(a) Performing bisulfite treatment on the interrupted or unbroken genome DNA to convert unmethylated C base into U base and form random AP site on the DNA;

(b) 5 'end dephosphorylation treatment is carried out on the reaction product of the previous step by adopting dephosphorylase so as to remove the phosphate group at the 5' end;

(c) Amplifying the denatured single strand of the reaction product of the previous step under the action of DNA polymerase and random primers to synthesize a second strand; and

(d) Removing the AP sites by adopting endonuclease with the AP site removing function;

wherein the step (d) is performed at any stage after the step (a).

Further, each base site of the above random primer is independently selected from one of A, T, C bases.

Further, the random primer is 4 to 20 bases, preferably 8 bases in length.

Further, the DNA polymerase is one or more selected from Klenow exo-, klenow fragment (3 '-5' exo-), bst DNA polymerase, vent DNA polymerase (3 '-5' exo-), vent DNA polymerase, phi29DNA polymerase, deep Vent DNA polymerase (3 '-5' exo-), deep Vent DNA polymerase and DNA polymerase I having a nick translation function having a strand displacement function.

Further, the endonuclease having an AP site-removing action is an FPG enzyme.

Further, the final concentration of the above FPG enzyme in the reaction system was 0.8U/. Mu.L.

Further, the above genomic DNA is a disrupted genomic DNA having a major band of more than 300 bp.

Further, the whole genome methylation library is used for sequencing by a BGI sequencing platform.

Further, the method further comprises one or more of the following steps: terminal repair, linker ligation, PCR amplification, single-stranded circularization, and preparation of DNA nanospheres.

According to a second aspect, in one embodiment there is provided a whole genome methylation library obtained according to the method of the first aspect.

According to the method disclosed by the invention, the 5 '-end of the genome DNA subjected to the heavy sulfite treatment is subjected to dephosphorylation treatment, so that a gap is formed between the 3' -end and the connector when the connector is added to the original chain, the original chain cannot be effectively amplified, and the synthesized two chains can be effectively amplified, so that the connector for constructing the library can adopt the connector without special modification such as methylation, and the PCR polymerase does not need to adopt special PCR enzyme capable of amplifying U bases, and the cost for constructing the library is greatly reduced. Meanwhile, an endonuclease with an AP site removing function is adopted to remove the AP site, so that single-stranded or double-stranded DNA is disconnected at the AP site, more effective templates with joints added and PCR (polymerase chain reaction) can be formed, and the whole genome coverage is improved.

In addition, in a further improved version, the random primer is degenerate and each position of the random primer is selected from A, T, C bases instead of the conventional A, T, C, G bases, thereby more efficiently annealing to the bisulfite treated template and thus more efficiently performing two-strand extension.

Drawings

FIG. 1 is a full flow chart of a single-stranded library construction method for a whole genome methylation library according to one embodiment of the present invention;

FIG. 2 is a schematic diagram of the repair mechanism of an AP site according to one embodiment of the present invention, wherein the left graph shows a single-stranded repair mechanism and the right graph shows a single-double-stranded chimeric repair mechanism.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present invention. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

In one embodiment of the present invention, a single-strand library construction method for a whole genome methylation library is provided, as shown in fig. 1, comprising the steps of:

wherein the step (d) is performed at any stage after the step (a).

FIG. 1 shows a full flow chart of a single-strand library construction method of a whole genome methylation library according to one embodiment. In the present invention, however, only the steps (a) to (d) described above belong to the essential steps, i.e. steps 1 to 4 in fig. 1, and the other subsequent steps belong to the optional steps, i.e. in a preferred embodiment, one or more of the following steps are included: terminal repair, linker ligation, PCR amplification, single-stranded circularization, and preparation of DNA nanospheres.

The whole genome methylation library single-chain library construction method can be applied to any sequencing platform, in particular to various second-generation high-throughput sequencing platforms. In a preferred embodiment of the invention, whole genome methylation libraries are used for BGI sequencing platform sequencing.

In embodiments of the invention, the pooling starting material is "broken or unbroken genomic DNA," wherein the unbroken genomic DNA may be genomic DNA extracted from the sample material by any extraction method. The disrupted genomic DNA may be fragmented DNA obtained by any disruption method, including physically disrupted DNA and enzymatically disrupted DNA, wherein the enzymatically disrupted DNA needs to be purified and then subjected to bisulfite treatment. The disrupted genomic DNA generally requires a major band of greater than 300bp, which is too short to generate efficient library fragments. However, in the case of changing the two-chain synthesis mode to the fixed sequence reverse extension method in the embodiment of the invention, the method of the invention can be applied to DNA fragments with various fragment lengths, and can be compatible with the construction of libraries of DNA with the length of 300bp or less.

In the embodiment of the invention, genome DNA is treated by Bisulfite (Bisulfite), unmethylated C base is converted into U base by deamination, and methylated C base has no structural change. More than 95% of the bisulfite treated DNA becomes fragmented single stranded DNA structure and forms random AP sites (apurinic/apyrimidinic site, abasic sites) on the DNA, which are randomly generated from any of the A, T, C, G bases.

Then, a dephosphorylase is used, e.g. Thermo Scientific ^TM FastAP ^TM Thermosensitive Alkaline Phosphatase (temperature sensitive alkaline phosphatase) to remove phosphate groups at the 5 'end of single-stranded, double-stranded DNA, for example, 5' dephosphorylation may be achieved by incubation at 37℃for 10 min. The dephosphorylation step can ensure that the 5 'end of the original chain treated by the bisulfite is not added with a joint in the joint connection step, and a gap (nick) is formed between the 5' end of the original chain and the joint because of lack of a phosphate group during joint addition, so that the original chain can not be effectively amplified in the subsequent PCR amplification process, and the synthesized two chains can be effectively amplified, so that the joint for constructing a library is a joint without special modification such as methylation, and the PCR polymerase is a special PCR enzyme without amplification of U bases, and the library construction cost is greatly reduced. Directly after dephosphorylation treatment, the dephosphorylating enzyme is heat-inactivated, e.g. inAnd carrying out heat inactivation reaction at 95 ℃ for 5min, inactivating the dephosphorylating enzyme and realizing complete melting of the bisulfite reaction product, so that the effective template amount for two-chain synthesis can be increased.

The products after bisulfite treatment, dephosphorylation treatment and high temperature denaturation can be subjected to two-chain synthesis reaction. In a preferred embodiment of the invention, in step (c), each base site of the random primer is independently selected from one of A, T, C bases, i.e., H base represents one of A, T, C bases as shown in FIG. 1. Since more than 99.5% of unmethylated C bases are converted to U bases in Bisulfite (bisufite) -treated DNA, the proportion of methylated C bases in the template is generally not high (specifically determined by the methylation rate of the sample), and thus the embodiment of the invention innovatively uses H bases instead of N (A, T, C or G) bases as random primers for two-strand synthesis, and can anneal more efficiently with the Bisulfite-treated template, thereby performing two-strand extension more efficiently.

In the embodiment of the invention, the length of the random primer can be 4-20 bases, however, when the primer is overlong, the specificity is too high, and the primer is difficult to be effectively matched with a template; and when the primer is too short, the annealing temperature is lower, the primer is difficult to anneal and combine with a template at the optimal temperature of enzyme, and the random primer (H8 primer) with 8 bases (8 nt) tested is more suitable for two-strand synthesis.

In the two-strand synthesis process, the DNA polymerase may be selected from one or more of Klenow exo-, klenow fragment (3 '-5' exo-), bst DNA polymerase, vent DNA polymerase (3 '-5' exo-), vent DNA polymerase, phi29DNA polymerase, deep Vent DNA polymerase (3 '-5' exo-), deep Vent DNA polymerase and DNA polymerase I having a nick translation function. In a preferred embodiment of the present invention, a DNA polymerase I (PolI enzyme) having a nick translation function is used, the optimum temperature of which is 37 ℃.

In the embodiment of the present invention, the step (d) of removing the AP site using an endonuclease having an AP site-removing function may be performed at any stage after the step (a). In the embodiment of the present invention, the "step (d) is performed at an arbitrary stage after the step (a)" means that the step (d) is not limited except for being performed after the step (a), for example, the step (d) may be performed between the step (a) and the step (b), between the step (b) and the step (c), or after the step (c), or the like, and the step (d) may be performed simultaneously with the step (b) or the step (c), or the like, without causing mutual influence.

Removal of the AP site is also referred to as "AP site repair", and as shown in the left diagram of FIG. 2, if AP site repair is performed before two-strand synthesis, single-stranded DNA is broken at the AP site by an endonuclease having an AP site removal action (i.e., a repair enzyme), and the broken single-stranded DNA synthesizes two strands, respectively, in subsequent amplification. As shown in the right graph of FIG. 2, if AP site repair is performed after two-strand synthesis, when the extension reaction proceeds to the AP site, the extension reaction cannot proceed due to lack of a base as a template, and the single-double stranded chimera is broken at the chimeric site by the action of endonuclease having AP site removal (i.e., repair enzyme).

In a preferred embodiment of the present invention, the endonuclease having an AP site removal action is FPG enzyme (formamidopyrazine [ fampy ] -DNA glycosidase, formyl pyrimidine DNA glycosidase), which is a base cleavage repair enzyme (base excision repair enzyme) having N-terminal glycosidase activity and AP site cleavage enzyme activity acting mainly on various oxidized bases, such as 7, 8-dihydro-oxo-guanine (7, 8-dihydro-8-oxo-guanine), oxo-guanine (8-oxo-adenine), 2, 6-diamino-4-hydroxy-5-carboxamide pyrimidine (fampy-guide), methyl-2, 6-diamino-4-hydroxy-5-carboxamide pyrimidine (methyl-fampy-guide), carboxamide adenine (fampy-adenine), xanthomycin B1-carboxamide pyrimidine (aflavip-guide), 5-hydroxy-cytosine (5-hydroxy-pyrimidine) and 5-hydroxy-uracil). The N-terminal glycosidase can cut off damaged purine from double chains to generate a purine-free site; and its AP site lyase activity cleaves deoxyribose and phosphate groups (dR 5P) at the AP site, leaving a 5 'terminal nucleic acid group and a 3' terminal hydroxyl group, resulting in cleavage in the strand.

The concentration of the FPG enzyme in the reaction system can be adjusted according to the requirement, in the preferred embodiment of the invention, the FPG enzyme with the final concentration of 0.8U/. Mu.L is tested, and the AP site in the sample after the bisulfite treatment can be effectively repaired after the two-chain synthesis, so that the single-chain and double-chain chimera is broken at the single-chain and double-chain connection position, and more templates for effective linker adding and PCR are formed. In other embodiments, such as those in which the AP site is removed prior to two-strand synthesis, the working concentration of the FPG enzyme may be adjusted as desired.

In summary, the embodiments of the present invention provide a method for preparing a genome-wide methylation library with low initial quantity, high library quality, high data utilization rate and data reliability. The beneficial effects are as follows: the number of effective templates is increased, and the initial quantity of library establishment is reduced. The bisulfite treatment is followed by dephosphorylation treatment, so that single-chain library establishment is realized, and the use of PCR enzyme capable of amplifying uracil is avoided, thereby reducing the cost; the simultaneous sequencing of the original chain and the synthesized two chains is avoided, and the data utilization rate is improved. The primer for two-chain synthesis creatively adopts H (A, T, C) base to replace conventional N (A, T, C, G) base, improves the probability of template which is mainly A, T, G after annealing to bisulfite treatment, and effectively improves the yield of warehouse building. And in the AP site repairing process, single-chain and double-chain chimera caused by the AP site after two-chain synthesis is innovatively and effectively removed, and the quality of the library and the PCR amplification efficiency are improved.

As shown in fig. 1, in a preferred embodiment, the library construction method of the present invention is based on a BGI sequencing platform, and therefore further comprises the steps of: end repair and 3' end addition A treatment; the joints are connected; polymerase Chain Reaction (PCR); high temperature denaturation and single strand cyclization of the PCR product; rolling circle replication of the single-stranded loops to form DNA nanospheres (DNA nanoball, DNB). Finally, the library was immobilized on an arrayed silicon chip by the cPAS principle and sequenced by camera acquisition of signals. These steps are performed according to the current routine operation of the BGI sequencing platform.

It is particularly emphasized that, as shown in FIG. 1, since the above-mentioned bisulfite treatment is followed by dephosphorylation treatment in the library creating step, a gap (nick) is formed between the original strand and the linker upon ligation of the linker, and since the original strand template contains a large number of U bases, the PCR polymerase used in the examples of the present invention cannot amplify the U bases and thus does not become a template for PCR.

In the library construction scheme based on the BGI library construction sequencing platform, single-joint (with different tag sequences) library construction is realized, and single tube end repair to joint addition reaction is carried out, so that the operation is convenient. The DNA Nanosphere (DNB) method and the matrix chip are adopted in the sequencing, so that the fidelity with the template can be greatly improved, the error rate can be reduced, and the data repetition rate can be reduced.

The following detailed description of the present invention is provided by way of example only, and should not be construed as limiting the scope of the invention.

Examples

The sample of this example is unbroken genomic DNA.

(1) Bisulfite treatment of DNA:

the reaction mixtures shown in table 1 below were prepared:

TABLE 1

The reaction conditions are shown in table 2 below:

TABLE 2

Bisulfite treatment conditions	Time
		98℃	10min
64℃	2.5h
		4℃	∞

After the reaction was completed, the mixture was purified by a purification column and dissolved back in 10. Mu.L.

(2) 5' -end dephosphorylation treatment:

the reaction mixtures shown in table 3 below were prepared:

TABLE 3 Table 3

The reaction conditions are shown in table 4 below:

TABLE 4 Table 4

And (3) placing the mixture on ice after the reaction is finished for 3 to 5 minutes.

(3) Two-chain synthesis reaction:

the reaction mixtures shown in table 5 below were prepared:

TABLE 5

Reagent(s)	Volume of
		10XNEB2 buffer	3μL
2.5mM dNTP	1μL
		40 mu M H primer	1μL
Klenow exo-	2μL
		Water and its preparation method	3μL
Total amount of	10μL

Immediately adding 10. Mu.L of the two-chain synthesis reaction solution to the reaction product of step (2), and carrying out the two-chain synthesis reaction.

The reaction conditions are shown in table 6 below:

TABLE 6

Treatment conditions	Time
		37℃	30min
4℃	∞

After the reaction, the mixture was purified using 1X magnetic beads and finally dissolved back in 20. Mu.L of TE solution.

(4) FPG enzyme treatment

The reaction mixtures shown in table 7 below were prepared:

TABLE 7

Reagent(s)	Volume of
		10XNEB2 buffer	3μL
FPG enzyme (8U/. Mu.L)	0.5μL
		Water and its preparation method	6.5μL
Total amount of	10μL

Immediately, 10. Mu.L of the FPG enzyme repair reaction solution was added to the product reconstituted after the purification in step (3), and the following reaction was carried out.

The reaction conditions are shown in table 8 below:

TABLE 8

Treatment conditions	Time
		37℃	30min
4℃	∞

After the reaction, the mixture was purified using 1X magnetic beads and finally dissolved back in 40. Mu.L TE solution.

(5) And (3) terminal repair:

the reaction mixtures shown in table 9 below were prepared:

TABLE 9

Reagent name	Volume of
		Nuclease-free Water (Water)	2.1μL
10 XPNK buffer	5μL
		5:1dATP:dNTP	0.6μL
Klenow fragment	0.1μL
		rTaq	0.2μL
T4DNA Polymerase (Polymerase)	2μL
		Total amount of	10μL

Immediately, 10. Mu.L of the end-repair reaction solution was added to the product which was reconstituted after the purification in step (4), and the following reaction was carried out.

The reaction conditions are shown in table 10 below:

table 10

Treatment conditions	Time
		37℃	30min
65℃	15min
		4℃	∞

(6) Joint connection

At the end of the reaction, 5. Mu.L of Ad153 linker was immediately added, the concentration of which was determined by the initial amount of library build in step (1).

The recommended amount to be added and the diluted concentration of the linker were as shown in Table 11 below (10. Mu.M of the linker stock):

TABLE 11

Sample DNA amount	Dilution factor	Dilution input (μL)
			200	Not diluting	5
100	2	5
			75	3	5
50	4	5
			25	8	5
10	20	5

The joint connection mixture was prepared as shown in table 12 below:

table 12

After adding an appropriate amount of linker to the end-repair product, 25. Mu.L of the linker ligation mixture was added and the following reaction was performed.

The reaction conditions are shown in table 13 below:

TABLE 13

Treatment conditions	Time
		23℃	30min
4℃	∞

(7) And (3) PCR amplification:

the PCR reaction solution was prepared on ice as shown in Table 14 below:

TABLE 14

Component (A)	Standard quantity of reaction
		PCR enzyme mixture	25μL
Ad153 primer mixture (20. Mu.M)	3μL
		Water and its preparation method	2μL
Total amount of	30μL

Immediately, 30. Mu.L of the reaction solution was added to the product which was reconstituted after the purification in step (6), and the following reaction of Table 15 was carried out:

TABLE 15

The cycle number was calculated from table 16 below:

table 16

(8) Denaturation (denaturation)

According to the main fragment distribution of the PCR product, 1pmol of the PCR product was taken into a new 0.2mL PCR tube, and the total volume was made up to 48. Mu.L with TE buffer.

The reaction of Table 17 below was performed under denaturing conditions by placing 0.2mL of the PCR tube on a PCR instrument:

TABLE 17

After the reaction was completed, 0.2mL of the PCR tube was immediately transferred to ice, and after standing for 2min, it was subjected to instantaneous centrifugation.

(9) Single-stranded cyclization

Single-stranded cyclization reaction solutions were prepared on ice as shown in Table 18 below:

TABLE 18

Component (A)	Standard quantity of reaction
		Splint Buffer (cyclization Buffer)	11.6μL
DNA Rapid enzyme (quick Ligase)	0.5μL
		Total amount of	12.1μL

And (3) sucking 12.1 mu L of the prepared single-chain cyclization reaction liquid by a pipette, adding the product in the step (8), vortex vibrating for 3 times, and collecting the reaction liquid to the bottom of a tube by instantaneous centrifugation each time for 3 s.

A0.2 mL PCR tube was set on a PCR instrument, and the reaction was performed under the single strand cyclization reaction conditions shown in Table 19 below:

TABLE 19

Temperature (temperature)	Time
		Thermal cover	ON
37℃	30 min
		4℃	Holding

After the reaction was completed, the reaction was centrifuged instantaneously, and a 0.2mL PCR tube was transferred onto ice, immediately followed by the next reaction.

(10) Digestion by enzyme digestion

The digestion reaction was prepared on ice in advance, as shown in table 20 below:

table 20

And (3) sucking 4 mu L of the prepared digestion reaction liquid, adding the prepared digestion reaction liquid into the PCR tube in the step (9), vortex vibrating for 3 times, and collecting the reaction liquid to the bottom of the tube by instantaneous centrifugation for 3s each time.

The reaction was performed according to the cleavage digestion conditions of table 21 below:

table 21

Temperature (temperature)	Time
		Thermal cover	ON
37℃	30min

The reaction solution was collected to the bottom of the tube by instantaneous centrifugation. To a 0.2mL PCR tube, 7.5. Mu.L of 0.2M EDTA solution was added, vortexed 3 times, each for 3s, and the reaction solution was collected to the bottom of the tube by instantaneous centrifugation, and the whole reaction solution was aspirated and transferred to a new 1.5mL EP tube. And (5) completing the construction of the library.

(11) Preparation and sequencing of DNB:

specific procedures may be referred to the MGI sequencing kit instructions.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A single-stranded library construction method for a whole genome methylation library, comprising the steps of:

(c) Amplifying the denatured single strand of the reaction product of the previous step under the action of a DNA polymerase and a random primer, each base site of the random primer being independently selected from one of A, T, C bases, to synthesize a second strand; and

(d) Removing the AP sites by adopting endonuclease with AP site removal effect, wherein the endonuclease with AP site removal effect is FPG enzyme;

wherein said step (d) is performed at any stage after said step (a).

2. The whole genome methylation library single strand pooling method according to claim 1, wherein the random primer is 4-20 bases in length.

3. The whole genome methylation library single strand pooling method according to claim 1, wherein the random primer is 8 bases in length.

4. The whole genome methylation library single-strand library construction method according to claim 1, wherein the DNA polymerase is selected from one or more of Klenow exo-, bst DNA polymerase, vent DNA polymerase, phi29DNA polymerase, deep Vent DNA polymerase, and DNA polymerase I having a nick translation function.

5. The single-stranded whole genome methylation library construction method according to claim 1, wherein the final concentration of said FPG enzyme in the reaction system is 0.8U/. Mu.l.

6. The whole genome methylation library single-strand banking method according to claim 1, wherein the genomic DNA is interrupted genomic DNA with a major band of more than 300 bp.

7. The whole genome methylation library single-stranded library construction method of claim 1, wherein the whole genome methylation library is used for BGI sequencing platform sequencing.

8. The whole genome methylation library single-strand pooling method of claim 1, further comprising one or more of the following steps: terminal repair, linker ligation, PCR amplification, single-stranded circularization, and preparation of DNA nanospheres.