HK1168393B - Method for accurate detection of whole genome methylation sites by utilizing trace genome dna (deoxyribonucleic acid) - Google Patents

Abstract

The present invention establishes a new method for precise detection of whole genome methylation sites using trace genomic DNA based on Illumina conventional tag library sequencing and methylation conventional sequencing, combined with conventional library tag sequencing methods.Moreover, the present invention innovatively utilizes exogenous NA for efficient co treatment with bisulfite when constructing methylation libraries using trace amounts of genomic DNA; At the same time, there is no need for fragment size selection, and PCR amplification can be directly performed after bisulfite treatment.The method of the present invention overcomes the disadvantages of conventional methylation sequencing, such as inability to mix samples, low PCR amplification efficiency, and inability to study trace DNA samples.

Description

Method for accurately detecting full-genome methylation sites by using trace genome DNA

Technical Field

The invention relates to the technical field of methylation high-throughput sequencing, in particular to the technical field of trace DNA whole genome methylation high-throughput sequencing. In addition, the invention also relates to a tag sequencing technology and a method for constructing a tag library by a plurality of samples in the same reaction system. The method of the invention is particularly suitable for second generation sequencing techniques, in particular solexa sequencing techniques.

Background

DNA methylation is the most deeply studied epigenetic mechanism, plays an important role in maintaining normal cell functions, inhibiting damage of parasitic DNA components to genome integrity, chromatin structure modification, X chromosome inactivation, genome imprinting, embryonic development and human tumorigenesis, and is one of the new research hotspots at present^[1]。

The main methods for DNA methylation research today are: hybridization of whole genome chip, restriction enzyme digestion of methylation sensitive (non-sensitive) restriction enzyme in whole genome, etc., and bisulfite treatment of specific site or partial range gene combined with methylation specific PCR^[2]. Bisulfite treatment (bisufite) in combination with sequencing is the most common and accurate method for studying methylation. Illumina GA is the most commonly applied new generation high-throughput sequencing instrument at present, and has been successfully applied to the whole genome methylation sequencing research^[2]The main process is as follows: the library construction firstly needs to randomly break genome DNA, then carry out end repair on a target fragment, connect 'A' base at the 3 'end of the target fragment, connect the target fragment with the' A 'base at the 3' end with a DNA adaptor (also called adapter) (C site methylation modification), then carry out bisulfite treatment, then carry out fragment selection, finally amplify the target fragment through PCR reaction, and finally recover the target fragment library containing the DNA adaptor^[2]See fig. 1. The main drawbacks or problems of this method are: 1. the inability to mix multiple samples for methylation library construction; 2. PCR amplification efficiency is not high, and a sufficient amount of library can be obtained after multiple cycles (more than 16 cycles) of amplification for high-throughput sequencing; 3. the initial construction of the library needs more than 5-10 mug of genomic DNA and is not suitable for the construction of a micro DNA sample.

Disclosure of Invention

Sequencing of Illumina conventional tag (also referred to as index) library in accordance with the present invention^[3](drawing)2) And methylation routine sequencing^[2](figure 1) three innovative changes were made: 1. the conventional library tag sequencing method is introduced into methylation sequencing research, a conventional library tag sequencing adaptor sequence (table 1) provided by Illumina company is modified, a base sequence (which can be used as a tag sequence) of 6bp is added, methylation modification is adopted during synthesis, the length of a subsequent PCR primer is increased, and the subsequent PCR amplification efficiency is effectively increased for DNA treated by bisulfite; 2. when a methylation library is constructed by using trace genome DNA, the bisulfite is efficiently co-processed by adding exogenous DNA innovatively, so that trace DNA fragments are protected, the damage of bisulfite to the trace DNA is reduced to the maximum extent, and the high-precision methylation detection of the entire level of a nanogram-level (30-100ng) genome is realized; 3. the method changes the flow that the conventional Illumina methylation sequencing needs to be subjected to fragment size selection before bisulfite treatment and then PCR amplification, and finds out a condition that the PCR amplification is directly carried out after bisulfite treatment without fragment size selection. Overcomes the defects that the samples can not be mixed in the conventional methylation sequencing, the PCR amplification efficiency is low and the trace DNA samples can not be researched. The flow chart of the construction of the trace DNA whole genome methylation high-throughput sequencing library is shown in figure 3.

TABLE 1 Illumina GA-based minigenome DNA Whole genome methylation high throughput sequencing related sequences (5 '- > 3')

In one aspect, the invention provides a method for whole genome methylation high-throughput sequencing, and in a specific embodiment, the method comprises the following steps:

step AFragmentation of target genomic DNA and exogenous genomic DNA

The starting research material of interest and the material used as the exogenous genomic DNA may be genomic DNA of any species (e.g., human, plant, insect), and the fragmentation is usually carried out by nebulization, ultrasonic fragmentation, HydroShear or enzyme digestion to break the genomic DNA into fragments of 100-200bp in size. Among the above-mentioned many common methods, the ultrasonic fragmentation method is preferably used, and the exogenous genomic DNA is preferably selected from Arabidopsis thaliana genomic DNA.

Step BTerminal modification of genomic DNA

Fragmented DNA requires end modification, the ends first being filled in with polymerases such as Klenow, T4 polymerase and T4 polynucleotide kinase and dntps to produce blunt-ended DNA. The "A" bases were then added to the 3 ' end of the filled-in sequence using Klenow fragment (3 ' -5 ' exo-) polymerase and dATP.

Step CJoint connection of micro-building warehouse and bisulfite treatment

The sequence with the base of ' A ' added at the 3 ' end is connected with a specially designed methylation-modified micro-library-building adaptor (methylation modification at the C site) under the action of T4 ligase. Then, 200ng of the fragmented Arabidopsis genomic DNA was added to the fragments with linkers at both ends, and then treated with bisulfite all at once, thereby converting unmethylated cytosine to uracil.

Step DPCR amplification and library gel cutting purification

Taking DNA converted by bisulfite as a template, adding a PCR primer sequence specially designed for a trace library construction joint sequence, carrying out PCR amplification (can be amplified by conventional r-taq or other polymerases) by using hot start taq enzyme aiming at the DNA converted by bisulfite, carrying out electrophoresis on an amplification product by using 2% agarose, cutting off a target band and purifying to obtain the library to be sequenced. PCR amplification preferably uses a hot start taq enzyme.

Compared with the prior methylation high-throughput sequencing technology, the invention has the advantages that: 1. the special adapter (Minim _ adapter) which has higher PCR amplification efficiency and aims at the methylation library construction of trace DNA replaces the adapter used by the conventional library, compared with the conventional adapter, a partial sequence is changed, the sequence length is increased by 6bp base (can be used as a tag sequence for the sequencing of a plurality of mixed samples), the subsequent PCR amplification efficiency and the product amount are increased for DNA treated by bisulfite, the library is built by the same material in an equal amount, and the product concentration is increased from 1.67 ng/muL to 20.04 ng/muL under the same PCR condition (see the partial results of the examples as shown in FIGS. 6-8); 2. when a methylation library is constructed by using trace genome DNA, the DNA added with the exogenous vector creatively and together with the target DNA is subjected to high-efficiency bisulfite co-treatment. The single-stranded DNA denatured under the dual actions of high temperature and bisulfite (high salt and low pH value environment) is easy to destroy and degrade, the addition of exogenous DNA has a certain buffer effect on the destruction effect, the destruction of bisulfite on trace DNA can be reduced to the maximum extent, and in addition, the subsequent purification efficiency is improved due to the increase of DNA amount, so that the methylation detection with high precision at the nanogram level (30-100ng) genome overall level becomes practical. 3. The method changes the flow that the conventional methylation sequencing of the Illumina needs to be subjected to fragment size selection and then PCR amplification before and after bisulfite treatment, finds out a condition that the PCR amplification is directly carried out after the bisulfite treatment without the fragment size selection, concretely refers to detailed parameters of an embodiment, and mainly changes the using amount of a terminal repair enzyme and the adding amount of a linker in a linker linking step (reduces to 1/10 of the using amount of a conventional library). The method overcomes the defects that the samples cannot be mixed in the conventional methylation sequencing, the PCR amplification efficiency is low and the trace DNA sample cannot be researched. Accurate studies of whole genome methylation can also be performed for low sample size samples.

In one aspect, the invention provides a method for constructing a genome-wide methylation high-throughput sequencing library, which is used for trace amounts of genomic DNA, preferably nanogram-scale genomes, and more preferably 30-100ng genomes.

In one embodiment of the present invention, the method comprises the steps of:

step AFragmentation of target genomic DNA and exogenous genomic DNA

The genomic DNA of interest and the material as the exogenous genomic DNA may be any species including various plant, animal, microorganism, such as human, plant, particularly Arabidopsis thaliana, insect, particularly mammalian including human, mouse genomic DNA; the fragmentation method comprises atomization, ultrasonic fragmentation, HydroShear or enzyme digestion treatment, so as to break the genome DNA into fragments with the size of preferably 100-200 bp; the fragmentation method preferably adopts an ultrasonic fragmentation method, and the exogenous genomic DNA is preferably selected from arabidopsis genomic DNA;

step BTerminal modification of genomic DNA

For fragmented DNA, the ends are first filled in with polymerases including, but not limited to, Klenow, T4 polymerase and T4 polynucleotide kinase, as well as dntps to produce blunt-ended DNA; the "A" bases are then added to the 3 ' end of the filled-in sequence, preferably using Klenow fragment (3 ' -5 ' exo-) polymerase and dATP.

Step CJoint connection of micro-building warehouse and bisulfite treatment

Connecting the obtained DNA sequence with the base of ' A ' added at the 3 ' end with a micro-library-building adaptor which is subjected to methylation modification, preferably C site methylation modification, under the action of ligase including but not limited to T4 ligase, and preferably connecting the micro-library-building adaptors at both ends of the sequence; then 100-500ng, preferably 200ng, was added to the fragments with linkers at both endsStep A(ii) the middle-fragmented Arabidopsis genomic DNA, which is then treated together with bisulfite, preferably for 2 hours, to convert unmethylated cytosine to uracil;

step DPCR amplification and library gel cutting purification

Adding a PCR primer sequence aiming at a trace library building joint sequence by taking the obtained DNA converted from the bisulfite as a template to carry out PCR amplification; PCR amplification preferably uses hot start taq enzymes including but not limited to conventional r-taq or other polymerases, and the library to be sequenced is obtained after the amplification product is electrophoresed using preferably 2% agarose and the band of interest is excised and purified.

In one embodiment of the invention, the methodStep CThe miniprep connectors used in (1) are Minim _ adapter1 and Minim _ adapter2 shown in table 1.

In one embodiment of the invention, the methodStep DThe PCR primers used in (1) were the Minim _ PCR primer1.1 and the Minim _ PCR primer2.1 shown in Table 1.

In another aspect the invention provides a sequencing library, preferably a minigenome DNA whole genome methylation high throughput sequencing library, constructed by the method described above.

In a further aspect the invention provides the use of a sequencing library, preferably a minigenome DNA whole genome methylation high throughput sequencing library, constructed by the method described above for performing sequencing, wherein the sequencing can be performed by a second generation sequencing platform.

In another aspect, the invention provides adaptors for micro sequencing libraries, particularly micro DNA whole genome methylation high throughput sequencing libraries, which are Minim _ adapter1 and Minim _ adapter2 shown in table 1.

In a specific embodiment of the invention, the use of the linker for constructing a micro-sequencing library, in particular a micro-DNA whole genome methylation high-throughput sequencing library.

In one embodiment of the invention, a microsequencing library, in particular a microsequencing DNA whole genome methylation high throughput sequencing library, is constructed using the linkers described above.

In another aspect, the invention also provides PCR primers for use in a mini sequencing library, in particular a mini DNA whole genome methylation high throughput sequencing library, which are Minim _ PCR primer1.1 and Minim _ PCR primer2.1 shown in table 1.

In a specific embodiment of the invention, the PCR primer is used for constructing a microscale sequencing library, in particular to the use of a microscale DNA whole genome methylation high-throughput sequencing library.

In one embodiment of the invention, a microsequencing library, in particular a microsequencing DNA whole genome methylation high throughput sequencing library, is constructed using the PCR primers described above.

Drawings

FIG. 1: routine methylation sequencing library preparation flow.

FIG. 2: a conventional DNA label library building flow chart of Illumina company.

FIG. 3: the invention relates to a construction flow chart of a micro DNA whole genome methylation high-throughput sequencing library based on Illumina GA.

FIG. 4: schematic diagram of conventional DNA tag library building principle of Illumina company.

FIG. 5: the invention discloses a principle schematic diagram for constructing a micro DNA whole genome methylation high-throughput sequencing library based on Illumina GA.

FIG. 6: 100ng of micro DNA initial micro library, and Agilent 2100 detection result of PCR amplification product according to the method of the invention.

FIG. 7: 30ng of micro DNA initial micro library, and according to the method, the Agilent 2100 detection result of the PCR amplification product.

FIG. 8: 100ng of starting genomic DNA, and adopting a conventional Illumina joint to establish a library according to the method of the invention to obtain a detection result of a PCR amplification product 2100.

FIG. 9: sequencing data of a trace database (100ng DNA) and a conventional database (5ug DNA) are compared with sequencing data of each chromosome of the whole genome to obtain a deep sequencing result.

FIG. 10: sequencing data of a trace library (100ng DNA) and a conventional library (5ug DNA) are compared with the result of chromosome coverage.

FIG. 11: the trace library (100ng D NA) was compared to the conventional library (5ug DNA) sequencing data methylation patterns.

FIG. 12: comparison analysis of methylation correlation between trace library construction (100ng DNA) and conventional library construction (5ug DNA) sequencing data. Wherein Methylation rate of YH-3.5G represents the Methylation rate of YH-3.5G, and Methylation rate of 100ng represents the Methylation rate of 100ng of DNA minipools.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.

Using the method of the invention, we constructed 2 trace whole genome methylation high-throughput sequencing libraries by using 30ng and 100ng of human peripheral blood whole genome DNA (extracted from the blood of a Chinese adult male) as starting materials, detected the quality of 2 libraries by using a sanger sequencing method, and performed high-throughput whole genome sequencing (Illumina GA) on 100ng of the libraries. Meanwhile, the difference of PCR amplification efficiencies after using 100ng of human peripheral blood whole genome DNA (genome DNA extracted from blood of a Chinese adult male) as a starting material and using conventional library construction linkers (Illumina adapter1 and Illumina adapter2) and micro linkers (Minim _ adapter1 and Minim _ adapter2) as linkers is compared.

1. Experimental part:

list of major laboratory instruments

List of relevant reagents

1.1DNA fragmentation

Human whole blood genomic DNA (30ng and 100ng) and Arabidopsis genomic DNA (5. mu.g) were fragmented into fragments of about 100-200bp in the main band (the major band shown in agarose gel electrophoresis) using an sonicator (covaris S2) according to the following parameter settings. Parameters for the ultrasound machine (covaris 2) (appropriately labeled in chinese in the table below) settings:

the broken DNA is directly subjected to the next operation.

1.2 end repair

The reaction mixture was prepared according to the following ratio:

disrupted DNA fragment of 100. mu.L

10x Polynucleotide Kinase Buffer 15μL

dNTP Solution Set 6μL

T4DNA polymerase 7.5. mu.L

Klenow DNA polymerase 1. mu.L

H₂O 13μL

T4Polynucleotide Kinase 7.5μL

Total volume 100. mu.L

Thermomixer (Eppendrf) was adjusted to 20 ℃ for 30min, then purified using the QIAquick PCR Purification Kit (Qiagen), and finally the sample was dissolved in 32. mu.L of Elution Buffer (EB).

1.3DNA fragment 3' end plus "A" base

The reaction mixture was prepared according to the following ratio:

32. mu.L of DNA after end repair

10x blue buffer 5μL

dATP(1mM) 10μL

Klenow exo-(3′to 5′exo-) 3μL

Total volume 50. mu.L

Thermomixer (Eppendrf) was adjusted to 37 ℃ for 30min, then purified using the MiniElute PCR Purification Kit (Qiagen), and finally the sample was dissolved in 10. mu.L of Elution Buffer.

1.4Minim adapter connection

10 μ L of each of the synthesized Minim _ adapter1 and Minim _ adapter2 of 100 μ M was mixed, placed in a water bath at 94 ℃ for 5 minutes and 65 ℃ for 15 minutes, and then naturally cooled to obtain a 50 μ M Minim _ adapter product, and the 50 μ M Minim _ adapter product was diluted 10 times to obtain a 5 μ M Minim _ adapter working solution.

Minim adapter ligation the reaction mixture was prepared according to the following ratio:

10. mu.L of the DNA obtained in the above step

T4DNA ligase buffer 25μL

Minim_adapter(5μM) 1μL

DNA ligase 5μL

ddH₂O 9μL

Total volume 50. mu.L

Thermomixer (Eppendrf) was adjusted to 20 ℃ for 15min, then purified using the QIAquick PCR Purification Kit (Qiagen), and finally the sample was dissolved in 30. mu.L EB.

1.5 ligation products were co-treated with exogenous DNA bisulfite

200ng of fragmented exogenous Arabidopsis genomic DNA was added to the ligation product, followed by bisulfite treatment for 2 h. Bisulfite treatment with ZYMO EZ DNAlation-Gold Kit^TMThe method comprises the following specific steps: A) preparation of CT Conversion Reagent: CT Conversion Reagent (solid mixture) was removed from the kit and 900. mu.l of water, 50. mu.l of M-dispensing Buffer and 300. mu.l of M-Dilution Buffer were added to one tube of CT Conversion Reagent. Dissolve at room temperature and shake for 10 minutes or shake on a shaker for 10 minutes.

B) Preparation of M-WASH BUFFER: M-Wash Buffer which can be finally used was prepared by adding 24ml of 100% ethanol to M-Wash Buffer.

C) The DNA to be converted is dispensed into PCR tubes, and water is supplemented to 20 ul.

D) To the PCR tube, 130. mu.l of CT Conversion Reagent was added and the sample was mixed by flick tube or pipette operation.

E) Placing the sample tube on a PCR instrument according to the following steps:

standing at 98 deg.C for 10 min

Standing at 64 deg.C for 2.5 hr

Immediately for the next operation or stored at 4 ℃ (up to 20 hours).

F) Add 600. mu.l of M-Binding Buffer to the Zymo-Spin IC^TMColumn, and put the Column in the Collection Tube as provided in the kit.

G) Filling of samples into Zymo-Spin IC^TMColumn contains M-Binding Buffer. The cap was closed and the column was inverted several times to mix the samples.

H) Centrifuge at full speed (> 10,000Xg) for 30 seconds to remove the effluent.

I) Add 200. mu.l of M-Wash Buffer to the column and centrifuge at full speed for 30 seconds.

J) 200. mu.l of M-depletion Buffer was added to the column and left at room temperature (20 ℃ C. -30 ℃ C.) for 15 minutes, after incubation, and centrifuged at full speed for 30 seconds.

K) Add 200. mu.l of M-Wash Buffer to the column and centrifuge at full speed for 30 seconds; 200. mu.l of M-Wash Buffer was added and centrifuged for 30 seconds.

L) Add 10. mu.l of M-Elution Buffer directly to the column matrix. The column was placed in a 1.5 ml tube and centrifuged at full speed to elute the DNA.

1.6PCR amplification and library size selection

The following reaction system prepared the reaction mixture and the reagents were placed on ice.

Bisulfite treated DNA 20. mu.l

Minim_PCR primer 1.1 1μl

Minim_PCR primer 2.1 1μl

dNTP Solution Set 4μl

10X PCR Buffer 5μl

JumpStart^TM Taq DNA Polymerase 0.5μl

ddH₂O 18.5μl

Total volume 50. mu.l

PCR reaction conditions

98℃ 30s

(Note: 30ng 12 cycles, 100ng 10 cycles)

72℃ 2min

Storing at 4 deg.C

The amplification products were purified using the PCR Purification Kit (Qiagen), followed by electrophoresis using 2% agarose gel, followed by gel selection of the library of the desired size, gel Purification recovery using the MiniElute PCR Purification Kit (Qiagen), and finally dissolution of the library in 20ul EB.

1.7 library assays

1) Library production using Agilent 2100Bioanalyzer^[4]。

2) Quantitative detection of library yield using QPCR^[4]。

2. Results section:

2.1 Agilent 2100Bioanalyzer assay results for PCR products

The PCR product Agilent 2100Bioanalyzer detection results of FIGS. 6 and 7 show that a methylation library for high-throughput sequencing by using a high-throughput next generation sequencing instrument can be constructed from 30ng and 100ng of starting genomic DNA, and the analysis results of the actual sequencing data show that the method is feasible and can be applied to actual research.

FIG. 8 shows the Agilent 2100Bioanalyzer detection results of PCR amplification products obtained by the microcantilever method of the present invention using conventional Illumina linker, and comparing FIG. 6 and FIG. 7, it is found that the PCR products have no significant bands, but the bands are not obvious, and the concentration does not meet the requirement of high throughput sequencing. The comparison shows that the method of the invention actually improves the PCR amplification efficiency and provides possibility for subsequent sequencing.

2.2 library quality test results (Sanger sequencing)

Table 230ng DNA and 100ng DNA library quality test results

76 and 78 clones are respectively selected from the 2 libraries for quality detection, and the conversion efficiency of the results is over 99 percent, which indicates that the bisulfite treatment realizes high-efficiency conversion. An alignment (also called a map rate) of 100ng of library above 30ng indicates that the initial amount has a large effect on the library building results, but an initial alignment of 30ng above 40% is within acceptable limits considering the initial amount of DNA. In addition, no repeated sequence exists in the view of sequence repetition rate, which shows that the PCR amplification has good randomness and generates no bias.

3. High-level analysis part of sequencing (Illumina GA) result information

Taking 100ng of the library constructed aboveHigh throughput sequencing was performed and high level alignment analysis was performed with the sequencing results of a conventional library (conventional library construction method: using 5ug of the same human whole blood genome as in the above example, library preparation was performed using the conventional methylation sequencing library preparation protocol, see FIG. 1), and the original data of 100ng of library sequencing data 2.52G was compared with the normal whole genome sequencing 1.99G data, which is denoted YH-3.4G.

3.1 comparison with Whole genome alignment

TABLE 3 basic alignment of the results of sequencing data for micro-pooling (100ng DNA) and conventional pooling (5ug DNA)

Sample (I)	Number of Primary bases (Gb)	Number of aligned bases (Gb)	Comparison ratio (%)
				100ng	2.52	1.33	52.65
YH_3.5G	1.99	1.19	59.74

3.2 comparison of Whole genome coverage

TABLE 4 comparison of sequencing data for miniprep (100ng DNA) and conventional prep (5ug DNA) for whole genome coverage

Sample (I)	Coverage of all sites (%)	Coverage of CG (%)
			100ng	32.13	19.16％
YH_3.5G	26.97	16.08％

3.3 comparison of coverage per chromosome (see Table 5 below and FIG. 9)

TABLE 5 comparison of coverage of sequencing data on each chromosome between miniprep (100ng DNA) and conventional prep (5ug DNA)

The results of the comparison show that the coverage trends of the micro-database construction and the conventional database construction on each chromosome are basically consistent.

3.4 Whole genome methylation Pattern ratio comparison (see Table 6, Table 7 and FIGS. 11 and 12)

TABLE 6 Overall analysis and comparison of methylation patterns of sequencing data from micro-library (100ng DNA) and conventional library (5ug DNA)

Mode(s)	C	CG	CHG	CHH
					100ng	4.05	71.46	0.8	0.89
YH_3.5G	4.49	77.5	0.28	0.36

TABLE 7 comparison of methylation patterns of sequencing data from mini-pools (100ng DNA) and conventional pools (5ug DNA) on each chromosome

The comparison shows that the methylation pattern distribution of the micro-pooling and conventional pooling data is substantially consistent both on the whole and on each chromosome.

3.5 data correlation comparison

The correlation between the 100ng library sequencing data and the corresponding sequencing data from the normal library was found to be good by comparison analysis of methylation correlation (correlation coefficient 0.9258572).

By mixing 100ng of libraryAs for the result of comparing the sequencing data with the corresponding data of the conventional library sequencing by high-level information analysis, the comparison condition of 100ng of sequencing data is more ideal from the aspects of comparison efficiency, coverage, methylation rate of each chromosome and correlation, the coverage rate in all aspects is relatively good, but the methylation change trend is still consistent, and the methylation rate is also good in consistency. These show that the methylation whole genome high-throughput sequencing research laboratory using trace DNA is feasible, and a good solution is provided for the bottleneck that methylation research samples are few and are not easy to obtain.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Reference to the literature

[1]Fraga MF，Ballestar E，Paz MF，et al.(2005)Epigenetic differences arise during the lifetimeof monozygotic twins.Proc Natl Acad.102：10604-9.

[2]Ryan Lister and Joseph R.Ecker(2009)Finding the fifth base：Genome-wide sequencing of cytosine.Genome Res.19：959-966.

[3]Multiplexing Sample Preparation Guide.(Illumina part#1005361).

[4]Bernd Buehler，Holly H.Hogrefe，Graham Scott，et al.(2010)Rapid quantification of DNA libraries for next-generation sequencing.Methods.50：S15-S18.

Claims (36)

1. A method for constructing a genome-wide methylation high-throughput sequencing library, said method being for minigenome DNA, said method comprising the steps of:

step AFragmentation of target genomic DNA and exogenous genomic DNA

The target genomic DNA and the material as the exogenous genomic DNA are any species including genomic DNAs of various plants, animals, and microorganisms; the fragmentation method comprises atomization, ultrasonic fragmentation, Hydroshear or enzyme digestion treatment, so that the genome DNA is broken; (ii) a

Step BTerminal modification of genomic DNA

For fragmented DNA, the ends are first filled in with polymerase and T4 polynucleotide kinase and dntps to produce blunt-ended DNA; then adding an "A" base at the 3' end of the filled sequence by using polymerase and dATP;

step CJoint connection of micro-building warehouse and bisulfite treatment

Will be provided withStep BConnecting the obtained DNA sequence with the base of ' A ' added at the 3 ' end with a methylation-modified micro-library-building joint under the action of ligase; then 100-500ng of linker fragments were addedStep AFragmenting the exogenous genomic DNA and then treating it together with bisulfite to convert unmethylated cytosine to uracil;

step DPCR amplification and library gel cutting purification

To be provided withStep CAdding a PCR primer sequence aiming at a trace library building joint sequence into the DNA converted by the bisulfite as a template to perform PCR amplification; and (4) carrying out electrophoresis on the amplification product by using agarose, and cutting and purifying a target band to obtain the library to be sequenced.

2. The method of claim 1, wherein the method is used for nanogram-grade genomic DNA.

3. The method of claim 1, wherein the method is used for 30-100ng of genomic DNA.

4. The method of claim 1, whereinStep AThe genomic DNA of interest and the material as the foreign genomic DNA are genomic DNAs of a plant, which is Arabidopsis thaliana.

5. The method of claim 1, whereinStep AThe genomic DNA of interest and the material as the foreign genomic DNA areGenomic DNA of an animal, said animal being an insect.

6. The method of claim 1, whereinStep AThe target genomic DNA and the material as the exogenous genomic DNA are genomic DNA of mammals including human and mouse.

7. The method of claim 1, whereinStep AThe genomic DNA was broken into fragments of 100-200bp in size.

8. The method of claim 1, whereinStep AThe middle fragment method adopts an ultrasonic fragmentation method.

9. The method of claim 1, whereinStep AThe Arabidopsis thaliana genomic DNA was selected as the foreign genomic DNA.

10. The method of claim 1, whereinStep BIn (3), the ends are filled in with Klenow polymerase or T4 polymerase.

11. The method of claim 1, whereinStep BIn (1), the "A" base is added using Klenow fragment (3 '-5' exo-) polymerase.

12. The method of claim 1, whereinStep CThe ligase is T4 ligase.

13. The method of claim 1, whereinStep CIn the method, the micro-library building joint is a C site methylation modified micro-library building joint.

14. The method of claim 1, whereinStep CIn the middle, micro library building joints are connected at both ends of the sequence.

15. The method of claim 1, whereinStep CIn (1), 200ng of the fragment with the linker at both ends is addedStep AFragmented exogenous genomic DNA.

16. The method of claim 1, whereinStep CIn (1), the treatment with bisulfite was carried out for 2 hours.

17. The method of claim 1, whereinStep DThe PCR amplification uses a hot start taq enzyme, which includes conventional r-taq or other polymerases.

18. The method of claim 1, whereinStep DIn (3), the amplification product was subjected to electrophoresis using 2% agarose.

19. The method of claim 1, whereinStep CThe miniprep connector used in (1) is Minim _ adapter1 shown in table 1: ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAAT and Minim _ adapter 2: TTGGTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC are provided.

20. The method of claim 1, whereinStep DThe PCR primers used in (1) were the Minim _ PCR primer1.1 shown in Table 1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAA and Minim _ PCR primer 2.1: CAAGCAGAAGACGGCATACGAGATAAGCAATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACCAA are provided.

21. A sequencing library constructed by the method of any one of claims 1-20.

22. The sequencing library of claim 21, which is a minim DNA whole genome methylation high throughput sequencing library.

23. Use of a sequencing library constructed by the method of any one of claims 1-20 for sequencing, wherein the sequencing is performable by a second generation sequencing platform.

24. The use of claim 23, wherein the sequencing library is a minim DNA whole genome methylation high throughput sequencing library.

25. An adaptor for a microsequencing library which is the Minim adapter1 shown in table 1: ACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAAT and Minim _ adapter 2: TTGGTTAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC are provided.

26. The linker of claim 25 wherein the sequencing library is a minim DNA whole genome methylation high throughput sequencing library.

27. Use of the linker of claim 25 to construct a microsequencing library.

28. The use of claim 27, wherein the sequencing library is a minim DNA whole genome methylation high throughput sequencing library.

29. A microsequencing library constructed using the linker of claim 25.

30. The sequencing library of claim 29 which is a minim DNA whole genome methylation high throughput sequencing library.

31. PCR primers for microsequencing libraries, which are Minim _ PCR primer1.1 shown in table 1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAACCAA and Minim _ PCR primer 2.1: CAAGCAGAAGACGGCATACGAGATAAGCAATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACCAA are provided.

32. The PCR primer of claim 25, wherein the sequencing library is a minim DNA whole genome methylation high throughput sequencing library.

33. Use of the PCR primers of claim 31 to construct a microsequencing library.

34. The use of claim 33, wherein the sequencing library is a minim DNA whole genome methylation high throughput sequencing library.

35. A microsequencing library constructed using the PCR primers of claim 31.

36. The sequencing library of claim 35, which is a minim DNA whole genome methylation high throughput sequencing library.