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WO2012019320A1 - 一种甲基化dna的高通量测序方法及其应用 - Google Patents

一种甲基化dna的高通量测序方法及其应用 Download PDF

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Publication number
WO2012019320A1
WO2012019320A1 PCT/CN2010/001219 CN2010001219W WO2012019320A1 WO 2012019320 A1 WO2012019320 A1 WO 2012019320A1 CN 2010001219 W CN2010001219 W CN 2010001219W WO 2012019320 A1 WO2012019320 A1 WO 2012019320A1
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Prior art keywords
dna
sequencing
enzyme
cleavage site
accessory
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English (en)
French (fr)
Inventor
王燕
叶明芝
韩旭
张秀清
孙中生
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BGI Shenzhen Co Ltd
Institute of Psychology of CAS
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BGI Shenzhen Co Ltd
Institute of Psychology of CAS
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Priority to US13/814,925 priority Critical patent/US9518295B2/en
Priority to CN201080067524.2A priority patent/CN102971434B/zh
Priority to PCT/CN2010/001219 priority patent/WO2012019320A1/zh
Publication of WO2012019320A1 publication Critical patent/WO2012019320A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to the field of genomics and biotechnology, and in particular to a combination of thiolated DNA immunoprecipitation, repeated sequence removal and bisulfite treatment high-throughput sequencing technology for cytosine 5' carbon atom in a genomic functional region.
  • a method for precise sequencing of the thiolation state further providing a device capable of performing such sequencing, thereby enabling a reduction in sequencing cost and a reduction in information throughput, and more efficient high-throughput sequencing of methylated DM.
  • DM thiolation is to change the spatial structure of the modified DNA without changing the type and quantity of DNA bases, resulting in silencing or overexpression of the gene, thereby presenting the phenotype of the organism. diversification.
  • thiolation occurs frequently in CpG sites in normal cells, but no thiolation occurs in CpG islands in the promoter region.
  • the overall DNA thiolation level of tumor cells is significantly reduced, and the regions with low gene abundance undergo significant dethiolation.
  • This low thiolation of DNA leads to chromosome instability and cancer.
  • testicular-specific genes, melanoma-associated genes, and genes involved in proliferation are silenced in somatic cells, and their promoter CpG islands are thiolated, and in the corresponding cancer cells, these promoters are demethylated. These genes enable expression of these genes.
  • a decrease in the level of methylation promotes the expression of certain genes, such as transcription factors associated with proliferation.
  • a decrease in the degree of DNA thiolation also further exacerbates the damage and promotes the transition from benign expansion to malignant expansion.
  • DNA thiolation plays a crucial role in gene expression patterns as well as genomic stability.
  • DNA thiolation plays a crucial role in gene expression patterns as well as genomic stability.
  • DNA thiolation In view of the important role of DNA thiolation in the occurrence and development of human diseases, it has been widely recognized by most researchers in the world and has become one of the hotspots of current research. And DM methylation modification at the genome-wide level, so the development of its detection technology is related to the research and understanding of thiolation, which in turn affects researchers' research on human diseases, especially cancer-related diseases. .
  • the existing sequencing-based DNA thiolation detection technology can be divided into direct bisulfite sequencing, MeDIP sequencing, MBD sequencing, enzymatic-bisulfite sequencing, etc. according to different methods of constructing library. .
  • the main steps of sulfite direct sequencing include cleavage of DNA, DNA fragment end repair, ligation of methylation sequencing linkers, bisulfite turnover, PCR amplification, sequencing and sequence comparison.
  • the ligated DM is directly ligated to the methylated sequencing adaptor (all the sites on the linker are modified to the thiolated state) after the terminal modification and the addition of the "A" base at the 3' end.
  • the amino group of the undenidylated cytosine is removed by using bisulfite to convert it into uracil, while the thiolated cytosine remains unchanged, ie, Bisulfite is turned over.
  • PCR amplification is then performed to convert all of the uracil to thymine.
  • the PCR product was sequenced and compared to the untreated sequence to determine if the CpG site was thiolated.
  • This sequencing method has been applied to Arabidopsis thaliana sequencing and human cell line sequencing, and the data of bi ll ion level are obtained.
  • the sounding depth reaches 2 ( ⁇ and 14 respectively), that is, the average sequencing depth of the genomic base reaches 20 times and 14 times.
  • this sequencing method solves the problem of high-throughput sequence scanning of DNA thiolation at the whole gene level, this method produces a large number of nucleotide sequences, which also brings new problems: First, the problem of massive data analysis. Especially for the analysis of sequencing data of such a large genome of higher mammals (20 X coverage, about 60 billion base pairs), splicing and re-aligning massive data after sequencing, huge workload, work steps complex. Followinged by Order cost issue. According to this method, even if the latest 3G sequencing chip is used, the cost of sequencing alone is very expensive, so it cannot be adapted to the requirements of most molecular biology laboratories as common experimental techniques.
  • thiolated DNA can be enriched by protein MBD or 5,-mercaptocytosine antibody MeDIP that specifically binds to thiolated DNA. Fragment.
  • the enriched DNA fragments were sequenced in conjunction with second-generation high-throughput sequencing. Specifically, the method of isolating a thiolated DNA fragment by the MDB method is called thiolated CpG immunoprecipitation (MCIp).
  • MCIp thiolated CpG immunoprecipitation
  • MeDIP is a highly specific enriched thiolated DNA fragment that can be used for immunoprecipitation by 5-mercaptopyrimidine antibody.
  • 5-methylcytosine antibody can also bind to a single thiolated cytosine at a non-CpG site. Therefore it has higher specificity than MBD.
  • This technique known as thiolated DNA immunoprecipitation, combines next-generation sequencing technology to enable high-throughput screening of aberrant methylated genes, a method that avoids the limitations of restriction enzymes at the cleavage site.
  • sequencing library preparation is performed. After genomic DNA is fragmented, a sequencing linker without chemical group modification is ligated. After ligation, MBD or 5-mercaptocytosine antibody is used to contain sulfhydryl groups. The DNA fragment of cytosine is separated from the unpurified DNA fragment. The thiolated DNA fragment was purified and directly subjected to PCR and sequencing without bisulfite inversion.
  • the purpose of bisulfite sequencing based on the enzyme digestion method is to enrich the DNA fragment to be tested and reduce it. Sequencing DNA library size and reducing sequencing costs.
  • the method was able to successfully enrich some CpG islands (8% of the measured data compared to different CpG islands). Moreover, the method reduces the size of the sequenced DNA library to a certain extent, and after the bisulfite is reversed, the subsequent identification of the thiolation site is not required.
  • Michael Zeschnigk utilizes four endonucleases for enrichment of DNA fragments in CpG-rich regions using bisulfite sequencing based on enzymatic cleavage [Smiragl ia DJ, Plass C. The s tudy of aberrant methylation In cancer via res tr ict ion landmark genomic scanning. Oncogene 2002; 21: 5414-5426].
  • the method is based on the principle that the fragmentation of DM is not by ultrasonic cleavage, but by using the endonuclease enzyme (Msel, Tsp 5091, Nlal ll and Hpy CH4V) to cleave DNA, MseI, Tsp509I, NlaI II and Hpy CH4V.
  • the restriction enzyme recognition sites are TTAA, AATT, CATG and TGCA. According to the computer prediction made by the author, the combined enzyme digestion of these four enzymes is superior to other enzymes in terms of the size of the DNA fragment and the number of CpG islands that can be cleaved. Therefore, the four enzymes are combined with the enzyme digestion. . After digestion, a fragment of 300 bp to 800 bp was selected for purification, and the thiolated sequencing linker was ligated and sequenced by bisulfite reverse PCR.
  • sequencing of the above various methods results in the production of a large number of non-biologically functional sequencing data.
  • the heterochromatin region data consisting of a large number of repeat sequences in the genome occupies a large proportion of the sequencing data, because the repeat region containing the thiolated CpG in the gene to be tested (For example, centromeres and telomeres contain repeats, especially highly repetitive sequences, which are thought to be involved in the structural composition of the chromosome, but have not been found It is directly involved in the expression and regulation of genes).
  • COT-1 DM is a closed sequence of repeat sequences in hybridization experiments such as fluorescence in situ hybridization and comparative genomic hybridization. The inventors believe that it can be used as a powerful means of removing repetitive sequences. It is known that COT-1 DNA is rich in high and moderate repeats, which can be renatured and single-copy and low-copy according to high- and medium-duration repeats. The principle that DNA is difficult to renature is made.
  • COT-1 DNA repeats The usual way to remove COT-1 DNA repeats is as follows: labeling COT-1 DNA with biotin, coating magnetic beads with avidin, and labeling biotin with the principle of binding of avidin magnetic beads to biotin.
  • the COT-1 DM is bound to the avidin-coated magnetic beads to obtain a complex of biotin-labeled COT-1 DNA-avidin-coated magnetic beads, and this complex may exist.
  • Repeated sequence of DM fragments to be tested, according to the principle of high-to-moderate repeat DNA denaturation, single-copy and low-copy DNA are difficult to renature, and repeats are hybridized to COT-1 DNA labeled with biotin.
  • the magnetic bead complex containing the repeat sequence-biotin-labeled COT-1 DNA-avidin-coated magnetic beads is obtained, and the magnetic bead complex is separated and discarded, and the DM to be tested by the magnetic beads is recovered and recovered.
  • the DNA is the DNA fragment from which the repeat sequence has been removed.
  • COT-1 DNA to remove repeats is characterized by the fact that the thiolated DNA fragments (promoter, exon, partial intron, etc.) located in the functional region are not captured and removed, but are located at the height of the heterochromatin region. And moderately thiolated repeats were removed. This method of repeat sequence removal meets the requirements for sequencing analysis of genomic DNA at the genome level.
  • the inventors envisaged that the redundant methylation sequence was removed by pre-processing, and then sequenced, thereby reducing the amount of post-processing information and reducing the cost of sequencing. After many choices and investigations, the inventor conceived the following technical route for the first time:
  • the thiolated DNA fragment obtained by enrichment in (1) is subjected to a COT-1 DNA repeat removal technique to obtain a DNA library containing a thiolated fragment containing only the functional region DNA.
  • the present invention provides the following:
  • a high-throughput sequencing method for thiolated DM comprising a step of building a library and a sequencing step, wherein the step of building a library refers to a step of obtaining a methylated D library to be detected, the step of building the library comprising:
  • helper linker which is a double-stranded DNA sequence designed to be linked to the repaired double-stranded DNA end portion, which allows The sulfonated single-stranded DNA becomes double-stranded DNA after PCR.
  • One end of the accessory link to the repaired double-stranded DNA is the terminating end and the other end is a non-ligating terminus;
  • the sequencing step refers to sequencing the library obtained by the above-mentioned database construction step, including steps:
  • auxiliary joint in step B) is selected from at least one of the following a-h:
  • a is an enzyme-free cleavage site, a non-ligated end is a chain protruding structure, the connecting end is a blunt end;
  • b is an enzyme-containing cleavage site, the non-joining end is a chain protruding structure, the connecting end is a blunt end;
  • c is not Containing the enzyme cleavage site, the non-ligated end is a tree scorpion structure, the connecting end is a blunt end;
  • d is an enzyme-containing cleavage site, the non-ligating end is a tree scorpion structure, and the connecting end is a blunt end;
  • e is an enzyme-free cleavage site, the non-ligated end is a chain protruding structure, the connecting end is a sticky end;
  • f is an enzyme-containing cleavage site, the non-ligating end is a chain protruding structure, the connecting end is a sticky end;
  • g is not Containing the enzyme cleavage site, the non-ligated end is a tree scorpion structure, the connecting end is a sticky end;
  • h is an enzyme-containing cleavage site, the non-ligating end is a tree scorpion structure, and the connecting end is a sticky end.
  • the accessory in step B), the primer in step F) and the cutting enzyme in step G) are designed as follows:
  • the primer is designed to be complementary to the sequence of the E) inverted accessory, and at 5, the restriction endonuclease is additionally ligated to the recognition site.
  • the recognition site of the endonuclease is located on the primer, and the cleavage site of the enzyme is ensured to be 5 bp upstream and downstream of the junction of the accessory and the DNA to be tested;
  • the restriction enzyme is selected from the group consisting of: EcoP15I and Mmel.
  • the restriction site is designed in the accessory 5 bp upstream of the junction of the accessory and the DNA to be tested.
  • the site needs to meet the following three characteristics: 1) The enzyme is cut.
  • the site contains at least one thiolated cytosine; 2) the cleavage site does not contain non-thiolated cytosine; 3) the cleavage site does not contain a CpG dinucleotide site.
  • restriction endonuclease is selected from the group consisting of: Alul, Bell, Bfal, Bgl ll, BsrGI, BspHI, CviAII, Fatl Hindl ll, HpyCH4V, Nlalll, Ns i l, Pci l, Seal, Spel, Xbal and the like.
  • step G the enzyme is cleaved with an enzyme that cleaves the designed methylation site; or, in primer 5 At the end, another restriction site is designed, and the two restriction sites are overlapped by adjusting the length of the helper; when step G is performed, the enzyme for the restriction enzyme site of the designed restriction enzyme is used. Double digestion.
  • auxiliary connectors When the auxiliary connectors are e and g, the design principles of the auxiliary connectors and primers are the same as those of the a and c schemes, except that they are connected differently to the DNA fragments of interest: a, c is connected by "TA”, and e and g are sticky. End connection. Therefore, the design principles of the auxiliary connectors and primers will not be described again.
  • auxiliary connectors are f and h
  • the design principle of the auxiliary connectors and primers is the same as that of the b and d schemes, except that the connection with the target DNA fragment is different: b, d is connected by "TA”, f and h are sticky End connection. Therefore, the design principles of the auxiliary connectors and primers will not be described again.
  • step A) includes:
  • step C The sequencing method of item 1 or 2, wherein the thiolated immunoprecipitation of step C) is selected from the group consisting of MeDIP and MBD.
  • step d) is repeated using C0T1
  • FIG 1 Schematic diagram of functional preparation of functionalized thiolated DNA fine-grain sequencing. Main experimental step Fragmentation of genomic DNA, DNA fragment end repair, DNA fragment 3, terminal A base, ligation accessory, methylation co-precipitation, removal of repeats in thiolated DNA fragments, bisulfite Flipping the DNA, using the primers designed according to the accessory adapter for PCR. The inverted DNA was double-stranded, the helper was removed, the PCR product of the terminal was removed, and the A-base was added at the 3' end, and the sequencing linker was ligated and sequenced.
  • Figure 2 Schematic diagram of the design of the auxiliary joint.
  • auxiliary linker The design of the auxiliary linker is divided into two types according to whether there are enzyme cleavage sites, an adaptor containing a restriction site and a linker containing no restriction site (b, d, f, h); the non-joining end of the auxiliary connector can be Designed in three forms: a treed structure (figure c and d), a chain protruding structure (a and b) and a flat end structure. In combination with the TA connection or other adhesive connection characteristics of the auxiliary connector connection, there are eight cases. The differences are as follows: a is a cleavage site, a non-joining end is a chain protruding structure, and the connecting end is a blunt end form;
  • b is an enzyme-containing cleavage site, the non-joining end is a chain protruding structure, and the connecting end is a flat end shape;
  • c is an enzyme-free cleavage site, the non-ligated end is a tree scorpion structure, the ligated end is a blunt end form;
  • d is an enzyme cleavage site, the non-ligated end is a tree scorpion structure, and the connecting end is a blunt end form;
  • e is no Containing an enzyme cleavage site, the non-joining end is a chain protruding structure, and the connecting end is a sticky end form;
  • f is an enzyme-containing cleavage site, the non-joining end is a chain protruding structure, and the connecting end is a sticky end shape;
  • Figure 3 Connection and removal of the flat end of the auxiliary joint (1), the auxiliary joint in this mode uses two of them in Figure 2: & and (; because the design is to remove the auxiliary joint by enzymatic cutting 1 , and the point of removal is removed after the bisulfite is turned over.
  • the accessory 1 is present in the form of a single strand at the end of the DNA to be tested.
  • the inventors have designed and After the bisulfate is turned over, the helper 1 sequence matches the primer (A), and at the same time, the restriction endonuclease (B) is designed near the 5, and the restriction endonuclease does not match the accessory sequence.
  • the selected restriction site is compatible with the restriction enzyme.
  • the restriction enzyme selected is characterized in that the DNA can be digested 20- 30 bp downstream of the restriction recognition site, by controlling the length of the accessory.
  • the restriction site was designed to be within 5 bp of the end of the DNA fragment to the accessory.
  • FIG. 4 Connection and removal of the flat end auxiliary joint 1 (2).
  • the auxiliary link 1 contains the attachment and removal of the auxiliary link 1 in the manner of a thiol cleavage site.
  • the auxiliary joint 1 in this mode can use two of the following two types: b and d.
  • Auxiliary linker 1 contains a methylated cleavage site, and the thiol cleavage site ensures the integrity of the DM fragment when the helper is removed (but allows for damage within 5 bp of the DNA fragment) and The restriction enzyme recognition sequence does not change after the bisulfite is turned over.
  • FIG. 5 Attachment and removal of the flat end of the flat end (3).
  • the accessory linker comprises a thiol cleavage site and the attachment and removal of an accessory link comprising a cleavage site in the primer.
  • the thiolated cleavage site is also designed in the accessory link near the junction.
  • another restriction site was designed, and the two restriction sites were overlapped by adjusting the length of the accessory. The enzymatic excision is ensured by two enzyme digestions.
  • Figure 6 Connection and removal of the auxiliary joint at the sticky end (1).
  • Two of the auxiliary connectors in this mode can be used: 6 and 8.
  • Primers that match the sequence of the helper sequence after bi sul f i te flipping are designed, and the restriction endonuclease recognition site is designed at the 5, near the primer.
  • Figure 7 Connection and removal of the auxiliary joint at the sticky end (2).
  • Two of the auxiliary joints in this mode can be used: f and h.
  • the accessory insert contains a methylated cleavage site to ensure that the DNA fragment is not cleaved and the restriction enzyme recognition sequence does not change after bisulfite turnover.
  • Figure 8 Connection and removal of the auxiliary joint at the sticky end (3).
  • the accessory linker comprises a thiolation cleavage site and an attachment and removal of an accessory link comprising a cleavage site in the primer.
  • a thiolated cleavage site is also designed in the accessory link near the junction.
  • Another restriction site was designed at the 5' end of the primer, and the two restriction sites were coincident by adjusting the length of the accessory. The enzymatic excision is ensured by two enzyme digestions.
  • FIG. 9 Schematic representation of the repetitive sequence for removal of methylation.
  • COT-1 DNA is labeled with biotin
  • the biotin-labeled COT-1 DNA is labeled on the avidin-coated magnetic beads and the unlabeled biotin is removed by the principle of binding of avidin magnetic beads to biotin.
  • the COT-1 DNA fragment The biotin-DNA-bound avidin magnetic beads are hybridized with the thiolated immunoprecipitated thiolated DNA fragment, and the methylated repeat sequence will be hybridized to the labeled biotin COT-1 DNA.
  • the magnetic beads are separated and the magnetic beads are discarded, and the DNA in the solution is recovered, and the recovered DNA is a methylated DNA fragment of the functional region.
  • Figure 10 Sequencing depth analysis of a single CpG locus that was aligned with whole-sequencing sulfonation sequencing and the sequencing results of this technology. The alignment indicates that the sequencing depth of individual CpG sites is consistent.
  • the left picture shows the results of MRERB sequencing; the right picture shows the results of whole gene sulfonation sequencing.
  • Figure 11 - Figure 18 are a description of all types of accessory and primer sequences:
  • Figure 11 Linker sequence and primer sequence for the blunt-end helper ligation and removal mode (1).
  • the linker sequence in the ligation and removal mode of this accessory does not contain a specific base sequence, but it is guaranteed that the linker is sulfonated. Can be successfully amplified.
  • the 3' end of the junction of this linker is designed with a "T" base protruding, and the 5' end is modified with a phosphate group to allow DNA to be attached.
  • the "TA" connection is connected.
  • an enzyme recognition sequence at the 5' end of the PCR primer, which is characterized by the ability to cleave the DNA fragment at a position greater than 20 bp downstream of the enzyme recognition site, and the accessory can be completely cleaved.
  • the biotin group can be introduced at the 5' end of the primer (the biotin group may not be modified, and the Ampure Beads may be removed when the small fragment is removed without modification). Therefore, the PCR product fragment before the removal of the linker includes the DNA fragment to be tested, the two cleavage positions symmetrically, the symmetric matching linker sequence, and Its two symmetric enzyme recognition site sequences.
  • Figure 12 Linker sequence and primer sequence for the blunt-end helper ligation and removal (2).
  • the linker sequence in the adaptor ligation and removal mode comprises a 20-30 bp sequence and a thiolated enzyme recognition sequence to ensure successful sulfation of the linker.
  • the enzyme is characterized by the same enzyme recognition site and cleavage site.
  • the 3' end of the connector end of this connector is designed
  • the "T” base is highlighted, and the 5' end modifies the phosphate group so that it can be linked to the DNA by a "T-A” linkage.
  • linker removal in the PCR product since the enzyme is methylated in the linker, since the sequence after sulfonation is unchanged, the enzyme can be removed by enzyme digestion.
  • the biotin group can be introduced at the 5' end of the primer (the biotin group may not be modified, and the Ampure Beads may be removed when the small fragment is removed without modification). Therefore, the PCR product fragment before removal of the linker includes the DNA fragment to be tested, the symmetric two enzyme recognition cleavage sequences, and the symmetric matching linker sequence.
  • Figure 13 Linker sequence and primer sequence for the blunt-end helper ligation and removal (3).
  • the linker sequence in the adaptor ligation and removal mode comprises a 20-30 bp sequence and a methylated enzyme recognition sequence to ensure successful sulfation of the linker.
  • the enzyme is characterized by the same enzyme recognition site and cleavage site.
  • the 3' end of the connector end of this connector is designed
  • the "T” base is highlighted, and the 5th end modifies the phosphate group so that it can be linked by a "T-A” linkage when connected to DM.
  • linker removal in the PCR product since the enzyme is thiolated in the linker, since the sequence after sulfonation is unchanged, the enzyme can be used to remove the linker; in addition, we simultaneously bind in the PCR product. Removal aspect, we are in PCR primers 5
  • the end-introducing enzyme recognition sequence is characterized in that the DNA fragment can be cleaved at a position greater than 20 bp downstream of the enzyme recognition site, and the accessory can be completely cleaved.
  • the biotin group can be introduced at the 5' end of the primer (the biotin group may not be modified, and the Ampure Beads may be removed when the small fragment is removed without modification). Therefore, the PCR product fragment before the removal of the linker includes the DNA fragment to be tested, the position where the two cleavage sites are symmetrically cleaved, the symmetric matching linker sequence, and the two enzyme recognition site sequences symmetric with the enzyme 2.
  • Figure 14 Linker sequence and primer sequence for the attachment and removal of the cohesive ends of the cohesive ends (1).
  • connection and removal of the auxiliary joint are similar to the way in which the auxiliary joint of the flat end is connected and removed (1).
  • the difference is that the 3' end of the connector is not connected to the "T-A" connection.
  • Also designed is a sequence that matches the sticky end sequence of the DNA end.
  • Figure 15 Linker sequence and primer sequence for the attachment and removal of the cohesive ends of the cohesive ends (2).
  • the connection and removal of the auxiliary joint are similar to the way in which the auxiliary joint of the flat end is connected and removed (2). The difference is that the 3' end of the connector end of this connector is not connected to the "T-A" connector. Also design a sequence that matches the sticky end sequence of the DNA end.
  • Figure 16 Linker sequence and primer sequence for the attachment and removal of the cohesive ends of the cohesive ends (3).
  • the connection and removal of the auxiliary joint are similar to the way in which the auxiliary joint of the flat end is connected and removed (3). The difference is that the 3' end of the connector end of this connector is not connected to the "T-A" connector. Also design a sequence that matches the sticky end sequence of the DNA end.
  • Figure 17 Description of other joint design options.
  • the series one ( bl, dl, fl, hl) auxiliary joint design is similar to the 1), d joint design, in the auxiliary joint near the auxiliary link and the DNA connection point of the test within 20-30 bp upstream design of the enzyme cleavage site,
  • the cleavage site needs to meet the following four characteristics: 1) the cleavage site contains at least one thiolated cytosine; 2) the cleavage site does not contain non-thiolated cytosine; 3) the cleavage site The site does not contain a CpG dinucleotide site; 4)
  • the cleavage site of the enzyme is located within 5 bp of the upstream and downstream of the junction of the accessory and the DNA to be tested.
  • the restriction endonuclease is selected from the group consisting of: EcoP15I and Mmel, and the like.
  • the enzyme is cleaved by the enzyme which cleaves the designed thiolated cleavage site.
  • the series two (b-2, d-2, f-2, h-2) auxiliary joint design is a combination of series one and b, d, f, h, and two enzyme cleavage positions are adjusted by adjusting the length of the auxiliary joint. Point coincidence, the program can use double enzyme digestion to remove the auxiliary joint.
  • the primer is designed to be complementary to the E) flipped accessory
  • the thiolation for the design is employed.
  • the enzyme cleavage site cleaves the enzyme for double digestion.
  • DNA fragmentation refers to: Cutting a genomic DNA into small DNA fragments (such as several hundred bases or several kilobases) by mechanical or enzymatic cleavage.
  • Repair of DNA fragments refers to: DNA fragments after shearing, especially those broken by mechanical means.
  • the double-stranded ends of DNA fragments may be as follows: The position of the ipsilateral end of the double strand is inconsistent, 3' end or 5 ' There is damage at the end, and the meaning of DNA fragment repair is to fill these ends or 3', 5, damaged fragments to 5, connect the phosphate group and the blunt end structure of the 3'-linked hydroxyl group.
  • a library of DNA fragments of appropriate length is prepared.
  • the D fragment is first fragmented into DNA fragments suitable for sequencing length of the sequencer.
  • the fragmentation effect is generally ideal for the size of the fragment of the fragmented DNA Smear in the desired size of the library fragment. For example, if a 100 bp Pa ired End library is required to be inserted, the DNA Smear main band after the cleavage can be at 100 bp, and if the rupture effect is not satisfactory, re-fracture is required.
  • Other fragmentation systems can also be used for the sample fragmentation step, and the specific parameters can be adjusted according to the requirements of the instrument.
  • Methods for breaking DM include methods of enzymatic cleavage and ultrasonic cleavage.
  • the digestion method can be performed by using a single endonuclease or a plurality of endonucleases without CpG sites. Due to the immobilization of the restriction enzyme recognition site, the restriction enzyme digestion method may cause some DNA fragments to be unsuitable and discarded outside the library.
  • the ultrasonic fragmentation DNA is randomly broken DNA, and the fragmentation site is not fixed. In theory, the entire genome may be included. Sequencing text In the library.
  • the fragmented genome can be either an enzymatic cleavage method or an ultrasonic cleavage method, but an example is an ultrasonic cleavage method.
  • D is broken by ultrasound
  • the end of the DNA fragment needs to be repaired to repair the flattened end.
  • the purpose of this repair is to phosphorylate the 5' end of the double-stranded DNA fragment, hydroxylate the 3' end of the damage, repair the blunt-ended DNA fragment 3, and add the "A" base to the end to form a "TA" when the auxiliary link is attached.
  • the auxiliary linker is:
  • the purpose of adding the accessory is to make the single-stranded DNA double-stranded after the DNA is subjected to the sulfonation of MeDIP and bi sulfite, so that the conventional library can be prepared.
  • thiolated bisulfite sequencing utilizes a thiolated linker (WO2009024019A1). Since all of the cytosine sites of the linker are thiolated, the sequence of the linker does not change after the DNA fragment is inverted by bisulfite after ligation of the methylated sequencing linker, and is still matched to the sequencing primer after PCR.
  • the thiolated linker affects the thiolated antibody-bound thiolated fragment in the MeDIP assay, so the linker is not suitable for bisulfite methylation sequencing after MeDIP.
  • a thiolated linker is ligated before MeDIP, bisulfite flipping will result in a change in the linker sequence that does not match the sequencing primer. If not before bisulfite With the addition of a linker, the double-stranded DNA becomes single-stranded DNA after MeDIP and bisulfite, and the sequence changes.
  • the Paired End adapter and the Paired End primer used in the conventional database cannot be used in this experiment.
  • the present invention introduces an auxiliary linker (Fig. 2, Fig. 17) to solve the contradiction between the sequence change caused by MeDIP and bisulfite flipping, and can successfully amplify the DNA fragment library after bisulfite without affecting MeDIP and can carry out subsequent routines. Building a library. Continuation and shearing of auxiliary joints
  • the role of the accessory is to resolve the contradiction between methylation co-immunoprecipitation and bisulfite-induced changes in the linker sequence leading to unmatched sequencing primers. Therefore, the accessory linker should be removed after bisulfite PCR to reduce sequencing costs.
  • the combination of the splicing and shearing of the accessory can be divided into the following cases: 1. Multiple cleavage of the DNA fragment or the cleavage fragment of the ultrasonic fragment and the enzyme cleavage method
  • the primer contains the cleavage site (Fig. 3).
  • the poly-sept DNA fragment or the ultrasonic fragment is end-repaired and 3, and the "A" base is added to the auxiliary link.
  • Two of the auxiliary connectors in this mode can be used: a and c.
  • the accessory is not required to be modified in addition to one of the 5, terminally modified phosphate groups to facilitate attachment to the hydroxyl group of the DNA fragment.
  • a series of treatments such as MeDIP are used to design a primer that matches the accessory sequence after bisulfite inversion (Fig. 3A), and a restriction enzyme recognition site is designed at the 5, near the primer. 3B), the restriction endonuclease recognition site does not match the accessory linker sequence and is in the extension of the primer.
  • the enzyme is characterized in that the DNA can be digested 20-30 bp downstream of the restriction endonuclease recognition site, and the restriction site is designed to be 5 bp in the end of the D fragment at the end of the D-fragment by controlling the length of the accessory.
  • the DNA fragment is digested with a recognition sequence at both ends of the DNA, and the DNA fragment can be digested at the restriction enzyme by recognizing the double-stranded sequence by the enzyme. Due to the 5 in the primer, labeled biotin, the enzyme-cleaved accessory part and not the enzyme The cut DNA fragment can be combined with avidin and can be removed quickly and thoroughly.
  • Enzymes that can be used in the primers include: EcoP15I and Mmel 0
  • the attachment and removal of the accessory link comprising the cleavage site in the primer is employed (Fig. 2).
  • Two of the auxiliary joints in this mode can be used: a and c.
  • the restriction recognition site does not match the accessory sequence.
  • the enzyme is characterized in that DM can be digested 20-30 bp downstream of the restriction endonuclease recognition site, and the restriction site is designed to be 5 bp in the end of the DNA fragment at the end of the DNA fragment by controlling the length of the accessory.
  • a multi-digested DNA fragment or ultrasound can be used in the end-repair of the fragment and 3, and the "A" base is added to the auxiliary link (Fig. 2) b and d.
  • the b and d auxiliary outer strands are designed to cleave the site near the junction, and the site is designed to cut off the helper enzyme after PCR. Therefore, the cleavage site is designed to be close to the junction of the accessory. Cytosine, which is not thiolated after bisulfite conversion, is converted to avidin and becomes thymine after PCR.
  • the cytosine when the inventors design a cleavage site, if the enzyme recognition sequence contains cytosine, the cytosine here needs to be thiolated to ensure that the enzyme recognition sequence does not change after bisulfite turnover. Since the adenine (A) and thymine (T) in the PCR product sequence after bisulfite conversion increased, the inventors did not cleave the DNA fragment when designing the restriction site, and the selected restriction enzyme recognition site contained C of a non-CpG site, and the cytosine is not at the end of the restriction endonuclease recognition sequence.
  • A adenine
  • T thymine
  • Enzymes useful herein include: Alul, Bell, Bfal, Bglll, BsrGI, BspHI, CviAII, Fatl, Hindlll, HpyCH4V, Nlalll, Nsil, Pcil, Seal, Spel, Xbal and the like.
  • the accessory linker also modifies the phosphate group in one of the 5' ends to facilitate attachment to the 3' hydroxyl group in the DNA fragment.
  • a primer matching the accessory sequence of the bi sul f i te flip is designed and biotin is modified at the 5' end (Fig. 4). Since biotin is modified at the end of the primer 5, the excised co-ligand moiety and the uncut DNA fragment can be removed rapidly and thoroughly by binding to avidin.
  • auxiliary link comprising a thiolation cleavage site in the accessory
  • Two of the auxiliary connectors in this mode can be used: b and d in Fig. 4.
  • the ligated linker contains a thiolated cleavage site to ensure that the DNA fragment is not cleaved and the digestion recognition sequence does not change after bisulfite turnover.
  • the tri-auxiliary linker contains a thiol cleavage site and a primer containing an enzyme cleavage site (Fig. 5).
  • Enzyme digestion by a single enzyme is often not thorough enough, and sometimes it is necessary to digest with two or more enzymes.
  • the multi-digested DNA fragment or ultrasound is ligated at the end of the fragment and 3, and the "A" base is added to the auxiliary link as in the second mode (Fig. 2) 1) and 3, and also in close proximity.
  • the methylation site was designed to be methylated.
  • Designing a primer that matches the sequence of the accessory link after bi sulf i te flipping is the same as mode 1.
  • DNA was digested at -30 bp, and two restriction sites were designed to be in the same position of the DNA fragment by controlling the length of the helper. Through two enzyme digestions, the cutting is thorough.
  • the labeled biotin, the excised explant portion and the undigested DM fragment can bind to avidin, Can be removed quickly and thoroughly.
  • an accessory link comprising a methylase cleavage site and a primer comprising a cleavage site in the accessory is employed (Fig.
  • a thiolated cleavage site is also designed in the accessory link near the junction.
  • another restriction site was designed at the 5' end, and the two enzymes were ligated by adjusting the length of the auxiliary link. The enzymatic excision is ensured by two enzyme digestions.
  • a DNA fragment with a single-stranded DNA fragment that is a sticky end does not have to be end-repaired and 3, and an "A" base is added to the end.
  • the auxiliary link that matches the sticky end can be directly designed and used.
  • the auxiliary joint in this mode can use two of them. : 6 and 8 .
  • the accessory does not have to be modified in any way other than in one of the 5, modified phosphate groups to facilitate attachment to the 3' hydroxyl group in the DNA fragment.
  • the primers matching the sequence of the helper after bi sul fi te flipping are designed (Fig. 6), and the restriction enzyme recognition site is designed at the 5, near the primer.
  • the restriction endonuclease recognition site does not match the accessory linker sequence and is in the extension of the primer.
  • the enzyme is characterized in that the DNA can be digested 20-30 bp downstream of the restriction endonuclease recognition site, and the restriction site is designed to be 5 bp in the end of the DNA fragment to the accessory.
  • the DNA fragment is digested with a recognition sequence at both ends of the DNA, and the DNA fragment can be digested at the restriction enzyme by recognizing the sequence of the silent strand by the enzyme. Since the biotin at the primer 5, the cut-off accessory part and the uncut DNA fragment can be combined with avidin, it can be removed quickly and thoroughly. Enzymes that can be used in the primers include: EcoP15 I and Mmel.
  • the attachment and removal of the cohesive linker at the cohesive end is employed (the primer comprises a cleavage site) (Fig. 6).
  • the primer comprises a cleavage site
  • Fig. 6 Two types of adapters can be used: 6 and g. Design primers that match the parental linker sequence after bisulfite flipping, and design the restriction endonuclease site at the 5, near the primer.
  • the accessory linker contains a thiol cleavage site (Fig. 7).
  • a DNA fragment with a single-stranded DNA fragment that is a sticky end does not have to be end-repaired and 3, and an "A" base is added to the end.
  • the auxiliary link that matches the sticky end can be directly designed and used.
  • the auxiliary joint in this mode can use two of them. : f and 11.
  • the accessory is not required to be modified in addition to one of the 5, terminally modified phosphate groups to facilitate attachment to the 3' hydroxyl group in the DNA fragment.
  • the auxiliary outer chain is designed to contain a thiol cleavage site near the junction end, and in order to not cleave the DNA fragment when the restriction site is ligated, the selected restriction enzyme recognition site contains a non-CpG site C, and the cell
  • the pyrimidine is not at the end of the restriction endonuclease recognition sequence.
  • the inventors methylated the cytosine in the recognition sequence, and the other bases were not modified.
  • the enzymes usable herein include: Alul, Bell, Bfal, Bglll, BsrGI, BspHI, CviAII, Fatl, Hindlll, HpyCH4V, Nlalll, Nsil, Pcil, Seal, Spel, Xbal, and the like.
  • the accessory is also modified at one of the 5' ends to facilitate attachment to the 3' hydroxyl group in the DNA fragment.
  • a primer matching the sequence of the accessory after the bisulfite inversion is designed and biotin is modified at the 5' end (Fig. 4). Since biotin is modified at the end of the primer 5, the excised co-ligand moiety and the uncut DNA fragment can be removed rapidly and thoroughly by binding to avidin.
  • auxiliary link comprising a thiolation enzyme cleavage site in the accessory linker
  • Fig. 7 Auxiliary connection in this mode
  • the head can use two of them: f and h.
  • the accessory insert contains a methylated cleavage site to ensure that the DNA fragment is not cleaved and the restriction enzyme recognition sequence does not change after bisulfite turnover.
  • Tri-A-Linker contains a methylase cleavage site and the primer contains an enzyme cleavage site (Fig. 8).
  • the single enzyme is often not sufficiently lysed, and it is sometimes necessary to perform digestion with two or more enzymes.
  • the multi-digested DNA fragment or ultrasound is ligated at the end of the fragment and 3, and the "A" base is added to the auxiliary link, as in the second mode (Fig. 8) f and h, and also in the vicinity of the junction. Design a thiolated cleavage site.
  • Designing a primer that matches the sequence of the accessory link after bi sulf i te flipping is the same as mode 1.
  • the DNA was digested at -30 bp, and the two restriction sites were designed to be in the same position of the DM fragment by controlling the length of the accessory. Through two enzyme digestions, the cutting is thorough.
  • labeled biotin labeled biotin
  • the excised part of the helper and the uncut DNA fragment can be combined with avidin and can be removed quickly and thoroughly.
  • the accessory and the removal of the accessory link comprising the thiol cleavage site and the cleavage site in the primer are included in the accessory (Fig. 8).
  • the methylation cleavage site is also designed in the accessory link near the junction.
  • another restriction site was designed at the 5' end, and the two restriction sites were coincident by adjusting the length of the accessory. The enzymatic excision is ensured by two enzyme digestions.
  • Methylated immunoprecipitation is a highly specific enriched methylated DNA fragment that can be used for immunoprecipitation by 5-methylcytosine antibody. High-throughput screening of aberrant methylation in combination with next-generation sequencing technology Gene, this method avoids application limits The limitations of the systemic enzyme at the cleavage site.
  • the methylated immunoprecipitation technique used in the present invention is slightly modified according to the co-immunoprecipitation technique of Michael Weber. In order to sequence the length of the DNA fragment, the size of the DNA fragment was adjusted from 200-1000 bp to 100-300 bp. In addition, the incubation time of the 5-mercapto-cytosine antibody was optimized and the optimal incubation time was 12 hours instead of the literature. Reported for 2 hours.
  • Sulfite inversion is the chemical modification of single-stranded DNA molecules with bisulfite, resulting in the undecylated cytosine (C) being converted to uracil (U) by bisulfite, and 5 mC. Can not be modified, still kept at 5mC, during the PCR reaction, uracil is paired with adenine (A), and uracil is replaced by thymine (T).
  • This chemical reaction process was first carried out by Frommer et al. (M. Frommer et al., A genomic sequencing protocol that yields a pos it ive di splay of 5-methylcytos ine res idues in individual DNA s trands.
  • the linker is cut off as completely as possible by using an appropriate amount of enzyme and by extending the digestion time.
  • the inventors modified the biotin at the 5' end of the primer and removed the short film by binding the biotin to the magnetic beads coated with avidin. Segment of the helper and DNA fragments that are not digested.
  • the end of the endonuclease-cleaved DNA fragment is first repaired to an end that matches the sequencing adapter (Solexa, 454, Sol id ).
  • the end of the enzymatically digested DNA fragment if it is a sticky end, is first repaired into a blunt end, and then 3' at the blunt end of the DNA.
  • the PE adapter forms a "T-A" connection.
  • Bisulfite-PCR After the DNA is sulfonated (bisulfite), the purpose of Bisulfite-PCR is to turn the single-stranded DNA fragment into a double strand to facilitate the ligation of the sequencing linker. At the same time, the amount of DNA is amplified.
  • the primers for Bisulfite PCR amplification were matched to the sequences of the accessory after sulfonation.
  • PE PCR is amplified after the restriction enzyme is removed, and then ligated with a PE linker.
  • the purpose of amplification is to convert the PE fork of the DNA fragment into a sequence that matches the sequencing primer.
  • the DNA fragment after removal of the accessory can be used directly in routine experimental procedures for subsequent high-throughput sequencing of the Genome Sequencer FLX system, such as the use of Roche's GS FLX Standard DNA library preparation kit; or Illumina's Genome Analyzer (GA) system high throughput
  • Conventional experimental procedures for sequencing such as the use of the Paired End DNA Library Preparation Kit, to construct a conventional Paired End DNA sequencing library; can also be used for routine high-throughput sequencing of the SOLiD Library Oligos kit of the AB SOLiD system.
  • the PCR amplification primers of the linker, qPCR detection primer and the sulfonated DNA were synthesized by Invitrogen; the used COT1 DNA was purchased from Invitrogen, and the qPCR was detected using AB SYBR and supporting reagent; EcoP15I The enzyme was purchased from NEB. The principle of operation of the embodiment is shown in FIG.
  • Blood sample 1 (taken from volunteer) 10 ml, and sample DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen). The extracted DNA was numbered as YH-1, and 10 ⁇ g of the DNA sample was taken as a starting material.
  • the library was constructed with reference to the flow of Fig. 1, and an Illumina GA System Paired End library was constructed in this example.
  • the DNA sample in the above step 1 was disrupted by Covaris system (AB company), and after the sample was broken, about 1/30 (by volume) of the fractured sample was taken on a 2% agarose gel. Electrophoretic detection, the DNA fragment to be tested is recovered from the electrophoresis gel. In order to control the sample DNA fragment in the range of 100 ⁇ 500 bp, the specific operation of the Covaris system is:
  • Methylated DNA co-immunoprecipitation (MeDIP)
  • step 5 Take the sample from step 5, refer to step 4 for NanoDrop 1000.
  • the DNA is about 4 g, and add TE to a volume of 450 ⁇ . Mix well. It was treated in a water bath of 98 ° for 10 minutes and then placed on ice for 10 minutes.
  • the magnetic beads were washed 3 times with 800 ⁇ l ⁇ IP buffer, vortexed on vertex for 2-4 seconds during the first wash, and vortexed 3 times; the latter two washes were repeatedly pipetted with a pipette. Remove the supernatant after collecting with a magnetic stand.
  • step (7) Recover the DNA in the reaction system of step (6) with ZYMO DNA Clean & Concentrator PTMP-5, using a binding buffer of 7 times the volume of the DNA solution (step 6), dissolved in 2 ( ⁇ L 601; ultra-pure water in a bath. l L in NanoDrop 1000 The OD value was measured and the sample concentration was recorded, and the MeDIP effect was detected by Q-PCR.
  • This experiment uses ZYMO EZ DNA Methylation-Gold Kit PTMP (ZYM0) for bisulfite treatment
  • step 2 (4) Load the sample (from step 2) to the Zymo-Spin ICPTMP column containing the M binding buffer. Cover the column by inverting the column several times to mix the sample.
  • the DNA in the purified reaction system was recovered by QIAquick PCR purification kit, and the product was dissolved in 50 ⁇ L of EB. Take 1 ⁇ L of the 0D value on the NanoDrop 1000 and record the sample concentration.
  • step 2 Resuspend the magnetic beads in 50 ⁇ L of 2 X B&W buffer and add 50 ⁇ L of the digested product (step 2 );
  • the DNA fragment after removal of the helper 1 in step 10 was directly used in the subsequent Illumina Genome Analyzer (GA) system for high-throughput sequencing.
  • GA Illumina Genome Analyzer
  • the enrichment effect of the MeDIP method used in the present invention was compared with the enrichment effect of the commercial MeDIP kit (Diagenode) (Table 1), wherein the thiolation enrichment effect (4994) was 62.71%, which was greater than that of Diagenode.
  • the recovery rate of this MeDIP method reaches the recovery requirement of the commercial MeDIP kit (Diagenode) and is relatively large compared to the commercial MeDIP kit. Therefore, the MeDIP method is considered to be comparable to the commercial kit, and the test results are parallel. Trustworthy.
  • the sequencing library prepared by the sequencing library sample preparation technique of the present invention was subjected to cloning treatment and subjected to small-scale sequencing detection using the Sanger method. 76% of the results of the library check (Table 3)
  • the reads can be compared to the genomic region, and its thiolation rate is as high as 87%; the turnover rate of sulfite (sulfonated) to unmethylated cytosine is 99%; the fragment amplification repetition rate is zero.
  • Embodiment 2 The principle of Embodiment 2 is as shown in FIG. 6, wherein all the steps are the same as those in Embodiment 1, except that the following steps are the same as Embodiment 1, and steps 5, 9 and 10 are replaced with Step 5 in Embodiment 1, respectively. 9 and 10:
  • the DNA in the purified reaction system was recovered by QIAquick PCR purification kit, and the product was dissolved in 50 L of EB. Take 1 ⁇ L of the 0D value on the NanoDrop 1000 and record the sample concentration.
  • the accessory contains a methylated cytosine
  • Use thiolation detection primer number 4994 and GAPDH two pairs, non-methylation detection primers The two pairs of No. 8804 and TSH2B were tested for the content of thiolated and non-thiolated auxiliary joints after MeDIP.
  • the enrichment ratio of the thiolated auxiliary linker corresponding to the non-thiolization was consistent. Therefore, the background effect of the thiolated auxiliary on MeDIP is negligible.
  • the MeDIP enrichment effect was detected by using the primers for detecting the methylation enrichment effect (4994) and the non-thiolization enrichment primers (8804) (Table 5), wherein the methylation enrichment effect was 45. 6 %, and the methylation enrichment effect of Diagenode (Table 2) is almost the same, the enrichment rate of non-thiolization is 2.9%, which is slightly higher than the non-thiolization enrichment rate of Diagenode, but far lower The enrichment effect of ⁇ ⁇ . Therefore, this MeDIP enrichment DM efficiency is successful.
  • the sequencing library prepared by the sequencing library sample preparation technique of the present invention was subjected to cloning treatment and subjected to a small-scale sequencing detection using the Sanger method. There are 85°/ in the library test results (Table 7).
  • the reads can be compared to the genomic region, and its methylation rate is as high as 93%; indicating that the DNA fragment linked to the Alu l linker can be enriched into the sequence of most thiolated regions of the genome after MeDIP treatment.
  • a conversion rate of up to 97% indicates that the effect of the sulfite (sulfonation) treatment is also very significant.
  • the DM fragment ligated by the Alu l linker can still obtain effective sequence information after MeDIP treatment, repeated sequence removal, sulfite treatment and Alu I digestion.
  • the library was tested and used to construct a paired End DM library for high-throughput sequencing of the Illumina GA system and to perform corresponding sequencing.
  • connection and removal methods (1) and (2) of the auxiliary joints using only the flat ends in the examples 1 and 2.
  • Description. Has a certain representativeness. Since the apex-end auxiliary joint connection and removal method (3) is a combination of the blunt end auxiliary joint connection and removal methods (1) and (2), the flat-end auxiliary joint connection and removal methods (1) and (2) The success of the implementation can indicate that (3) can also be successful.
  • the principle of the flat end relative to the sticky end is the difference in the way the joint is connected, and the flat end is less efficient to join the sticky end, so the joint connection and removal of the sticky end in the case of successful flat end (4-6) Will be successful.
  • High-throughput sequencing was performed on the Illumina GA system, which was a 45-cycle Paired End sequencing. Specific sequencing procedures and reagents are used in I I lumina standard procedures and kits. The sequencing results in 2.53 Gb of data, which can be mapped to half of the reference genomics (homo sapienss), and the ratio of other bisulfite sequencing is similar [H. Xiang et al., Single base-resolut ion methylome of the Si lkworm reveal sa sparse epigenomic map. Nat Biotechnol 28 516-20] , the specific results can be seen in Table 8. The average DNA thiolation level was 84.2%, and the about 70% thiolation level of the whole genome methylation sequencing showed higher specificity of MeDIP binding to thiolated cytosine.
  • the filtration rate is 0. 80%.
  • the logarithm is 30, 287, 175.
  • the ratio is 53. 09%.
  • the amplification rate is 5.83%.
  • the valid data is 28, 619, 720 ( 1. 27Gb).
  • the data efficiency is 50. 17%. 97. 18% thiolization rate 84. 20%
  • the present technology can detect DNA thiolation of the genomic functional region and accurately align the single cytosine thiolation state.
  • the amount of data of the whole genome bisulfite sequencing can be 80%-90% less than the data of the whole genome bisulfite sequencing, and the cost of each sample detection is 70-80%.

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Description

一种甲基化 DNA的高通量测序方法及其应用 技术领域
本发明涉及基因组学、 生物技术领域， 具体而言涉及将曱基化 DNA 免疫共沉淀、 重复序列去除以及亚硫酸氢盐处理高通量测序技术结合， 进行基因组功能区的胞嘧啶 5' 碳原子的曱基化状态进行精确测序的方 法； 进一步还提供了能够实施这种测序的装置， 进而能够使测序成本降 低、 并且减少信息处理量， 更高效地对甲基化 DM进行高通量测序。 背景技术
DNA甲基化与基因调控或疾病间的关系
在高等真核生物基因组中， DM曱基化是在不改变 DNA碱基种类与 数量的前提下使得被修饰 DNA的空间结构发生改变导致基因的沉默或过 度表达， 从而使生物体表型呈现出多样化。
例如， 在正常细胞中常在 CpG位点发生曱基化， 但在启动子区域的 CpG岛不发生曱基化。 肿瘤细胞的整体 DNA曱基化水平显著降低， 基因 丰度低的区域发生显著的去曱基化。这种 DNA的低曱基化导致染色体的 不稳定性与癌症的发生。 如像睾丸特异性基因、 黑素瘤相关基因和与增 殖相关的基因在体细胞中被沉默， 其启动子 CpG岛被曱基化， 而在相应 的癌细胞中， 这些启动子发生去甲基化， 从而使得这些基因能够表达。 另外， 甲基化水平的降低促使了某些基因（如： 与增殖相关的转录因子） 的表达。 在肿瘤发育过程中， DNA曱基化程度的降低还会使得损伤进一 步的加剧， 促进从良性扩增到恶性扩增的转变。
DNA曱基化对基因表达模式以及基因组稳定性均起着至关重要的作 用。 鉴于 DNA曱基化在人类疾病发生、 发展过程中的重要作用已经得到 了世界上大多数研究者的普遍重视， 已经成为当前研究的热点之一， 并 且 DM甲基化修饰作用于全基因组水平， 因此其检测技术的发展左右着 对曱基化的研究与认识， 进而在很大程度上影响着研究者们对于人类疾 病特别是癌症相关疾病的研究。
DNA曱基化测序方法的现状
目前， 现有的基于测序仪的 DNA曱基化检测技术根据建序库方法的 不同可分为直接亚硫酸氢盐测序、 MeDIP测序、 MBD测序、 酶切 -亚硫酸 氢盐测序等几种方法。
• 亚硫酸氢盐 （bi sulf i te ) 直接测序
亚硫酸盐直接测序法的主要步骤包括断裂 DNA、 DNA片段末端修复、 连接甲基化测序接头、 亚硫酸氢盐翻转、 PCR扩增、 测序和序列比较。 具体而言是将断裂的 DM在经过末端修饰和 3，端加 "A" 碱基后， 直接 连接到甲基化的测序接头 （adapter ) (接头上所有位点修饰成曱基化 状态）上， 在合适的反应条件下， 针对单链 DNA分子， 使用亚硫酸氢盐 脱去未曱基化胞嘧啶的氨基而使之转变成尿嘧啶， 而曱基化的胞嘧啶保 持不变， 即进行亚硫酸氢盐翻转。 然后进行 PCR扩增使尿嘧啶全部转化 成胸腺嘧啶。最后，对 PCR产物进行测序，并且与未经处理的序列比较， 判断 CpG位点是否发生曱基化。
这种测序方法已经应用在拟南芥曱基化测序和人细胞系测序中， 均 得到了 bi l l ion级的数据， 测深分别达到 2(^和 14 ， 即基因组碱基 平均测序深度分别达到 20倍和 14倍。
该测序方法虽然解决了在全基因水平对 DNA曱基化谱扫描的序列高 通量问题， 但是该方法产生了海量核苷酸序列， 这也带来了新问题： 首 先是海量数据分析问题。 尤其是对于高等哺乳动物这种庞大基因组的测 序数据分析（20 X的覆盖率， 大约达到 600亿碱基对的数量） ， 测序后 对海量数据的拼接和重新比对， 工作量巨大， 工作步骤复杂。 其次是测 序成本问题。 按此方法即便使用最新型的 3G测序芯片， 仅测序成本一 项就很昂贵， 所以无法适应大多数分子生物学实验室作为常用实验技术 的要求。
• MeDIP测序和 MBD测序
由于在哺乳动物中曱基化一般发生在 CpG的胞嘧啶 5位碳原子上， 所以可通过特异性结合曱基化 DNA 的蛋白 MBD 或 5，-曱基胞嘧啶抗体 MeDIP富集高曱基化的 DNA片段。 并结合第二代高通量测序， 对富集到 的 DNA片段进行测序。 具体而言， MDB方法分离曱基化 DNA片段的方法 被称为曱基化 CpG免疫沉淀 (MCIp) 。 MeDIP是通过 5-曱基胞嘧啶抗体可 用来进行免疫沉淀高特异性的富集曱基化 DNA片段， 5-甲基胞嘧啶抗体 也可以结合非 CpG位点的单个曱基化的胞嘧啶， 因此比 MBD有更高的特 异性。 这项技术被称为曱基化 DNA免疫沉淀， 结合新一代测序技术可以 高通量的筛选异常甲基化的基因， 此法避免了应用限制性酶在酶切位点 上的局限性。
当采用 MeDIP测序、 MBD测序时，要进行测序文库制备，基因组 DNA 经片段化之后， 连接没有化学基团修饰的测序接头， 连接好后先用 MBD 或者 5-曱基胞嘧啶抗体将含有曱基化胞嘧啶的 DNA 片段与未曱基化的 DNA片段分离。 曱基化的 DNA片段纯化后不经亚硫酸氢盐翻转直接进行 PCR和测序。
例如， David Serre对 HCT116结肠癌细胞系 DM进行 MBD测序， 测 序结果显示将近一千九百万（占用芯片中两道）的数据可检测到所有已 知的曱基化和一些未知甲基化区域， 大大降低了测序费用。 但是由于该 技术没有在测序前进行亚硫酸氢盐的磺酸化处理， 必需对甲基化位点进 行鉴定， 大大增加了后续的工作量。
• 酶切-亚硫酸氢盐测序
基于酶切方法的亚硫酸氢盐测序的目的是富集待测 DNA片段， 降低 测序 DNA文库大小并降低测序费用。 该方法能够成功地富集到一些 CpG 岛（所测数据的 8%比对到了不同的 CpG岛）。 而且该方法在一定程度上 降低了测序 DNA文库大小， 并且利用亚硫酸氢盐翻转之后， 不用再做后 续的曱基化位点的鉴定工作。
例如， 已知 Michael Zeschnigk利用基于酶切方法的亚硫酸氢盐测 序时利用 4种内切酶对 CpG富集区进行 DNA片段的富集 [Smiragl ia DJ, Plass C. The s tudy of aberrant methylat ion in cancer via res tr ict ion landmark genomic scanning. Oncogene 2002; 21: 5414-5426]。 该方法的制作原理是 DM的片段化不是用超声断裂， 而是 利用多内切酶联合酶 ( Msel, Tsp 5091， Nlal l l和 Hpy CH4V )切方法 断裂 DNA，MseI， Tsp509I，NlaI I I和 Hpy CH4V酶切识别位点分别是 TTAA, AATT, CATG和 TGCA。 根据作者做的计算机预测， 这四种酶的联合酶切 在 DNA片段大小， CpG岛能被切割的个数等因素方面比其他酶联合酶切 的优势大， 因此选该四种酶联合酶切。 酶切后选择 300bp- 800bp的片段 进行纯化，连接曱基化的测序接头，经亚硫酸氢盐翻转 PCR后进行测序。
但是该方法由于对基因组 DN A的片段化是利用酶切完成的， 酶切识 别位点的固定性会导致片段大小的分布差异很大， 小于 300bp 和大于 800bp的 DNA被舍弃将使部分基因组 DNA不可能被测序到； 另外， 测序 的读长只有 130bp、 300bp-800bp的片段不能被测通。 因此， 该方法使 部分有生物学意义和功能的 DNA甲基化不能被检测到。
无论是上述哪一种测序方法， 都存在一个值得注意的问题， 就是以 上各种方法的测序均会致使大量无生物学功能的测序数据的产生。 其原 因在于在现有的建库和测序方法中，基因组由大量重复序列组成的异染 色质区域数据占据测序数据的很大比例， 这是因为在待测基因中含有高 曱基化 CpG的重复序列区（例如， 丝粒和端粒含有重复序列尤其是高度 重复序列， 这些重复序列被认为参与染色体的结构组成， 但是尚未发现 它直接参与基因的表达和调控）。 而重复序列尤其是高度重复序列及其
DNA 曱基化谱与目的基因表达关系的分析相对联系较弱 [Herman JG 等 人， Methylat ion - spec i f ic PCR: a novel PCR as say for methyla t ion s tatus of CpG i s lands. Proc Nat l Acad Sci U S A 1996; 93: 9821-9826] , 因此， 去除掉重复序列后只对功能区 DNA的甲基化片段进 行测序可大大节省测序成本。
重复序列去除技术
如何去除重复序列， 目前已有研究人员对此进行了研究， 例如将亚 硫酸氢盐测序技术与高密度芯片结合， 先通过芯片技术将待测甲基化 DNA的范围进行控制。 已知 Ag i lent和 NimbleGen公司设计出的芯片探 针位置集中于基因组的启动子和第一外显子以控制目标 DNA的范围， 这 种方法可以去除如来自异染色质 DNA片段的冗余。 另外， 还有以高通量 外显子 DM 片段以降低测序文库的大小来降低每个样本测序的检测成 本。但是由于外显子捕获芯片的捕获 DNA量有限，对后续的实验有影响, 其去除冗余曱基化 DM 的能力根本不足以满足在基因组水平的曱基化 DNA的测序分析。
此外， COT-1 DM 是一种荧光原位杂交和比较基因组杂交等杂交试 验中重复序列的封闭的序列。发明人认为其可以作为去除重复序列的一 个有力手段， 已知 COT-1 DNA富含高度和中度重复序列， 是根据高、 中 度重复序列 DM变性后可以复性而单拷贝和低拷贝的 DNA很难复性的原 理制成。
COT- 1 DNA重复序列去除的常用方式如下所述：用生物素标记 C0T-1 DNA, 用亲和素包被磁珠， 利用亲和素磁珠与生物素结合的原理， 将标 记有生物素的 COT-1 DM结合在包被有亲和素的磁珠上， 得到生物素标 记 COT- 1 DNA -亲和素包被磁珠四者的复合体， 将此复合体与可能存在 重复序列的待测 DM片段杂交， 根据高、 中度重复序列 DNA变性后可以 复性而单拷贝和低拷贝的 DNA很难复性的原理， 重复序列被杂交在标记 生物素的 COT-1 DNA上， 得到含有重复序列 -生物素标记 COT-1 DNA - 亲和素包被磁珠五者的磁珠复合体， 分离磁珠复合体并舍弃， 同时回收 经过磁珠处理的待测 DM,回收的 DNA即是去除了重复序列的 DNA片段。
利用 COT-1 DNA去除重复序列的特点在于位于功能区的曱基化 DNA 片段（启动子， 外显子， 部分内含子等区域）不会被捕获移除， 而位于 异染色质区的高度和中度曱基化重复序列被移除。 这种重复序列去除方 式能够满足基因组水平的曱基化 DNA的测序分析的要求。
发明内容
发明人面对现有技术的缺陷， 设想通过前期处理， 去除冗余的甲基 化序列，然后再进行测序，以此减少后期处理的信息量并降低测序成本。 经过多方面的选择和调查， 发明人首次构思了下述技术路线：
( 1 )先通过 MeDIP或 MBD技术从待测 DNA中富集曱基化 DNA片段；
( 2 )将（1 ) 中富集得到曱基化 DNA片段通 COT-1 DNA重复序列去 除技术处理， 得到去除冗余序列的仅含有功能区 DNA 的曱基化片段的 DNA文库；
( 3 ) 然后对（2 ) 中的文库进行亚硫酸氢盐测序的高通量测序。 在该技术路线中首次将 COT- 1 DNA重复序列去除技术应用到高通 量测序当中。
但是在实际实施时， 遇到了很多技术问题。 最关键的就是接头的匹 配衔接问题。
目前， 曱基化 DNA的亚硫酸氢盐测序利用的曱基化的接头， 由于该 接头的所有胞嘧啶位点都是曱基化的， DNA片段在连接甲基化的测序接 头后经亚硫酸氢盐翻转之后， 接头的序列不发生改变， PCR之后仍和测 序引物匹配。 但是将 MeDIP和亚硫酸氢盐测序法联合应用的话，会出现下述几种 情况： （1)先亚硫酸氢盐测序后 MeDIP处理， 在构建亚硫酸氢盐测序文 库时， 在亚硫酸氢盐磺酸化处理前引入曱基化修饰的接头的话， 曱基化 的接头的引入会造成后续的 MeDIP曱基化免疫共沉淀出现 "假阳性" 的 曱基化 DNA富集。 （2)先进行 MeDIP处理然后进行亚硫酸氢盐测序， 并 在 MeDIP处理前不加接头， 双链 DNA经 MeDIP和亚硫酸氢盐之后变成单 链 DNA， 并且序列发生改变， 常规建库所用的测序接头与测序引物无法 使用。（3) 先进行 MeDIP处理然后进行亚硫酸氢盐测序，如果在 MeDIP 前连接没有曱基化的接头， 亚硫酸氢盐翻转会致使接头序列发生 改变而和测序引物不匹配。
为实现以上两种甲基化 DNA富集技术和曱基化 DNA的亚硫酸氢盐测 序技术结合， 发明人通过^ ^理设计辅助接头及引物， 合理安排该接头的 连接和去除与各技术的使用顺序解决了上述难题。 由此首次提供了曱基 化 DNA免疫共沉淀与重复序列去除的亚硫酸氢盐处理的高通量测序方法 ( MeDIP- repet i t ive elements remova卜 bi sulf i te,简称 MRERB技术)， 这种方法既满足了对待测 DNA片段检测的要求又降低了每个样本测序文 库的大小和测序量， 减少了测序后期处理信息量进而节约了检测费用， 实现了低成本对功能区 DM曱基化片段进行曱基化精细图谱的绘制的目 标。
具体而言， 本发明提供了下述内容：
1. 一种曱基化 DM的高通量测序方法， 包括建库步骤和测序步骤， 其中建库步骤是指获得待检测的甲基化 D 文库的步骤， 所述建库 步骤包括：
A)基因组 DNA的片段化及双链 DNA片段末端修复；
B)将 A )得到的双链 DNA与辅助接头连接， 所述的辅助接头是指设 计成能与修复后的双链 DNA末端部分相连接的双链 DNA序列， 它可以使 经磺酸化处理的单链 DNA经 PCR后变成双链 DNA。 辅助接头与修复的双 链 DNA连接的一端为连接末端， 另一端为非连接末端；
C)将 B ) 的产物进行曱基化免疫共沉淀；
D)去除 C ) 的产物中的中度， 高度重复序列；
E)将 D )得到的产物进行亚硫酸氢盐翻转处理；
F)将 E )得到的单链 DNA通过引物 （根据辅助接头序列设计）进行 PCR扩增获得双链 DNA, G )酶切去除辅助接头；
其中测序步骤是指对前述建库步骤获得的文库进行序列测定， 包括 步骤：
H )对 G ) 中获得的双链 DNA进行末端修复， 连接测序接头；
J )对 H ) 中的产物进行 DNA测序。
2.项目 1的测序方法：
其中， 步骤 B ) 中辅助接头选自下述 a-h中的至少一种：
a为不含酶切位点， 非连接端为一条链突出结构，连接端为平末端； b为含酶切位点， 非连接端为一条链突出结构， 连接端为平末端； c为不含酶切位点， 非连接端为树杈结构， 连接端为平末端； d为含酶切位点， 非连接端为树杈结构， 连接端为平末端；
e为不含酶切位点，非连接端为一条链突出结构，连接端为粘末端； f 为含酶切位点， 非连接端为一条链突出结构， 连接端为粘末端； g为不含酶切位点， 非连接端为树杈结构， 连接端为粘末端； h为含酶切位点， 非连接端为树杈结构， 连接端为粘末端。
步骤 B ) 中的辅助接头，步驟 F ) 中的引物和步骤 G ) 中切割酶如下 设计：
当辅助接头为 a和 c时， 引物设计成互补于经过 E )翻转的辅助接 头的序列， 且在 5，末端额外连接有限制性内切酶的识别位点， 所述的限 制性内切酶的识别位点位于引物上， 并保证该酶的切割位点位于辅助接 头与待测 DNA连接点的上下游 5bp; 所述的限制性内切酶选自： EcoP15I 和 Mmel。 当进行步骤 G时， 釆用针对所设计的限制性内切酶的酶切位 点的酶进行单酶切；
当辅助接头为 b和 d时，在辅助接头中靠近辅助接头与待测 DNA连 接点的上游 5bp内位置设计酶切位点，该酶切位点需符合以下三个特点： 1 )该酶切位点至少包含一个曱基化胞嘧啶； 2 )酶切位点内不含有非曱 基化的胞嘧啶； 3 )该酶切位点不含有 CpG二核苷酸位点。 所述的限制 性内切酶选自： Alul， Bell, Bfal, Bgl l l, BsrGI, BspHI, CviAII, Fatl Hindl ll, HpyCH4V, Nlalll, Ns i l, Pci l, Seal, Spel, Xbal等等。 在引物设计成与经过 E )翻转的辅助接头互补的序列， 当进行步骤 G时， 釆用针对所设计的甲基化的酶切位点进行切割的酶进行单酶切； 或者， 在引物 5， 端设计另外一个酶切位点， 通过调整辅助接头的长度使两个 酶切位点重合； 当进行步骤 G时， 釆用针对所设计的限制性内切酶的酶 切位点的酶进行双酶切。
当辅助接头为 e和 g时， 辅助接头和引物的设计原理与 a、 c方案 相同，不同处在于与目的 DNA片段连接方式不同： a、 c连接方式为 "T-A" 连接， e和 g为粘性末端连接。 因此， 对辅助接头和引物的设计原理不 再赘述。
当辅助接头为 f 和 h时， 辅助接头和引物的设计原理与 b、 d方案 相同，不同处在于与目的 DNA片段连接方式不同： b、d连接方式为" T-A" 连接， f 和 h为粘性末端连接。 因此， 对辅助接头和引物的设计原理不 再赘述。
3.项目 1或 2的测序方法， 其中步骤 A ) 包括：
a将基因组 DNA片段化成为双链 DNA片段； b将 a中的双链 DNA片段末端修复为平末端；
c将 b中的平末端的 3， 末端加上 A碱基。
4. 项目 1或 2的测序方法，其中步骤 C)的曱基化免疫共沉淀选自 MeDIP和 MBD。
5. 项目 1或 2的测序方法，其中步骤 d)的重复序列去除使用 C0T1
DNA,
6. 项目 2的测序方法， 其中步骤 F)中酶切位点曱基化是指 d、 f、 h方案， 具体如以上方案所述。
7.项目 1的测序方法，其中的辅助接头设计成防止自体连接的结构， 在 5， 末端标记有生物素。
8.—种执行项目 1或 2的方法的自动装置。
9.项目 8的装置， 其包括
A)基因组 DNA的片段化单元和末端修复单元；
B)将 A)得到的双链 DNA与辅助接头连接的连接单元；
C)将 B)的产物进行曱基化免疫共沉淀的单元；
D)去除 C)的产物中的中度， 高度重复序列的去除单元；
E)将 D)得到的产物进行亚硫酸盐翻转处理的单元；
F)将 E )得到的单链 DNA通过引物进行 PCR扩增获得双链 DNA的单 元；
G)酶切去除辅助接头的单元；
H)对 G) 中获得的双链 DM进行末端修复的单元
J)对 H) 中修复的 DNA进行测序接头连接的单元；
K)对 J) 中的产物进行曱基化 DNA测序的测序单元。 附图说明：
图 1: 功能区曱基化 DNA精细图测序样本制备原理图。 主要实验步 骤依次为基因组 DNA的片段化、 DNA片段末端修复、 DNA片段 3， 端加 A 碱基、 连接辅助接头、 甲基化免疫共沉淀、 去除曱基化 DNA片段中的重 复序列、 亚硫酸氢盐翻转 DNA、 使用根据辅助接头设计的引物进行 PCR 将经翻转的 DNA变成双链、 去除辅助接头、 末端修去除辅助接头的 PCR 产物并在 3， 端加 A碱基、 连接测序接头、 测序。 图 2: 辅助接头的设计示意图。 辅助接头的设计按是否含有酶切位 点分为两类，含有酶切位点的辅助接头和不含有酶切位点的接头（b， d, f, h ) ； 辅助接头的非连接端可以设计成三种形式： 树杈（forked )结 构（图 c和 d )、 一条链突出结构（a和 b )和平端结构。 结合辅助接头 连接端的 TA连接或其他粘性连接特性， 共分八种情况。 分别如下所示： a为不含酶切位点， 非连接端为一条链突出结构， 连接端为平末端 形式；
b为含酶切位点， 非连接端为一条链突出结构， 连接端为平末端形 式；
c为不含酶切位点， 非连接端为树杈结构， 连接端为平末端形式； d为含酶切位点， 非连接端为树杈结构， 连接端为平末端形式； e为不含酶切位点， 非连接端为一条链突出结构， 连接端为粘末端 形式；
f 为含酶切位点， 非连接端为一条链突出结构， 连接端为粘末端形 式；
g为不含酶切位点， 非连接端为树杈结构， 连接端为粘末端形式； h为含酶切位点， 非连接端为树杈结构， 连接端为粘末端形式。 图 3: 平末端的辅助接头的连接和移除方式（1 ) , 本方式中的辅助 接头使用其中图 2中的两种： &和(；。 因为设计成采用酶切方式移除辅 助接头 1， 而且移除的时间点在经过亚硫酸氢盐翻转之后， 所要移除的 辅助接头 1以单链的形式存在于待测 DNA的末端， 此时为了有效移除所 述辅助接头 1同时使单链 DNA成为双链 DNA以方便后续测序接头连接， 发明人设计出与经过亚硫酸氢盐翻转后辅助接头 1序列相匹配的引物 ( A ) ， 同时在靠近引物的 5，位点处设计酶切识别位点（B ) ， 酶切识别 位点不与辅助接头序列相匹配， 处于引物的突出延伸部分。 选用的酶切 位点是与限制切割酶相适应的， 所选用的限制性内切酶的特点为能在酶 切识别位点下游 20- 30bp处对 DNA进行酶切，通过控制辅助接头的长度 将酶切位点设计在 DNA片段末端连接辅助接头处 5bp范围内。
图 4: 平末端的辅助接头 1的连接和移除方式（2 ) 。 辅助接头 1 中包含曱基化酶切位点方式的辅助接头 1的连接和去除。 本方式中的辅 助接头 1可以使用图 2中的两种： b和 d。 辅助接头 1中含有甲基化的 酶切位点， 通过曱基化酶切位点可以保证在酶切去除辅助接头时 DM片 段的完整性（但可允许 DNA片段末端 5bp内的损伤）和经亚硫酸氢盐翻 转之后酶切识别序列不改变。
图 5: 平末端的辅助接头连接和移除方式（3 )。 辅助接头中包含曱 基化酶切位点和引物中包含酶切位点方式的辅助接头的连接和去除。辅 助接头中同样在靠近连接处设计曱基化的酶切位点。 引物中在 5， 端设 计另外一个酶切位点， 通过调整辅助接头的长度使两个酶切位点重合。 通过两次酶切保证酶切的彻底性。
图 6: 粘性末端的辅助接头的连接和去除方式（1 )。 本方式中的辅 助接头可以使用其中两种： 6和8。 设计与 bi sul f i te翻转后辅助接头 序列相匹配的引物， 同时在靠近引物的 5， 位点处设计酶切识别位点。
图 7: 粘性末端的辅助接头的连接和去除方式（2 )。 辅助接头中包 含曱基化酶切位点方式的辅助接头的连接和去除。 本方式中的辅助接头 可以使用其中两种： f 和h。 辅助接头中含有甲基化的酶切位点， 保证 DNA片段不被切割和经亚硫酸氢盐翻转之后酶切识别序列不改变。 图 8: 粘性末端的辅助接头的连接和去除方式（3 )。 辅助接头中包 含曱基化酶切位点和引物中包含酶切位点方式的辅助接头的连接和去 除。辅助接头中同样在靠近连接处设计曱基化的酶切位点。 引物中在 5' 端设计另外一个酶切位点， 通过调整辅助接头的长度使两个酶切位点重 合。 通过两次酶切保证酶切的彻底性。
图 9: 去除甲基化的重复序列示意图。 COT-1 DNA 标记生物素后， 利用亲和素磁珠与生物素结合的原理， 将标记有生物素的 COT-1 DNA标 记在包被有亲和素的磁珠上并去除未标记生物素的 COT-1 DNA片段。 将 与生物素 -DNA结合的亲和素磁珠与经曱基化免疫共沉淀后的曱基化 DNA 片段进行杂交， 甲基化的重复序列将会被杂交在标记生物素的 C0T-1 DNA上， 分离磁珠并舍弃磁珠， 将溶液中的 DNA收回， 回收的 DNA即功 能区甲基化的 DNA片段。
图 10:全基因磺酸化处理测序与本技术测序结果共同比对的单个 CpG 位点的测序深度分析。 比对说明单个 CpG位点的测序深度趋势一致。 其 中左图为 MRERB测序结果； 右图为全基因磺酸化测序结果。
图 11-图 18为所有类型的辅助接头和引物序列说明：
图 11 : 平末端的辅助接头连接和去除方式 （1 ) 的接头序列和引物 序列， 本辅助接头连接和去除方式中的接头序列不包含特殊的碱基序 列， 但要保证该接头经磺酸化后能成功扩增。 本接头的连接端的 3 '端 设计了 "T" 碱基的突出、 5， 端修饰了磷酸基团以便和 DNA连接时能以
"T-A" 连接式相连接。 在 PCR产物中接头去除方面， 我们在 PCR引物 的 5 '端引入酶识别序列，该酶的特点是能在酶识别位点下游的大于 20bp 的地方切割 DNA片段， 可以将辅助接头完全切去。 另外可以引物的 5， 端引入生物素基团 （也可以不修饰生物素基团， 不修饰的情况下去除小 片段时可以用 Ampure Beads去除。 ） 。 因此， 去除接头前的 PCR产物 片段包括待测 DNA片段、 对称的两个酶切位置、 对称的匹配接头序列及 其对称的两个酶识别位点序列。
图 12: 平末端的辅助接头连接和去除方式（2 ) 的接头序列和引物 序列。 本辅助接头连接和去除方式中的接头序列包含要保证该接头经磺 酸化后能成功扩增的 20- 30bp的序列和一个曱基化的酶识别序列。该酶 的特征是酶识别位点和切割位点相同。 本接头的连接端的 3 '端设计了
"T"碱基的突出、 5， 端修饰了磷酸基团以便和 DNA连接时能以 "T - A" 连接式相连接。 在 PCR产物中接头去除方面， 由于该酶在接头中做了甲 基化修饰， 因为经过磺酸化之后序列不变， 用该酶进行酶切可把接头去 除。 另外可以引物的 5， 端引入生物素基团（也可以不修饰生物素基团， 不修饰的情况下去除小片段时可以用 Ampure Beads去除。 ） 。 因此， 去除接头前的 PCR产物片段包括待测 DNA片段、对称的两个酶识别切割 序列、 对称的匹配接头序列。
图 13: 平末端的辅助接头连接和去除方式（3 ) 的接头序列和引物 序列。 本辅助接头连接和去除方式中的接头序列包含要保证该接头经磺 酸化后能成功扩增的 20-30bp的序列和一个甲基化的酶识别序列。该酶 的特征是酶识别位点和切割位点相同。 本接头的连接端的 3 '端设计了
"T"碱基的突出、 5， 端修饰了磷酸基团以便和 DM连接时能以 "T-A" 连接式相连接。 在 PCR产物中接头去除方面， 由于该酶在接头中做了曱 基化修饰， 因为经过磺酸化之后序列不变， 用该酶进行酶切可把接头去 除； 另外， 我们同时在 PCR产物中接头去除方面， 我们在 PCR引物的 5
'端引入酶识别序列， 该酶的特点是能在酶识别位点下游的大于 20bp 的地方切割 DNA片段， 可以将辅助接头完全切去。 另外可以引物的 5， 端引入生物素基团（也可以不修饰生物素基团， 不修饰的情况下去除小 片段时可以用 Ampure Beads去除。 ） 。 因此， 去除接头前的 PCR产物 片段包括待测 DNA片段、 对称的两个酶切位共同切割的位置、 对称的匹 配接头序列及酶 2对称的两个酶识别位点序列。 图 14: 粘性末端的辅助接头连接和去除方式（ 1 )的接头序列和引物 序列。本辅助接头连接和去除方式类同平末端的辅助接头连接和去除的 方式（1 )。 不同的是本接头的连接端的 3 '端非 "T- A"连接式相连接。 而且设计和 DNA末端的粘性末端序列匹配的序列。
图 15: 粘性末端的辅助接头连接和去除方式（2 ) 的接头序列和引 物序列。本辅助接头连接和去除方式类同平末端的辅助接头连接和去除 的方式（2 ) 。 不同的是本接头的连接端的 3 '端非 "T- A" 连接式相连 接。 而且设计和 DNA末端的粘性末端序列匹配的序列。
图 16: 粘性末端的辅助接头连接和去除方式（3 ) 的接头序列和引 物序列。本辅助接头连接和去除方式类同平末端的辅助接头连接和去除 的方式（3 ) 。 不同的是本接头的连接端的 3 '端非 "T-A" 连接式相连 接。 而且设计和 DNA末端的粘性末端序列匹配的序列。
图 17: 其他接头设计方案说明。
系列一 ( b-l，d-l，f-l，h-l )辅助接头设计方案类似1)、 d接头设计， 是在辅助接头中靠近辅助接头与待测 DNA连接点的上游 20- 30bp内 位置设计酶切位点， 该酶切位点需符合以下四个特点： 1 )该酶切 位点至少包含一个曱基化胞嘧啶； 2 )酶切位点内不含有非曱基化 的胞嘧啶； 3 ) 该酶切位点不含有 CpG二核苷酸位点； 4 ) 该酶的 切割位点位于辅助接头与待测 DNA连接点的上、 下游 5bp内。 所述的限制性内切酶选自： EcoP15I和 Mmel等等。 在引物设计 成与经过 E )翻转的辅助接头互补的序列， 当进行步骤 G时， 采 用针对所设计的曱基化的酶切位点进行切割的酶进行单酶切。
系列二（ b-2， d-2, f-2, h-2 )辅助接头设计方案是系列一方案和 b、 d、 f、 h的结合， 通过调整辅助接头的长度使两个酶切位点重合， 该方 案可以使用双酶切去除辅助接头。在引物设计成与经过 E )翻转的辅 助接头互补的序列， 当进行步骤 G时， 采用针对所设计的曱基化 的酶切位点进行切割的酶进行双酶切。 发明详述
定义
为了更清楚地阐述本发明， 在本发明中的各个定义解释如下。
DN A片段化及修复
DNA片段化是指： 将基因组用机械或者酶切等方法将基因组 DNA剪切成小的 DNA片段（如几百碱基或几千碱基） 。
DNA片段的修复是指： 剪切后的 DNA片段， 尤其是机械方法打 断的 DNA片段， DNA片段双链末端可能会出现以下情况： 双链同 侧末端断裂位置不一致、 3' 末端或者 5 ' 末端有损伤， DNA片段 修复的意义在于将这些末端补齐或者 3' 、 5， 有损伤的片段修复 成 5， 连接磷酸基团和 3' 连接羟基的平末端结构。
根据高通量测序仪对测序文库的要求 （W02008096146A1 ) ， 需制备合适长度的 DNA片段文库。 首先将 D 片段化成适合测序 仪测序长度大小的 DNA片段。 片段化效果一般以所要求制备的文 库片段大小在所片段化 DNA Smear的主带位置较为理想。 例如， 若要求制备插入 100 bp的 Pa ired End文库， 则断裂后 DNA Smear 主带在 100 bp处即可， 若断裂效果不理想则需要进行重新断裂。 样品片段化步骤也可使用其他断裂系统， 具体参数可根据仪器的 要求进行调整。
用于断裂 DM的方法包括酶切和超声断裂的方法。
酶切的方法可选用单一内切酶或多种不含 CpG位点的内切酶 联合酶切。 酶切方法由于酶切识别位点的固定性， 可能致使部分 DNA片段长度不适合而舍弃在文库之外； 而超声断裂 DNA是随机 断裂 DNA， 断裂位点不固定， 理论上整个基因组可包含在测序文 库之中。 本发明实验方案中片段化基因组既可用酶切的方法， 也 可选用超声断裂的方法， 但实例用超声断裂方法。
如果是由超声断裂的 D ， 需对 DNA片段末端修复， 修复成 平末端。 该修复的目的是将双链 DNA片段损伤的 5， 端磷酸化， 损伤的 3 '端羟基化， 修复成平末端的 DNA片段 3， 末端加 "A" 碱基使连接辅助接头时形成 "TA" 的粘性末端连接； 如果是多种 酶联合酶切方法片段化的基因组 DNA， 由于酶切末端序列不一样， 首先将 DNA片段修饰成平末端后在 3， 端加 "A" 碱基使连接辅助 接头时形成 "TA" 的粘性末端连接； 如果是单一酶切， 若酶切末 端是平末端在末端的 3，端加" A"碱基使连接辅助接头时形成 "TA" 的粘性末端连接； 如果是粘性末端， 设计辅助接头的时候可设计 成与 DNA片段粘性末端匹配的接连末端， 进行粘性末端的连接。 辅助接头 （adapter) 的连接和切除
辅助接头是指： 加入辅助接头的目的是在 DNA在进行 MeDIP和 bi sulf ite磺酸化后能经过 PCR反应后使单链 DNA变成双链，从而能 进行常规文库制备
辅助接头的类型： 根据 DNA片段化的方法不同以及碱基修饰的 不同的分类。 目前， 曱基化的 bisulfite测序利用的曱基化的接头 (WO2009024019A1) 。 由于该接头的所有胞嘧啶位点都是曱基化 的， DNA片段在连接甲基化的测序接头后经 bisulfite翻转之后， 接头的序列不发生改变， PCR之后仍和测序引物匹配。
但是曱基化接头影响了 MeDIP实验中曱基化抗体绑定曱基化 片段， 因此该接头不适用于 MeDIP后 bisulfite甲基化测序。 而 如果在 MeDIP前连接没有曱基化的接头， bisulfite翻转会致使 接头序列发生改变而和测序引物不匹配。 如果在 bisulfite前不 加接头， 双链 DNA经 MeDIP和 bisulfite之后变成单链 DNA, 并 且序列发生改变， 常规建库 （以 solexa 测序文库为例） 所用的 Paired End adapter与 Paired End primer无法在本实验中使用。 因此本发明引入辅助接头（图 2、 图 17)解决 MeDIP与 bisulfite 翻转导致序列改变之间的矛盾， 能在不影响 MeDIP的情况下， 经 bisulfite后成功扩增 DNA片段库并能进行后续的常规建库。 辅助接头的的接连和剪切
辅助接头的作用是解决甲基化免疫共沉淀与 bisulfite致使 接头序列改变导致测序引物无法匹配的矛盾， 因此在 bisulfite PCR之后要把辅助接头剪切去掉， 以减少测序成本。
辅助接头的接连和剪切结合起来考量可分为以下几种情况： 1. 多酶切 DNA片段或超声断裂片段的连接辅助接头及酶切方 式 引物中包含酶切位点 （图 3) 。
多酶切 DNA片段或超声断裂片段经末端修复和 3， 端加 "A" 碱基后需连接辅助接头，本方式中的辅助接头可以使用其中两种： a和 c。该辅助接头除了在其中一条 5，端修饰磷酸基团便于和 DNA 片段中 3， 的羟基连接外不必做任何其他修饰。
连接辅助接头之后的 DNA片段经 MeDIP等一系列处理后， 设 计与 bisulfite翻转后辅助接头序列相匹配的引物（图 3A) ， 同 时在靠近引物的 5， 位点处设计酶切识别位点 （图 3B) ， 酶切识 别位点不与辅助接头序列相匹配， 处于引物的延伸部分。 该酶的 特点为能在酶切识别位点下游 20-30bp处对 DNA进行酶切， 通过 控制辅助接头的长度将酶切位点设计在 D 片段末端连接辅助接 头处 5bp范围内。 DNA片段经 PCR之后在 DNA的两端多了酶切识 别序列， 通过酶对双链序列的识别便可在酶切处酶切 DNA片段。 由于在引物的 5， 标记生物素， 酶切掉的辅助接头部分和未被酶 切的 DNA片段可与亲和素的结合， 能迅速、 彻底的被去除。 可用 于引物中酶包括： EcoP15I和 Mmel0
在本发明的一个实施方式中， 采用引物中包含酶切位点方式 的辅助接头的连接和去除（图 2) 。 本方式中的辅助接头可以使 用其中两种： a和 c。设计与 bisulfite翻转后辅助接头序列相匹 配的引物（A) ， 同时在靠近引物的 5， 位点处设计酶切识别位点 (B) , 酶切识别位点不与辅助接头序列相匹配， 处于引物的延伸 部分。 该酶的特点为能在酶切识别位点下游 20-30bp处对 DM进 行酶切， 通过控制辅助接头的长度将酶切位点设计在 DNA片段末 端连接辅助接头处 5bp范围内。
2.多酶切 DNA片段或超声断裂片段的连接辅助接头及酶切方 式二一-辅助接头中包含甲基化酶切位点 （图 3) 。
本方式中多酶切 DNA片段或超声在断裂片段经末端修复和 3， 端加 "A" 碱基后连接辅助接头可用 （图 2 ) b和 d两种。 b和 d 辅助外链子在靠近连接端设计酶切位点， 设计该位点的目的是为 了 PCR之后能在此处将辅助接头酶切掉去除。 因此， 该酶切位点 设计在辅助接头的靠近连接处。 由于经过亚硫酸氢盐转化之后未 曱基化的胞嘧啶会转化为鸟嘧啶， 并在 PCR后变成胸腺嘧啶。 发 明人在设计酶切位点时如果酶识别序列中含有胞嘧啶， 需将此处 的胞嘧啶做曱基化修饰以保证经亚硫酸氢盐翻转后酶识别序列不 发生改变。 由于亚硫酸氢盐转化后的 PCR产物序列中腺嘌呤（A) 和胸腺嘧啶（T)增多， 发明人在设计酶切位点时为了不切割到 DNA片段，选择的酶切识别位点中含有一个非 CpG位点的 C, 并且 该胞嘧啶不在酶切识别序列的末位。可用在此处的酶包括： Alul， Bell, Bfal, Bglll, BsrGI, BspHI, CviAII, Fatl, Hindlll, HpyCH4V, Nlalll, Nsil, Pcil, Seal, Spel, Xbal等等。 另外， 该辅助接头也在其中一条 5， 端修饰磷酸基团便于和 DNA片段中 3 ' 的羟基连接。
连接辅助接头之后的 DNA片段经 MeDIP等一系列处理后， 设 计与 bi sul f i te翻转后辅助接头序列相匹配的引物并在 5， 端修 饰生物素（图 4 )。 由于在引物 5， 端修饰了生物素， 酶切掉的辅 助接头部分和未被酶切的 DNA片段可与亲和素的结合， 能迅速、 彻底的被去除。
在本发明的一个实施方式中， 采用辅助接头中包含曱基化酶 切位点方式的辅助接头的连接和去除（图 4 ) 。 本方式中的辅助 接头可以使用其中两种： 图 4的 b和 d。 辅助接头中含有曱基化 的酶切位点， 保证 DNA片段不被切割和经亚硫酸氢盐翻转之后酶 切识别序列不改变。
3. 多酶切 DNA片段或超声断裂片段的连接辅助接头及酶切方 式三一-辅助接头中包含曱基化酶切位点和引物中包含酶切位点 (图 5 ) 。
单一种酶进行酶切时往往不够彻底， 有时候有必要利用两种 或多种酶进行酶切处理。 本方式中多酶切 DNA片段或超声在断裂 片段经末端修复和 3， 端加 "A"碱基后连接辅助接头如同方式二 用 （图 2 ) 1)和3两种， 并同样在靠近连接处设计甲基化的酶切位 点。
设计与 b i sulf i te翻转后辅助接头序列相匹配的引物时如同 方式一， 在靠近引物的 5， 位点处设计酶切识别位点（图 5 ) ， 该 酶在酶切识别位点下游 20-30bp处对 DNA进行酶切， 通过控制辅 助接头的长度将两个酶切位点设计在 DNA片段的同一个位置。 通 过两次酶切， 保证切割的彻底性。 在引物的 5， 标记生物素， 酶 切掉的辅助接头部分和未被酶切的 DM片段可与亲和素的结合， 能迅速、 彻底的被去除。
在本发明的一个实施方式中，采用辅助接头中包含甲基化酶切 位点和引物中包含酶切位点方式的辅助接头的连接和去除（图
5 )。 辅助接头中同样在靠近连接处设计曱基化的酶切位点。 引物 中在 5， 端设计另外一个酶切位点， 通过调整辅助接头的长度使 两个酶切^ i点重合。 通过两次酶切保证酶切的彻底性。
4. 单酶切 DNA片段的连接辅助接头及酶切方式 引物中 包含酶切位点 （图 6 ) 。
单酶切 DNA片段为粘性末端的 DNA片段不必做末端修复和 3， 端加 "A"碱基， 可直接设计和酶切粘性末端匹配的辅助接头， 本 方式中的辅助接头可以使用其中两种： 6和8。该辅助接头除了在 其中一条 5， 端修饰磷酸基团便于和 DNA片段中 3' 的羟基连接外 不必做任何其他修饰。
连接辅助接头之后的 DNA片段经 MeDIP等一系列处理后， 设 计与 bi sul f i te翻转后辅助接头序列相匹配的引物 （图 6 ) ， 同 时在靠近引物的 5， 位点处设计酶切识别位点（图 6 )， 酶切识别 位点不与辅助接头序列相匹配， 处于引物的延伸部分。 该酶的特 点为能在酶切识别位点下游 20-30bp处对 DNA进行酶切， 通过控 制辅助接头的长度将酶切位点设计在 DNA片段末端连接辅助接头 处 5bp范围内。 DNA片段经 PCR之后在 DNA的两端多了酶切识别 序列， 通过酶对默链序列的识别便可在酶切处酶切 DNA片段。 由 于在引物的 5， 标记生物素， 酶切掉的辅助接头部分和未被酶切 的 DNA片段可与亲和素的结合， 能迅速、 彻底的被去除。 可用于 引物中酶包括： EcoP15 I和 Mmel。
在本发明的一个实施方式中， 采用粘性末端的辅助接头的连 接和去除（引物中包含酶切位点方式） （图 6 ) 。 本方式中的辅 助接头可以使用其中两种： 6和 g。设计与 bisulfite翻转后辅助 接头序列相匹配的引物， 同时在靠近引物的 5， 位点处设计酶切 识别位点。
5.单酶切 DNA片段的连接辅助接头及酶切方式 辅助接头 中包含曱基化酶切位点 （图 7) 。
单酶切 DNA片段为粘性末端的 DNA片段不必做末端修复和 3， 端加 "A"碱基， 可直接设计和酶切粘性末端匹配的辅助接头， 本 方式中的辅助接头可以使用其中两种： f 和11。该辅助接头除了在 其中一条 5， 端修饰磷酸基团便于和 DNA片段中 3' 的羟基连接外 不必做任何其他修饰。
辅助外链子在靠近连接端设计包含一个曱基化酶切位点， 酶 切位点时为了不切割到 DNA片段， 选择的酶切识别位点中含有一 个非 CpG位点的 C, 并且该胞嘧啶不在酶切识别序列的末位。 为 了经亚硫酸氢盐翻转后酶切识别序列不发生改变， 发明人将酶切 识别序列里的胞嘧啶甲基化， 其他碱基不做修饰。 可用在此处的 酶包括： Alul, Bell, Bfal, Bglll, BsrGI, BspHI, CviAII, Fatl, Hindlll, HpyCH4V, Nlalll, Nsil, Pcil, Seal, Spel, Xbal等 等。 另外， 该辅助接头也在其中一条 5， 端修饰磷酸基团便于和 DNA片段中 3' 的羟基连接。
连接辅助接头之后的 DNA片段经 MeDIP等一系列处理后， 设 计与 bisulfite翻转后辅助接头序列相匹配的引物并在 5， 端修 饰生物素（图 4)。 由于在引物 5， 端修饰了生物素， 酶切掉的辅 助接头部分和未被酶切的 DNA片段可与亲和素的结合， 能迅速、 彻底的被去除。
在本发明的一个实施方式中，采用辅助接头中包含曱基化酶切 位点方式的辅助接头的连接和去除（图 7) 。 本方式中的辅助接 头可以使用其中两种： f 和 h。辅助接头中含有甲基化的酶切位点， 保证 DNA片段不被切割和经亚硫酸氢盐翻转之后酶切识别序列不 改变。
6.单酶切 DNA片段的连接辅助接头及酶切方式三一-辅助接头 中包含甲基化酶切位点和引物中包含酶切位点 （图 8 ) 。
在本发明的一个实施方式中， 单一种酶进行酶切时往往不够彻 底， 有时候有必要利用两种或多种酶进行酶切处理。 本方式中多 酶切 DNA片段或超声在断裂片段经末端修复和 3， 端加 "A"碱基 后连接辅助接头如同方式二用 （图 8 ) f 和 h两种， 并同样在靠近 连接处设计曱基化的酶切位点。
设计与 bi sulf i te翻转后辅助接头序列相匹配的引物时如同 方式一， 在靠近引物的 5， 位点处设计酶切识别位点（图 8 ) ， 该 酶在酶切识别位点下游 20-30bp处对 DNA进行酶切， 通过控制辅 助接头的长度将两个酶切位点设计在 DM片段的同一个位置。 通 过两次酶切， 保证切割的彻底性。 在引物的 5， 标记生物素
( biot in ) ， 酶切掉的辅助接头部分和未被酶切的 DNA片段可与 亲和素的结合， 能迅速、 彻底的被去除。
在本发明的一个实施方式中釆用辅助接头中包含曱基化酶切 位点和引物中包含酶切位点方式的辅助接头的连接和去除（图 8 )。 辅助接头中同样在靠近连接处设计甲基化的酶切位点。 引物 中在 5' 端设计另外一个酶切位点， 通过调整辅助接头的长度使 两个酶切位点重合。 通过两次酶切保证酶切的彻底性。
甲基化免疫共沉淀
甲基化免疫共沉淀（MeDIP )是通过 5-甲基胞嘧啶抗体可用 来进行免疫沉淀高特异性的富集甲基化 DNA片段， 结合新一代测 序技术可以高通量的筛选异常甲基化的基因， 此法避免了应用限 制性酶在酶切位点上的局限性。 本发明所使用甲基化免疫共沉淀技术 根据 Michael Weber的免疫共沉淀技术略有改动。 为了测序 DNA片段长 度的需要，将 DNA片段大小由原来的 200-1000bp调整为 100-300bp; 另 外， 5-曱基胞嘧啶抗体孵育时间经过优化后发现孵育效果最优时间为 12小时而非文献报道的 2小时。
亚硫酸盐翻转
亚硫酸盐翻转是釆用亚硫酸氢盐对单链 DNA分子进行化学修饰， 导 致未曱基化胞嘧啶 (C)可被亚硫酸氢盐脱去氨基而转变成尿嘧啶 (U)， 而 5mC不能被修饰， 仍保持为 5mC， 在 PCR反应过程中， 尿嘧啶与腺嘌呤 (A)配对， 尿嘧啶则被胸腺嘧啶（T)取代。 这一化学反应过程首先由 Frommer等 (M. Frommer等人， A genomic sequencing protocol that yields a pos i t ive di splay of 5-methylcytos ine res idues in individual DNA s trands. Proc Nat l Acad Sci U S A 89 (1992) 1827-31) 报导。 具体过程为： 第一步， 亚硫酸氢钠使胞嘧啶磺酸化； 第二步， 对 苯二酴脱氨基； 第三步， 在碱性环境下， 磺基消失， 成为尿嘧啶。 目前， 该实验可由天漠（zymo ) bi sulf i te- Golden试剂盒进行实施。
辅助接头的酶切和去除
根据辅助接头的不同设计方案中选择不同的酶， 利用合适量的酶 和通过延长酶切时间使接头尽可能彻底被酶切掉。 考虑到酶切的不彻底 性以及短片段辅助接头用普通试剂盒难去除干净， 发明人在引物的 5， 端修饰生物素， 通过用亲和素包被的磁珠结合生物素的方法去掉短片段 的辅助接头和未被酶切的 DNA片段。
UDNA末端修复和测序接头连接
经内切酶酶切的 DNA片段末端首先进行修复成可以和测序 adapter ( Solexa, 454, Sol id ) 匹配的末端。 以 Solexa为例， 经酶酶切的 DNA 片段末端， 若为粘性末端， 首先修复成平末端， 然后在 DNA平末端的 3' 端加 "A" 碱基， 和 Solexa公司提供的 PE adapter形成 "T-A" 连接； 若酶切末端为平末端，直接在 DM平末端的 3，端加" A"碱基后和 Solexa 公司提供的 PE adapter形成 "T- A" 连接。
PCR
在 DNA经过磺酸化 ( bisulfite )之后， Bisulf ite-PCR的目的是将 单链的 DNA 片段变成双链便于测序接头的连接。 同时扩增 DNA 的量。
Bisulfite PCR扩增用引物和磺酸化之后的辅助接头的序列进行匹配。
PE PCR在辅助接头酶切去除之后， 加连接 PE接头后扩增， 扩增的 目的是将 DNA片段两端的 PE分叉接头 （forked PE adapter) 变成可与 测序引物匹配的序列。
测序
去除辅助接头后的 DNA片段可直接用于后续 Genome Sequencer FLX 系统高通量测序的常规实验步骤如使用罗氏公司的 GS FLX Standard DNA 文库制备试剂盒； 或 Illumina公司 Genome Analyzer ( GA) 系统 高通量测序的常规实验步骤如使用 Paired End DNA文库制备试剂盒构 建常规 Paired End DNA测序文库； 也可以用于 AB公司 SOLiD系统的 SOLiD Library Oligos试剂盒常规实验步骤进行高通量测序。
实施例
下面通过实施例对本发明的具体实施方式进行阐述， 所述的实施方 式仅仅用来解释和说明本发明， 其并不限制本发明的保护范围。 任何本 领域技术人员根据公知的知识和现有技术的教导能够想到的等价的变 体都包含在本发明的保护范围中。
实施例 1
实施例中接头、 qPCR检测引物与磺酸化处理后 DNA的 PCR扩增引物 为人工合成序列由 Invitrogen公司合成； 所使用的 C0T1 DNA购买于 Invitrogen公司， qPCR检测使用 AB公司的 SYBR与配套试剂； EcoP15I 酶购买于 NEB公司。 实施例的操作原理如图 5所示。
1.获取基因组 DM
取血样 1 (取自志愿者） 10毫升， 用 QIAamp DNA Blood Mini Kit (Qiagen公司）提取样品 DNA。将提取得到的 DNA编号为 YH- 1，取其中 10微克 DNA样品作为起始材料，参照图 1的流程构建文库，本实施例中 构建的是 Illumina GA System Paired End文库。
2. 片段化基因组 DNA
采用 Covaris system (AB公司 )将前述步骤 1中的 DNA样品进行 断裂， 样品断裂结束后， 取约总量的 1/30 (体积比）的断裂后的样品在 2%琼脂糖凝胶上进行 ΙχΤΑΕ 电泳检测， 从所述电泳凝胶回收待测 DNA 片段。 为将样品 DNA片段控制在 100~ 500 bp范围内， Covaris system 断裂具体操作为：
1)双击打开 Covaris 主程序 "SonoLAB S- Series V2.54" 后点击 "Start [Enter]" ， 待仪器排气 30 分钟， 且水温降为 10 左右后方 可使用。 打开主程序之前须先确定打碎仪和冷却仪的电源开关是否打 开， 否则主程序会提示错误并重试。
2 )将样品加入待用的 lOOul的 covaris micro Tube中， 样品加 入量 5μ8， 最后将终体积用 ΤΕ补至 100 μ1， 用移液器将样品溶液吹打
^击 "Configure" ， 设置.
Figure imgf000028_0001
选择模式为 "Frequency Sweeping" 。 所有设置完成后点击 "Save" 或 "Save As..." 保存好程序， 再点击 "Return to Main Panel" 回到主界面。 将断裂管放入 Covaris装置中， 选择已保存 程序， 开始打碎。 按所设置的程序打碎完成后关闭打碎仪和制冷 仪的电源， 然后关闭打碎主程序和电脑。
3)将前述 2)中断裂后的样品从玻璃管中吸出，放入 1.5ml EP 管中。取出 3 μΐ的样品用于 2%琼脂糖凝胶进行 1 xTAE电泳检测， 并用 QIAquick PCR 纯化试剂盒进行回收， 产物溶于 32 μΐ的洗 脱緩沖液（Elution buffer, EB)中。
3.末端修复
先从 保存的试剂盒（ Illumina Paired End 测序文库构 建试剂盒）中取出 10 X多聚核苷酸激酶緩沖液和 lOmMdNTPs mix, 将其置于冰上融解并充分混勾 10 X多聚核苷酸激酶緩沖液。 在 1.5 mL的离心管中配制 ΙΟΟμΙ^末端修复反应体系： 取前述步骤 2 最后获得的 30 μΐ 的片段化回收产物、 45uL超纯水、 10 μΐ 10x多 聚核苷酸激酶緩冲液 (B904 ) 、 4 μΐ dNTP solution set (稀释混合为 lOmM each) 、 5 μί T4 DNA 聚合酶、 1 Klenow Fragment与 5 T4 多聚核苷酸激酶。 20Χ温浴 30分钟后， 用 QIAquickPCR纯化试剂盒回 收纯化经过末端修复的 DNA, 将产物 DNA溶于 34 L的 EB中。
4.添加 A碱基
先从 -20X:保存的试剂盒 ( Illumina Paired End 测序文库构建 试剂盒） 中取出 10x blue buffer 和 1 mM dATP, 将其置于冰上融解 并使其充分混匀。 在 1.5 mL的离心管中配制 50μΙ 的 DM末端加 "A" 反应的体系： 32μί前述步骤 3获得的末端修复纯化回收产物、 5μΙ 10χ blue buffer, 10 μΐ dATP (稀释为 ImM, GE公司)与 3 μΐ Klenow (3， -5， exo-)。 37 :温浴 30分钟后用 QIAquick PCR纯化试剂盒回收纯化 加 A体系中的 DM，将产物溶于 32μί的 EB中。取 1μΙ于 NanoDrop 1000 上测 0D值并记录样品浓度、 OD260/280比值以及 OD260/230比值等参数。
5.辅助接头 1的连接
从 -20 保存的试剂盒 ( Illumina Paired End 测序文库构建试 剂盒） 中取出 2 x Rapid 连接緩冲液和 Alu Linker, 将其置于冰上融解 并充分混匀 2 X Rapid 连接緩冲液。 在 1.5 mL的离心管中配制 ΙΟΟμΙ^的 连接反应体系： 30μΙ步骤 3得到的加 Α回收产物、 50 μΐ 2x Rapid连接 緩 冲 液 、 6 L ( 辅 助 接 头 ） （50uM) ( 5，- AGCTGGGCACCGCTCATGCCACTCCGGCT ， 5，- pGCCGGAGTGGCA TGAGCGGTGCCCAG ) 、 10 LT4 DNA连接酶与 4 L超纯水。 201；温浴 15 分 钟后用 ZYMO DNA Clean & ConcentratorPTMP-5 ( ZYMO公司 ) 回收纯化 加有辅助接头 1的 DNA， 产物溶于 40 的 TE中。
6.甲基化 DNA的免疫共沉淀 ( MeDIP )
(1)取来自步骤 5的样品， 参考步骤 4 NanoDrop 1000测定结果， 此 时 DNA为 4 g左右， 加 TE补至体积为 450μΙ 充分混匀。 在 98 ；的水 浴锅中处理 10分钟， 然后置于冰上 10分钟。
(2)在样品中加入 51 L 的 ΙΟ χ ΙΡ 緩冲液和 lO L Anti- 5 - Methylcytosine Mouse mAb (Abeam公司）， 充分混匀； 将样品竖直放置 在 rotator上， 孵育 12小时。
(3) 30μί Dynabeads M-280 山羊抗小鼠 IgG(Invitrogen公司）， 用 800μί 0.1% PBS-BSA将磁珠洗涤 2次， 加入 30 L 1 IP 緩沖液混匀。
(4)将 Dynabeads加到 DM- Antibody混合物中， 4 震荡混合 3小时。
(5)用 800μ11 χ IP緩冲液将磁珠洗涤 3次， 第一次洗时在 vertex上 涡旋 2-4秒， 涡旋 3次； 后两次洗涤用移液器反复吹打混合。 用磁力架 收集后将上清移除。
(6)加入 20(^L 蛋白水解酶 K 消化緩冲液和 3 L 蛋白水解酶 K (50μ8/μ1 ) ， 50 烘箱中旋转 3小时。
(7)用 ZYMO DNA Clean & ConcentratorPTMP-5回收步骤（6)这一反应 体系中的 DNA, 用 7倍 DNA溶液体积 （步驟 6 ) 的结合緩沖液（binding buffer) ， 溶于 2(^L经 601；水浴过的超纯水中。 l L于 NanoDrop 1000 上测得 OD值并记录样品浓度， 并 Q-PCR检测 MeDIP效果。
7. C0T1 DNA去除重复序列
(1)重复序列的生物素标记： 在 1.5mL 管中加入 4μΙ COT 1 DNA
( i g/μΐ ) (Invitrogen 公司） 、 8μί 随机引物（8N， lug/ L) (Invitrogen)与 25 L 超纯水后 97 处理 10分钟并置于冰上 10分钟。 然后继续加入 5 L 10 X Klenow 緩冲液、 5μί 生物素 /dNTP mix
(Biotin-16-dUTP: 0.35mM; dTTP: 0.65mM; dCTP: ImM; dGTP: ImM; dATP: ImM. ) 与 3μί Klenow Enzyme (exo-, Fermentas)使最终反应体 积为 50μ 371；处理过夜， 第二天继续反应并补加 Ιμΐ Klenow Enzyme, 继续反应 3-5小时。 反应结束用 QIAquick PCR纯化试剂盒回收纯化反 应体系中的 DNA, 产物溶于 ΙΟΟμΙ^的 ΕΒ中并用 Nano-droplOOO检测浓 度。 若总质量小于 2μ8， 可认为标记失败， 需重新标记。
(2)吸取 lOO L of Streptavidin— Dynabeads® M— 280 (Invitrogen) 到 1.5mL不粘管，用 80(^LTE 緩冲液或者 lxB&W緩冲液（5mM Tris- HCL pH 7.5, 0.5mM EDTA, 1M NaCl)洗磁珠两次。
(3)将磁珠重新悬浮于 ΙΟΟμΙ^ 2xB&W緩冲液（lOmM Tris-HCL pH 7.5, ImM EDTA, 2M NaCl).
(4)将用生物素标记好的 COT-1 DNA (预先变性， 971：处理 10分钟， 后置于水上 10分钟） 。 加入 悬浮磁珠 中 （步骤（3) ) ；
(5)常温低速旋转 1小时；
(6)移除上清， lxB&W緩冲液轻洗两次。
(7)抽干 MeDIP后的 DNA样品， 加入 65 ；预热的杂交液 ΙΟΟμΙ^, 移至 含有磁珠的不粘管， 651C翻转过夜。
(8)将磁珠上清移至新的 1.5mL Eppendorf 管， 用 ZYM0-5 纯化， 结 合緩冲液量为 7倍， 22 L 洗脱。 Ιμί于 NanoDrop 1000上测得 0D值并 记录样品浓度。 8·亚硫酸氢盐处理（Bisulfite treatment )
本实验使用 ZYMO EZ DNA Methylation-Gold Kit PTMP ( ZYM0 公 司）进行亚硫酸氢盐处理
(1)在 PCR管中添加 130 的 CT 翻转试剂到每 20μΙ DNA样品 中。 如果 DNA样品的体积小于 20μΙ 则用水来弥补差量。 通过轻弹试 管或移液器操作来混合样品。
(2)将样品管放到循环变温器并按以下步骤操作： 放置 10分钟， 641C放置 2.5 小时后立刻进行下述操作或者在 4 C下存储 (最多 20 小 时)。
(3)吸取 600 L的 Μ结合緩冲液到 Zymo-Spin ICPTMP 柱中， 并将 柱放如试剂盒所提供的收集管中。
(4)装填样品 (从步骤 2)到 Zymo-Spin ICPTMP 柱含有 M结合緩冲 液。 盖上盖子将柱颠倒数次来混合样品。
(5)全速 （>10，000 xg)离心 30秒， 去除流出液。
(6)吸取 200 μί的 M洗涤緩冲液到柱中， 全速离心 30 秒。
(7)吸取 200 μΐ的 M-Desulphonation 緩冲液到柱中并且在室温 (20Ό-30Χ )下放置 15-20分钟。 在培养后， 全速离心 30秒。
(8)吸取 200 μί的 M洗涤緩冲液到柱中， 全速离心 30 秒。 再添加 200 的 M洗涤緩冲液并且离心 30秒。
(9)吸取 20μί的 M洗脱緩沖液到柱基质中，将柱放置在 1.5mL 的 管中， 全速离心来洗脱 DNA。
9. 亚硫酸氢盐磺酸化处理后 DNA的 PCR扩增
(1)从 -20Ό保存的试剂盒中取出， 将其置于冰上化冻并充分混匀 JumpStartPTMP Taq DNA聚合酶。 在 0.2 mL的 PCR管中配制 50μί 的 PCR反应体系： 5μί 亚硫酸氢盐处理纯化后产物、 5yL 10 x PCR緩冲 液（Sigma)、 4 μ L dNTP、 0.5 μ L JumpStartPTMP Taq DNA 聚合酶（ Sigma )、 2 L辅助接头引物 1.0 (10 pmol/μυ (Invitrogen) 2 L辅助接头 引物（10 pmol/ μ L) ( Biotin-5' -GGTCAGCAGCTAAACACCACTCATACCACTC CA， Biotin- 5， - GGTCAGCAGTTGGGTATTGTTTATGTTATTTTGGT ) (Invitrogen) 与 35.5 超纯水。
(2)在热循环仪中运行下列扩增程序： 94Ό 10秒、 10个循环的 94 30秒 / 52Ό30秒 /72 30秒、 72匸 5分钟于 4 X存储。
(3)反应结束后用 QIAquick PCR 纯化试剂盒回收纯化反应体系中的 DNA, 产物溶于 50yL的 EB中。 取 1 μ L于 NanoDrop 1000上测得 0D值 并记录样品浓度。
10.酶切去除辅助接头 1
(1)在 1.5 mL的离心管中配制 100 L反应体系： PCR扩增产物 （ ~ 350ng, 不超过 400ng) ， 10 L 10 χ緩冲液 3 (NEB)， ΙμΙ lOOxBSA, 2 sinefungine ( lOmM) , 20μ 0 χΑΤΡ， 3 μ L EcoP15I ( 10U/ μ L ) , 补超纯水至 ΙΟΟμί;
(2) 37 酶切过夜，第二天补 lyL酶 继续酶切 2小时。 ZYM0-5纯化， 结合緩冲液用量为 5倍， 50μί洗脱；
(3)吸取 50 μΐ of Streptavidin-DynabeadsP®P M-280 (Invitrogen) 到 1.5mL 不粘管， 用 800 L TE緩冲液或 l xB&W緩沖液洗磁珠两次；
(4)将磁珠重悬于 50 μ L 2 X B&W緩冲液，加 50 μ L酶切产物（ step 2 );
(5)常温旋转 30分钟 (转速小不超过 600rpm)；
(6)将磁珠上清移至新的 1.5mLEppendorf 管中，用 Qiagen(Mini) 纯 化， 取 l L纯化产物于 NanoDrop 1000上测得 0D值并记录样品浓度。
11. 测序
将步骤 10中去除辅助接头 1后的 DNA片段直接用于后续 Illumina 公司 Genome Analyzer (GA) 系统， 进行高通量测序， 其采用的实验步 骤参见使用 Paired End DNA文库制备试剂盒构建常规 Paired End DNA 测序文库。
实施例 1的结果评价和分析
1. MeDIP富集效果的检测
对本发明使用的 MeDIP 方法的富集效果与商业 MeDIP 试剂盒 (Diagenode)的富集效果进行比较（表 1 )，其中曱基化富集效果 ( 4994 ) 为 62. 71%, 大于 Diagenode 的曱基化富集效果， 非甲基化的富集率 ( 8804 )为 0. 33%, 小于 Diagenode的非曱基化富集率。
因此， 本 MeDIP方法的回收率达到商业 MeDIP试剂盒（Diagenode) 的回收率要求范围， 并相对商业 MeDIP 试剂盒富集量大， 因此认为该 MeDIP方法可与商用试剂盒相媲美， 其试验结果平行可信。
表 1. YH-1 DNA样品的 MeDIP富集效率
引物 Diagenode YH-adapter-U
4994 回收率 （ 43. 65 62. 71
8804 0. 76 0. 33
2. 对 EcoP15I酶切效果的检测
对 EcoP15I酶切产物分别进行挑克隆后利用 Sanger法测序， 检测 结果表明 （表 2 ) ， EcoP15I 的酶切效率达 86%。 该酶的高酶切效率结 果表明该 Linker可被高效切除， 对后续接头连接等实验没有干扰。
表 2. EcoP15 /酶切效率检测
克隆 EcoP 15 7
醉切 24
未醉切 4
总量 28
酶切率（ ) 86
3. Sanger法测序库检
对用本次发明的测序库样本制备技术所制备的测序库进行克隆处 理并使用 Sanger法进行小范围的测序检测。 库检结果（表 3 ) 中有 76% 的 reads能比对回基因组区， 且其曱基化化率高达 87%; 亚硫酸盐 （磺 酸化）对非甲基化胞嘧啶的翻转率为 99%; 片段扩增重复率为零。
表 3: YH-1 基因组曱基化免疫共沉淀后重复序列去除 结合磺酸化处理文库 Sanger法测序结果
文库 测序 比对 比对 曱基化 转换率 扩增重复 GC碱
数量 数量 比率 率 率 基含量
YH-1 71 54 0.76 0.87 0.99 0 27.81% 实施例 2
实施例 2的原理如图 6所示，其中除了下述步骤与实施例一不同外， 所有步骤与实施例 1相同， 用下述步骤 5、 9和 10分别替代实施例一中 的步骤 5、 9和 10:
5. 辅助接头的连接
从- 20 保存的试剂盒中取出 2x Rapid连接緩冲液和 Alu Linker, 将其置于冰上融解并充分混匀 2x Rapid连接緩冲液。 在 1.5 mL的离心 管中配制 ΙΟΟμί的连接反应体系: 30μΙ加 A回收产物、 50 μ12χ Rapid 连 接緩冲液、 6μί 辅助接头（50uM) (5，- CTGGGCACCGCTCATGCCACTCCGGC TAAG5mCT, 5'-pG5mC TTAGCCGGAGTGGCATGAGCGGTGCCCAG)、 10 LT4 DNA连 接酶与 4 L超纯水。 20 温浴 15 分钟后用 ZYMO DNA Clean & ConcentratorPTMP-5回收纯化连接有辅助接头中的 DNA，产物溶于 40 μΐ 的 ΤΕ中。
9. 亚硫酸氢盐处理后 DNA的 PCR扩增 （Alul linker介导的 PCR反应）
(1)从 -20 保存的试剂盒中取出， 将其置于冰上化冻并充分混匀 JumpStartPTMP Taq DNA聚合酶。 在 0.2 mL的 PCR管中配制 50 L 的 PCR 反应体系： 5 μ ί 亚硫酸氢盐处理纯化后产物、 5 μ 10 x PCR buffer (Sigma)、 4μΙ dNTP、 0.5 μ L JumpStartPTMP Taq DNA 聚合酶 (Sigma) 、 2ML Alu I 引物 1.0 (10 pmol/ μ L) (Invitrogen)、 2μ L 辅助接头引物（lOpmol/ L) (Biotin-5' - CTAAACACCACTCATACCACT CCA, Biotin-5' — TTGGGTATTGTTTATGTTATTTTGGT ) (Invitrogen)与 35.5 μ L超纯氷。
(2)在热循环仪中运行下列扩增程序：94 10秒、 10个循环的 941； 30秒 / 52Ό30秒 /72匸30秒、 72 5分钟与 4 存储。
(3)反应结束后用 QIAquick PCR纯化试剂盒回收纯化反应体系中的 DNA, 产物溶于 50 L的 EB中。 取 1 μ L于 NanoDrop 1000上测得 0D值 并记录样品浓度。
10.酶切去除辅助接头
(1)在 1.5 mL 的离心管中配制 100 反应体系： PCR 扩增产物 (<400ng) 、 10yL 10x緩冲液 2(ΝΕΒ)、 3μΙ Alu I, 补超纯水至 100
(2) 371；酶切过夜， 第二天补 l L酶 继续酶切 2小时。 ZYM0-5纯化， 结合緩沖液用量为 5倍， 50μΙ洗脱；
(3)吸取 100 μ L 的 Streptavidin - Dynabeads P ® P M - 280 (Invitrogen) 到 1.5mL不粘管，用 800 μ L TE緩冲液或者 1 x B&W緩冲液 洗磁珠两次；
(4)将磁珠重悬于 50 2 xB&W緩冲液。 加入 50 (2)的酶切产物；
(5)常温旋转 30分钟 (转速小不超过 600rpm) ；
(6)将磁珠上清移至新的 1.5mLEppendorf 管， Qiagen (Mini) 纯化， 取 1 μ L于 NanoDrop 1000上测得 0D值并记录样品浓度。
实施例 2的结果评价和分析
1. 辅助接头效率检测
由于辅助接头中含有一个甲基化胞嘧啶， 因此首先检测该甲基化位 点是否对 MeDIP有影响。对比辅助接头，设计和其对应的非曱基化接头。 使用曱基化检测引物编号为 4994与 GAPDH两对、 非甲基化检测引物编 号为 8804与 TSH2B两对分别在 MeDIP后对曱基化与非曱基化的辅助接 头含量进去检测。 检测结果（表 4 )所示， 曱基化的辅助接头与非曱基 化对应的富集率一致。因此对曱基化的辅助接头对 MeDIP的本底影响可 忽略。
表 4. 曱基化与非甲基化辅助接头对 MeDIP的影响验证 引物 辅助接头 -U 回收率（W 辅助接头- M 回收率 0
4994 3. 560 3. 560
8804 0. 023 0. 028
TSH2B 0. 014 0. 015
GAPDH 13. 810 11. 390
2. MeDIP富集效果的检测
分别利用检测甲基化富集效果的引物（ 4994 )和非曱基化富集效果 的引物（ 8804 )对 MeDIP富集效果进行检测 （表 5 ) , 其中甲基化富集 效果为 45. 6%, 和 Diagenode的甲基化富集效果（表 2 )相差无几， 非 曱基化的富集率为 2. 9%,尽量略高于 Diagenode的非曱基化富集率，但 远远低于曱基化的富集效果。 因此， 本次 MeDIP富集 DM效率成功。
表 5. YH-1 DNA样品的 MeDIP富集效率
引物 YH-adapter-M
4994 回收率（％) 45. 6
8804 2. 9
3. 对 Alu/酶切效果的检测
对 Alu/酶酶切产物分别进行挑克隆后利用 Sanger法测序， 检测结果表明（表 6 )，两次 Alu/酶的酶切效率分别为 94%和 100%。 该酶切效率结果表明该 Linker可被高效切除，对后续接头连接等 实验没有干扰。 表 6. Alul的酶切效率检测
克隆 AluJ(l s t) A lu /(2nd)
醉切 30 79
未酶切 2 0
总量 32 79
酶切率 （％) 94 100
4. Sanger法测序库检
对用本次发明的测序库样本制备技术所制备的测序库进行克隆处 理并使用 Sanger法进行小范围的测序检测。 库检结果（表 7 ) 中有 85°/。 的 reads能比对回基因组区，且其甲基化化率高达 93%;说明连接 Alu l 接头的 DNA片段经过 MeDIP处理能富集到基因组中大部分曱基化区域的 序列。 另外， 高达 97%的转化率说明亚硫酸盐 （磺酸化）处理的效果也 是非常明显的。 因此，通过 Alu l接头连接的 DM片段经过 MeDIP处理、 重复序列去除、亚硫酸盐处理与 Alu I酶切后仍可得到有效的序列信息。 文库检测合格并用于构建 I l lumina公司 GA系统高通量测序的 Paired End DM文库与进行相应的测序。
表 7: YH-1 基因组曱基化免疫共沉淀后重复序列去除结合
磺酸化处理文库 Sanger法测序结果
文库 测序 比对 比对 曱基 转换率 扩增 GC碱
数量 数量 比率 化率 重复率 基含量
YH-1 75 64 0. 85 0. 93 0. 97 0 30. 64% 发明人仅仅在实施例 1和 2是仅用了平末端的辅助接头连接和去除 方式（1 )和（2 )进行了说明。 具有一定的代表性。 由于平末端的辅助 接头连接和去除方式（ 3 )是平末端的辅助接头连接和去除方式（ 1 )和 ( 2 ) 的综合， 因此， 平末端的辅助接头连接和去除方式（1 )和 （2 ) 实施的成功可以说明 （3 )也可以成功。 平末端相对粘性末端的原理在 于连接接头的方式的不同， 而平末端相对粘性末端连接效率低， 因此， 在平末端实施成功的情况下粘性末端的辅助接头连接和去除方式（4-6) 定能实施成功。
5. 与亚硫酸氢盐测序法的有效数据比较
在 I l lumina公司 GA系统进行高通量测序， 所述的测序是 45cycle Paired End测序。 具体测序流程与试剂均釆用 I I lumina公司标准流程 与试剂盒。 测序结果产生 2. 53Gb数据， 能比对（mapping )到参考基因 组 （homo sapienss ) 达一半， 和其他亚硫酸氢盐测序比对率近似 [H. Xiang 等人， Single base-resolut ion methylome of the s i lkworm reveal s a sparse epigenomic map. Nat Biotechnol 28 516-20] , 具 体结果可以参见表 8。 其中 DNA平均曱基化水平达 84. 2%， 全基因组甲 基化测序的约 70%曱基化水平相比说明 MeDIP结合曱基化胞嘧啶的特异 性较高。
表 8: YH-1 基因组曱基化免疫共沉淀后重复序列去除
结合磺酸化处理文库 GA 系统测序结果
文库 YH-1 总数据 57, 510, 098 ( 2. 53Gb ) 过滤后总数据 57, 049, 532
过滤率 0. 80% 比对数 30, 287, 175 比对率 53. 09% 扩增重复率 5. 83% 有效数据 28， 619, 720 ( 1. 27Gb ) 数据有效率 50. 17% 转化率 97. 18% 曱基化率 84. 20%
6.与 Sanger测序法比较文库的重复序列含量
对 45cycle Paired End测序数据的比对至基因组的序列进行高级 生物信息分析， 结果如表 9所示， 重复序列为 30. 87%， 和 Sanger测序 法检测的 MeDIP的重复序列含量比 （75% ) 降低了一半多。 以上结果说 明该 MeDIP结合 bisulf i te方法有很高的特异性， 能有效的分离 DNA曱 基化片段和非曱基化片段。 GA测序序列比对到重复序列区比率（ 30. 8% )， 重复序列去除将近一半， 降低了测序文库大小和冗余数据。
表 9 YH-1 基因组曱基化免疫共沉淀后重复序列去除结合
磺酸化处理文库 GA 系统测序其他参数结果
基因特征 数据量 百分比 （½)
重复序列 2927604 30. 83
转座子 2173324 22. 89
CpG 144614 1. 52
基因元件 1678027 17. 66
ncRNA 24037
总数 9493842
7.全基因磺酸化处理测序与本技术测序结果共同比对的单个 CpG 位点的测序深度分析
对 1号染色体采用 MRERB方法和全基因组磺酸化处理测序结果进行 比较，针对在基因组功能区共同比对到的 CpG位点做测序深度比较发现， 二者测序深度的趋势基本一致， 具体参见图 10。 但是测序深度差 4倍， 因此， 利用本发明的 MRERB方法只需 10G的数据将达到 70G全基因组 亚硫酸氢盐测序的水平， 整体数据量降低了 7倍。
因此， 通过对 MRERB文库制备技术的 Solexa测序数据初步分析表 明该方法能够从全基因组中特异性的分离出曱基化 DNA片段， 并能去除 大部分重复序列； 经亚硫酸氢盐翻转之后， 能对 GA系统读取的序列进 行比对、 定位， 并可以进行单个胞嘧啶的曱基化分析， 可用于不同样本 的曱基化检测。
综上所述， 本技术既能检测到基因组功能区 DNA曱基化， 又能精 准比对单个胞嘧啶曱基化状态。 在技术成本上， 在功能区曱基化测序 数据饱和的情况下可比全基因组亚硫酸氢盐测序的数据量少 80%-90%， 节约每个样本检测成本 70-80%。

Claims

权 利 要 求
1. 一种曱基化 DNA的高通量测序方法， 包括建库步骤和测序步骤， 其中建库步骤是指获得待检测的曱基化 DNA文库的步骤， 所述建库 步骤包括:
A)基因组 DNA的片段化及双链 DNA片段末端修复；
B)将 A)得到的欢链 DNA与辅助接头连接，所述的辅助接头是被设计 成能与修复后的双链 DNA末端相连接的双链 DNA序列， 经由该辅助接头 使经后续 E)步骤处理的单链 DNA经 PCR后变成双链 DNA,辅助接头与修 复的双链 DNA连接的一端为连接末端， 另一端为非连接末端；
C)将 B)的产物进行甲基化免疫共沉淀；
D)去除 C)的产物中的中度， 高度重复序列；
E)将 D)得到的产物进行亚硫酸盐翻转处理；
F)将 E)得到的单链 DNA通过引物进行 PCR扩增获得双链 DNA;
G)酶切去除辅助接头；
其中测序步骤是指对前述建库步骤获得的文库进行序列测定， 包括 步骤：
H)对 G)中获得的双链 DM进行末端修复， 连接测序接头；
J)对 H)中的产物进行曱基化 DNA测序。
2. 权利要求 1的测序方法：
其中， 步骤 B ) 中辅助接头选自下述 a-h中的至少一种：
a为不含酶切位点， 非连接端为一条链突出结构， 连接端为平末端； b为含酶切位点， 非连接端为一条链突出结构， 连接端为平末端； c为不含酶切位点， 非连接端为树杈结构， 连接端为平末端； d为含酶切位点， 非连接端为树杈结构， 连接端为平末端；
e为不含酶切位点， 非连接端为一条链突出结构， 连接端为粘末端； f 为含酶切位点， 非连接端为一条链突出结构， 连接端为粘末端； g为不含酶切位点， 非连接端为树杈结构， 连接端为粘末端； h为含酶切位点， 非连接端为树杈结构， 连接端为粘末端。
步驟 B)中的辅助接头，步骤 F)中的引物和步骤 G)中切割酶的设计 和组合应用选自下述（I) - (VI)中的至少一种：
(I)当辅助接头为 a和 c时， 引物设计成互补于经过 E)翻转的辅助 接头的序列， 且在 5，末端额外连接有限制性内切酶的识别位点， 所述的 限制性内切酶的识别位点位于引物上， 并保证该酶的切割位点位于辅助 接头与待测 DNA连接点的上下游 5bp的范围内； 当进行步骤 G)时，采用 针对所设计的限制性内切酶的酶切位点的酶进行单酶切；
(I I)当辅助接头为 b和 d时，在辅助接头中靠近辅助接头与待测 DNA 连接点的上游 5bp内位置设计酶切位点， 该酶切位点需符合以下三个特 点： 1)该酶切位点至少包含一个曱基化胞嘧啶； 2)酶切位点内不含有非 曱基化的胞嘧啶； 3)该酶切位点不含有 CpG二核苷酸位点；引物设计成 与经过 E)翻转的辅助接头互补的序列， 当进行步骤 G)时， 釆用针对所 设计的曱基化的酶切位点进行切割的酶进行单酶切； 任选地， 在所述引 物 5' 端设计另外一个酶切位点， 通过调整辅助接头的长度使两个酶切 位点重合； 当进行步骤 G)时，采用针对所设计的限制性内切酶的酶切位 点的酶进行双酶切；
(I I I)当辅助接头为 e和 g时， 辅助接头和引物的设计与 a、 c方案 相同，不同处在于与待测 DNA片段连接方式不同： a、 c连接方式为 "T-A" 连接， e和 g为粘性末端连接；
(IV)当辅助接头为 f 和 h时， 辅助接头和引物的设计与 b、 d方案 相同，不同处在于与待测 DNA片段连接方式不同： b、 d连接方式为 "T-A" 连接， f 和 h为粘性末端连接；
(V)辅助接头设计同 1>、 d接头设计， 进一步在辅助接头中靠近辅助 接头与待测 DNA连接点的上游 20-30bp内位置设计酶切位点， 该酶切位 点同时满足以下四点： 1 )该酶切位点至少包含一个甲基化胞嘧啶； 2 ) 酶切位点内不含有非曱基化的胞嘧啶； 3 )该酶切位点不含有 CpG二核 苷酸位点； 4 )该酶的切割位点位于辅助接头与待测 DNA连接点的上、 下游 5bp内； 在引物设计成与经过 E )翻转的辅助接头互补的序列， 当 进行步骤 G)时，采用针对所设计的曱基化的酶切位点进行切割的酶进行 单酶切
(VI)是 (V)和 b、 d、 f、 h的组合， 通过调整辅助接头的长度使两个 酶切位点重合， 该方案可以使用双酶切去除辅助接头；在引物设计成与 经过 E )翻转的辅助接头互补的序列， 当进行步骤 G)时， 釆用针对所设 计的甲基化的酶切位点进行切割的酶进行双酶切。
3. 权利要求 1或 2的测序方法， 其中步骤 A)包括：
a将基因组 DNA片段化成为双链 DNA片段；
b将 a中的欢链 DNA片段末端修复为平末端；
c将 b中的平末端的 3， 末端加上 A碱基。
4. 权利要求 1或 2的测序方法， 其中步骤 C)的曱基化免疫共沉淀 选自 MeDIP和 MBD。
5. 权利要求 1或 2的测序方法， 其中步驟 D)的重复序列去除使用 C0T1 DNA0
6. 权利要求 2的测序方法， 其中步骤 F)中酶切位点曱基化是指辅 助接头中设定的酶切位点里的的胞嘧啶位点是甲基化的。
7. 权利要求 1的测序方法，其中的辅助接头设计成防止自体连接的 结构， 在 5， 末端标记有生物素。
8. 一种执行权利要求 1或 2的方法的自动装置。
9. 权利要求 8的装置， 其包括
A)基因组 DNA的片段化单元和末端修复单元； B)将 A)得到的双链 DNA与辅助接头连接的连接单元；
0将 B)的产物进行曱基化免疫共沉淀的单元；
D)去除 C)的产物中的中度， 高度重复序列的去除单元；
E)将 D)得到的产物进行亚硫酸盐翻转处理的单元；
F)将 E)得到的单链 DM用引物进行 PCR扩增获得双链 DNA的单元;
G)酶切去除辅助接头的单元；
H)对 G)中获得的双链 DNA进行末端修复的单元
J)对 H)中修复的 DNA进行测序接头连接的单元；
K)对 J)中的产物进行甲基化 DM测序的测序单元。
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