WO2017181880A1 - 构建待测基因组的dna测序文库的方法及其应用 - Google Patents

构建待测基因组的dna测序文库的方法及其应用 Download PDF

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WO2017181880A1
WO2017181880A1 PCT/CN2017/080142 CN2017080142W WO2017181880A1 WO 2017181880 A1 WO2017181880 A1 WO 2017181880A1 CN 2017080142 W CN2017080142 W CN 2017080142W WO 2017181880 A1 WO2017181880 A1 WO 2017181880A1
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treatment
digestion
stranded dna
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颉伟
张冰洁
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Tsinghua University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • 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
    • 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
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to the field of biotechnology.
  • the invention relates to methods of constructing DNA sequencing libraries of a genome to be tested and uses thereof. More specifically, the present invention relates to a method of constructing a DNA sequencing library of a genome to be tested, an apparatus for constructing a DNA sequencing library of a genome to be tested, a method of determining DNA sequence information of a genome to be tested, and a system for determining DNA sequence information of a genome to be tested. And a method for determining sequence information of a chromatin target region of the genome to be tested.
  • chromatin immunoprecipitation techniques include several important steps such as cross-linking, breaking DNA, antibody incubation, de-crosslinking, and eluting DNA.
  • traditional chromatin immunoprecipitation techniques are commonly used to study readily available Common types of cells of the order of magnitude above 10 6 are incapable of studying this extremely difficult, small number of types of cells in the early stages of embryonic development.
  • the process of cross-linking and cleavage of DNA in the traditional chromatin immunoprecipitation technique causes a large amount of DNA loss, and the subsequent several processes of purifying DNA make the DNA recovery efficiency a key factor affecting the amount of DNA obtained.
  • the inventors Based on the discovery by the inventors of the above factors affecting the amount of DNA obtained in the traditional chromatin immunoprecipitation technique, the inventors have creatively improved the traditional co-immunoprecipitation technique and proposed a new chromatin-based immunoprecipitation.
  • Library and sequencing technology The database construction technology and sequencing technology can build and efficiently sequence the DNA of primary cells or cells as low as 500, which brings hope for the study of fine regulation of epigenetics in early embryonic development.
  • the invention proposes a method of constructing a DNA sequencing library of a genome to be tested.
  • the method comprises: (1) performing a first digestion treatment on the genome to be measured using the microsphere nuclease, In order to obtain the first digestion treatment product; (2) subjecting the first digestion treatment product to co-immunoprecipitation treatment to obtain an immunoprecipitation treatment product; (3) using the proteinase K to perform the second embodiment of the immunoprecipitation treatment product Digesting treatment to obtain a second digestion treatment product; (4) directly dephosphorylating the second digestion treatment product with shrimp alkaline phosphatase to obtain a dephosphorylated product; (5) The phosphorylated product is directly subjected to a denaturation treatment to obtain a denatured treatment product containing a single-stranded DNA molecule; and (6) a sequencing library is obtained according to the TELP method based on the denatured-treated product containing the single-stranded DNA molecule.
  • the method for constructing a DNA sequencing library for constructing a genome uses microspheres to cleave the chromatin, thereby avoiding local damage of DNA caused by ultrasonication, and greatly improving the recognition efficiency of the antigen-antibody;
  • the method for constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention does not require purification and recovery of the second digestion treatment product, directly performing dephosphorylation using shrimp alkaline phosphatase, and passing through shrimp alkaline phosphatase
  • the dephosphorylated product also does not require purification and recovery, but can be directly denatured and a sequencing library is obtained, thereby significantly increasing the amount of DNA available for library construction.
  • the method proposed by the embodiments of the present invention can efficiently establish a DNA sequencing library of a genome having a starting cell amount as low as about 500 cells or a genome having a starting DNA amount as low as 2.5 nanograms, according to a specific embodiment of the present invention, A DNA flanking library of genomes with a starting cell volume as low as 200 cells or a DNA starting amount as low as 1 ng can be efficiently established.
  • the method proposed in the embodiments of the present invention is particularly suitable for constructing a DNA sequencing library of a micro-genome of primary cells or exfoliated tissues, and can be applied to high-throughput sequencing technology efficiently and sensitively, based on data analysis of sequencing results. , the gene sequence information can be obtained efficiently.
  • the invention proposes an apparatus for constructing a DNA sequencing library of a genome to be tested.
  • the apparatus comprises: a first digestion processing device for digesting the genome to be measured by the microsphere nuclease to obtain a first digestion treatment product; co-immunoprecipitation a treatment device, the co-immunoprecipitation treatment device is connected to the first digestion treatment device, and is configured to perform co-immunoprecipitation treatment on the first digestion treatment product to obtain an immunoprecipitation treatment product; and a second digestion treatment device
  • the second digestion treatment device is connected to the co-immunoprecipitation treatment device, and is configured to perform a second digestion treatment on the co-co-precipitation treatment product with proteinase K to obtain a second digestion treatment product; dephosphorylation treatment a device, the dephosphorylation treatment device is connected to the second digestion treatment device, the dephosphorization treatment device is configured to directly dephosphorylate the second digestion treatment
  • an apparatus for constructing a DNA sequencing library for constructing a genome to be tested it is possible to establish a genome having a starting cell volume as low as about 500 cells or a starting DNA amount as low as 2.5 nanoliters.
  • a DNA sequencing library of grams of genome can even efficiently establish a DNA sequencing library of a genome having a starting cell amount as low as 200 cells or a DNA starting amount as low as 1 nanogram.
  • the device proposed in the embodiment of the invention retains the specificity of the single-stranded DNA strand, has less DNA loss, and the genetic information remains intact. It is especially suitable for the construction of DNA sequencing libraries of the same micro-genome of primary cells or exfoliated tissues.
  • the invention proposes a method of determining DNA sequence information of a genome to be tested.
  • the method comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the site based on the sequencing result Describe the DNA sequence information of the genome.
  • DNA sequence information of the genome to be tested it is possible to sensitively, accurately and efficiently determine a genomic or microgenome of a trace amount of cells (initial amount of cells as low as 500 or even as low as 200) Sequence information for single-stranded DNA molecules with a starting DNA amount as low as 2.5 ng, or even as low as 1 ng.
  • the invention proposes a system for determining DNA sequence information of a genome to be tested.
  • the system comprises: a sequencing library construction device, the sequencing library construction device being the aforementioned; a sequencing device, the sequencing device being connected to the sequencing library construction device for use in The genome sequencing library of the genome is subjected to sequencing to obtain a sequencing result of the genomic DNA; and an analysis device analyzes the sequencing result of the genomic DNA to obtain the DNA sequence information.
  • a system for DNA sequence information of a genome to be tested it is possible to sensitively, accurately, and efficiently determine a genomic or microgenome of a trace amount of cells (initial amount of cells as low as 500 or even as low as 200) Sequence information for single-stranded DNA molecules with a starting DNA amount as low as 2.5 ng, or even as low as 1 ng.
  • the invention proposes a method for determining sequence information of a chromatin target region of a genome to be tested.
  • the method comprises: constructing a target region sequencing library of a genome to be tested according to the method described above, wherein the histone antibody specifically recognizes the target region; for the genome to be tested The target region sequencing library is sequenced to obtain sequencing results; and based on the sequencing results, sequence information of the chromatin target region of the test genome is determined.
  • Using the method for determining sequence information of a chromatin target region of a test genome according to an embodiment of the present invention it is possible to sensitively, accurately, and efficiently determine a genome of a trace amount of cells (initial amount of cells as low as 500 or even as low as 200) Or the sequence information of the chromatin target region of the microgenome (starting DNA amount as low as 2.5 ng, or even as low as 1 ng), thereby enabling detection of the chromatin target region of the genome of the micro-cell.
  • the genome to be tested proposed by the present invention refers to a whole genome or a partial genome of a cell or a tissue, and the genome is composed of chromatin or chromosome.
  • the source of the genome is not particularly limited and can be obtained from any possible route, either directly from a commercial market, directly from other laboratories, or directly from a cell or tissue. Extracted from the sample.
  • FIG. 1 is a flow chart of a method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention
  • FIG. 2 is a flow chart of a method of constructing a DNA sequencing library of a genome to be tested according to still another embodiment of the present invention
  • FIG. 3 is a flow diagram of performing a first digestion process on a genome to be measured using a micrococcal nuclease according to an embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of an apparatus for constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention
  • FIG. 5 is a schematic structural diagram of an apparatus for constructing a DNA sequencing library of a genome to be tested according to still another embodiment of the present invention.
  • FIG. 6 is a schematic structural view of a first digestion processing device according to an embodiment of the present invention.
  • FIG. 7 is a schematic structural view of an immunoprecipitation device according to an embodiment of the present invention.
  • Figure 8 is a schematic structural view of a second digestion processing apparatus according to an embodiment of the present invention.
  • FIG. 9 is a schematic structural diagram of a TELP device according to an embodiment of the present invention.
  • FIG. 10 is a schematic structural diagram of a TEL apparatus according to still another embodiment of the present invention.
  • FIG. 11 is a schematic structural view of a joint connecting unit according to an embodiment of the present invention.
  • FIG. 12 is a schematic structural diagram of a system for determining DNA sequence information of a genome to be tested according to an embodiment of the present invention
  • FIG. 13 is a flowchart of a method of determining sequence information of a chromatin target region of a genome to be tested according to an embodiment of the present invention
  • Figure 14 is a DNA gel electrophoresis pattern of a dephosphorylation enzyme screening experiment according to an embodiment of the present invention.
  • Figure 15 is a DNA gel electrophoresis pattern of a dephosphorylation enzyme screening experiment according to still another embodiment of the present invention.
  • 16 is a diagram showing the results of verifying the validity of the method of building a database according to an embodiment of the present invention.
  • FIG. 17 is a diagram showing the result of verifying the validity of the database construction method proposed by the embodiment of the present invention according to still another embodiment of the present invention.
  • the invention proposes a method of constructing a DNA sequencing library of a genome to be tested.
  • the method comprises: (1) performing a first digestion treatment on a genome to be measured by a microsphere nuclease to obtain a first digestion treatment product; (2) performing a first digestion treatment product Co-immunoprecipitation treatment; in order to obtain co-immunoprecipitation treatment product; (3) second digestion treatment of the co-immunoprecipitation treatment product with proteinase K to obtain a second digestion treatment product, wherein proteinase K is used for digestion and co-immunoprecipitation treatment a corresponding histone antibody in the product so as to expose the DNA, whereby the second digestion treatment product here is naked DNA; (4) directly dephosphorylating the second digestion treatment product with shrimp alkaline phosphatase to obtain Phosphorylation of the product; (5) direct dephosphorization of the product to obtain a denature
  • the method for constructing a DNA sequencing library of a genome to be tested adopts microsphere nuclease cleavage to cut chromatin, thereby avoiding local damage of DNA caused by ultrasonication, and greatly improving antigen-antibody recognition efficiency; According to the present invention
  • the method for constructing a DNA sequencing library of the genome to be tested does not require purification and recovery of the second digestion product, directly dephosphorylation with shrimp alkaline phosphatase, and dephosphorylation by shrimp alkaline phosphatase
  • the product also does not require purification and recovery, but can be directly denatured and a sequencing library is obtained, thereby significantly increasing the amount of DNA available for library construction.
  • the method proposed by the embodiments of the present invention can efficiently establish a DNA sequencing library of a genome having a starting particle amount as low as about 500 cells or a starting amount of DNA as low as 2.5 nanograms, according to a specific embodiment of the present invention, It is even possible to efficiently establish a DNA flanking library of genomics with a starting cell volume as low as 200 cells or a genome with a DNA starting amount as low as 1 ng.
  • the method proposed in the embodiments of the present invention is particularly suitable for the construction of a DNA sequencing library of the genome of a primary cell or a stripped tissue, and can be applied to high-throughput sequencing technology efficiently and sensitively, based on the data of the sequencing result. Analysis can effectively obtain gene sequence information.
  • the genome to be tested is obtained by lysing cells or tissues, wherein the cells are cell lines or primary cells, thereby releasing a genome in cells or tissues, thereby facilitating The micrococcal nuclease directly digests the chromatin.
  • the method of the present invention directly cleaves the cells or tissues in a natural state, and in the prior art, the cells are fixed by formaldehyde cross-linking, and the problem of masking the epitope information is presented, which is proposed by the embodiment of the present invention.
  • the method greatly improves the recognition efficiency of subsequent antigen-antibody.
  • the method for constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention is a state in which a chromatin is broken into a mononuclear body or a dinuclear body by using a micrococcal nuclease, that is, the first digestion treatment product described above is Fragmented chromatin, which effectively exposes histone-associated antigens, further enhances subsequent antigen-antibody reaction efficiency.
  • performing the first digestion treatment on the genome to be tested using the micrococcal nuclease may further comprise: (1-1) subjecting the test genome to the micrococcal nuclease working solution Precontact to obtain a precontacted mixture.
  • the product after the cleavage treatment is pre-contacted with the micrococcal nuclease working solution, so that the chromatin can be adapted to the enzyme digestion system in advance, thereby facilitating the improvement of the enzyme digestion efficiency; (1-2) using the micrococcal nuclease after the precontacting The mixture is subjected to a first digestion treatment to obtain a first digestion treatment product; (1-3) the digestion treatment is terminated using a micrococcal nuclease stop solution.
  • the immunoprecipitation treatment is carried out by incubating the first digestion treatment product with a histone antibody to obtain an immunoprecipitation treatment product, optionally further comprising:
  • the co-immunoprecipitation treatment product is contacted with magnetic beads carrying protein A, optionally, further comprising: washing the co-immunoprecipitation treated product with RIPA buffer and lithium chloride buffer.
  • the histone antibody specifically recognizes the specific antigenic site of the histone on the nucleosome, and then enriches the chromatin by immunoprecipitation of the antigen-antibody.
  • the immunoprecipitated product contacts the magnetic beads carrying the protein A.
  • the chromatin can be further enriched to improve the purity of the chromatin.
  • the immunoprecipitated product is washed with the RIPA buffer and the lithium chloride buffer to remove unintentional binding and further enhance the enriched target product (staining Quality).
  • the second digestion treatment is carried out by performing a second digestion treatment on the co-immunoprecipitation treatment product with an elution buffer and a proteinase K to obtain a second digestion treatment product.
  • the second digestion treatment is carried out at 55 degrees Celsius for 1 hour, optionally, further comprising: inactivating the proteinase K.
  • the above elution buffer is used for co-immunoprecipitation The substance is eluted from the magnetic beads, and then the naked DNA containing no histone is released under the digestion of histone antibody by proteinase K.
  • the optimum concentration of proteinase K is 55 degrees Celsius, and one reaction at 55 degrees Celsius.
  • the inventors found that naked DNA release is completely shorter than one hour, and that digestion is insufficient for more than one hour, causing non-specific degradation of DNA.
  • the proteinase K digestion treatment 1 the proteinase K was further subjected to inactivation treatment, and according to a specific example, the inventors used a heat inactivation treatment to cause the proteinase K to terminate the reaction in a timely manner.
  • the dephosphorylation treatment with shrimp alkaline phosphatase is carried out for 1 hour at 37 degrees Celsius.
  • shrimp alkaline phosphatase has the highest enzyme activity, and dephosphorylation for 1 hour can ensure sufficient reaction of the reaction substrate and ensure non-specific product increase without excessive reaction.
  • the dephosphorization treatment can inactivate the alkaline phosphatase of the shrimp, and the inactivation treatment is performed at 65 degrees Celsius for 10 minutes, and the inactivation treatment can effectively terminate the dephosphorylation reaction.
  • the denaturation treatment described above may be, but is not limited to, thermal denaturation treatment.
  • the denaturation treatment is to convert double-stranded DNA into single-stranded DNA, and further obtain a sequencing library according to the TELP method.
  • the heat denaturation treatment has high denaturation efficiency, small damage to DNA, low probability of DNA mutation, and no factor affecting subsequent construction. The advantages.
  • the applicant has elaborated on the TELP method in the patent 201410466261.2.
  • the TELP method is implemented as follows:
  • a denatured product containing a single-stranded DNA molecule is directly formed at the 3' end to form a poly(C) n tail to obtain a single-stranded DNA molecule having a Poly(C) n tail, wherein n represents the number of bases C, And n is an integer between 5 and 30.
  • the amount of the single-stranded DNA molecule is ⁇ 5 pg.
  • the initial amount of the sequencing library obtained by the present invention is significantly smaller than the initial amount of other second-generation sequencing libraries, and is suitable for constructing a sequencing library for a small sample, especially for a rare and scarce sample or a clinical patient specimen or a primary cell to construct a sequencing library. .
  • the amount of the single-stranded DNA molecule is from 5 pg to 10 ng. Therefore, the efficiency of constructing the sequencing library is high and the accuracy is good.
  • n of the tail of Poly(C) n may be an integer between 15 and 25. According to a specific example, n may be 20. Thereby, the effect of combining with the extended primer is good.
  • the manner in which the Poly(C) n tail is added to the 3' end of the single-stranded DNA molecule is not particularly limited.
  • the poly(C) n tail may be formed using a terminal transferase.
  • Single-stranded DNA can be linked to several polycytosine deoxynucleotides Poly(C) n at the 3' end of the DNA strand under the action of terminal transferase (TdT).
  • the reaction process is: pre-mixing 28 ⁇ l DNA solution, 1 ⁇ l 10x EX buffer (Takara, supplemented with RR006A), 1 ⁇ l of 1 mM dCTP (NEB, N0446S), denatured the DNA at high temperature to obtain a single-stranded DNA molecule. Then, 1 ⁇ l of terminal transferase (TdT; NEB, M0315S) was added and reacted at 37 ° C for 35 min. After completion of the reaction, the mixture was heated to 75 ° C for 20 minutes to inactivate TdT to obtain a single-stranded DNA molecule in which the 3' end was linked to the oligomeric Poly(C) n .
  • TdT terminal transferase
  • the extension primer includes a H(G) m unit at its 3' end, and H is a base A , base T or base C, m is the number of bases G, and m is an integer between 5 and 15.
  • the extension primer can initiate an annealing pair at a suitable position on the Poly(C) n tail.
  • m of the H(G) m unit may be 9.
  • the position at which the H(G) m unit is initially annealed with the oligomeric Poly(C) n is suitable.
  • the sequence of the extension primer is not particularly limited as long as DNA extension can be ensured.
  • the extension primer may be composed of the nucleotide set forth in SEQ ID NO: 1.
  • the extension primer is easily paired with the poly(C) n tail, and the extension reaction efficiency is high.
  • the specific sequence of SEQ ID NO: 1 is as follows:
  • a single-stranded DNA molecule can be extended using KAPA 2G Robust HS to obtain the double-stranded DNA molecule.
  • the extended reaction system is: in the single-stranded DNA molecule of the 3'-end oligo-Poly(C) n obtained in the previous step, 6.2 ⁇ l of water, 0.8 ⁇ l of KAPA 2G Robust HS (KAPA, KK 5515), 12 ⁇ l 5 ⁇ KAPA Buffer A (KAPA, KK 5515), 4.8 ⁇ l of 2.5 mM dNTP (Takara, RR006A), and 6 ⁇ l of 2 ⁇ M extension primer.
  • the procedure for the extension reaction is: (1) 95 ° C for 3 min; (2) 47 ° C for 1 min, 68 ° C for 2 min, 16 cycles; (3) 72 ° C for 10 min.
  • exonuclease I Exo I
  • the end of the extended chain obtained by the extension reaction that is, the 3' end of the extended strand is the base A, thereby facilitating the attachment to the half-linker with the base T as the head end.
  • the extension primer can have a screening marker, wherein the screening marker is formed at the 5' end of the extension primer.
  • the screening marker can be biotin.
  • the process of binding biotin to magnetic beads is as follows: magnetic streptavidin C1 magnetic beads (Invitrogen, 650.01) were previously washed with 1x Binding & Wash (B&W) buffer (10 mM Tris-HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl). Then, after mixing with the extended product, the incubation was carried out on a temperature-controlled mixing device under the following conditions: shaking at 1400 rpm at 23 ° C (oscillation frequency: shaking for 10 s, stopping for 10 s) for 30 min.
  • B&W Binding & Wash
  • the DNA-bound magnetic beads were washed once with 100 ⁇ l of 1 ⁇ B&W buffer, washed three times with 150 ⁇ l of EBT buffer (10 mM Tris-HCl pH 8.0, 0.02% Triton X-100), and finally 8.4 ⁇ l of elution buffer (EB). ; 10 mM Tris-HCl pH 8.0) was resuspended for subsequent ligation reactions.
  • EBT buffer 10 mM Tris-HCl pH 8.0, 0.02% Triton X-100
  • EB elution buffer
  • the step may further comprise: annealing the single-stranded nucleic acid molecule having the nucleotide sequence shown by SEQ ID NO: 2-3, respectively, to obtain a half-joint, and the half-joint One end of the double-stranded DNA molecule is ligated to obtain a double-stranded DNA molecule to which a half-linker is ligated.
  • the half-linker SEQ ID NO: 2-3 has a phosphate group modification to prevent self-ligation, and the nucleotide sequence shown by the semi-linker-ligation primer is as follows:
  • Adp_A GACGCTCTTCCGATCT (SEQ ID NO: 2);
  • Adp_B GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT (SEQ ID NO: 3)
  • the conditions of the ligation reaction are: 1 ⁇ l of rapid ligase (NEB, M2200L), 10 ⁇ l of 2x rapid ligation buffer, 8.4 ⁇ l of resuspended magnetic beads bound with extension product, and 0.6 ⁇ l of 10 mM adaptor, placed in a centrifuge tube Medium, mix well.
  • the centrifuge tube was placed on a rotary incubator to prevent the magnetic beads from settling, and the entire reaction was carried out overnight at 4 ° C (about 15 hours) to obtain a double-stranded DNA molecule to which the linker was attached. Thereby, the background of the connection is small and the connection efficiency is high.
  • the double-stranded DNA molecule to which the linker is ligated is purified after the double-stranded DNA molecule is ligated to the linker, prior to amplification.
  • the manner of purification is not particularly limited according to a specific embodiment.
  • the purification may be carried out using beads that specifically recognize biotin.
  • the beads may be magnetic beads, wherein streptavidin is attached to the magnetic beads.
  • the ultra-pure distilled water can be eluted by heating at 72 ° C, and the double-stranded DNA molecule to which the half-linker is ligated is obtained in the eluate to obtain a purified double-stranded DNA molecule linked with a half-linker.
  • an eluate containing a double-stranded DNA molecule to which a half linker is attached can be directly used for the next amplification product, reducing intermediate steps and avoiding DNA loss.
  • the manner of amplification is not particularly limited, and one round of PCR or multiple rounds of PCR may be employed as needed.
  • the method employs two rounds of PCR, the first round of PCR, using the nucleotides represented by SEQ ID NOS: 4-5 as primers to amplify the double-stranded DNA molecule to which the half-linker is ligated, thereby The amplification efficiency of the DNA molecule is high; in the second round of PCR, according to an embodiment of the present invention, the second round of PCR utilizes the nucleotide represented by SEQ ID NOS: 4-5 as a primer. Thereby, the amplification efficiency of the DNA molecule is high.
  • the specific nucleotide sequence of the amplification primer is as follows:
  • Amplification primers :
  • MP24_G5 GTGACTGGAGTTCAGACGTGTGCTGGGGG (SEQ ID NO: 4),
  • P1_FL AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC TTCCGATCT (SEQ ID NO 5);
  • P1_Sh AATGATACGGCGACCACCGA (SEQ ID NO: 6)
  • the ligation product in the amplification unit, may be amplified using a primer comprising a tag sequence, whereby a plurality of different samples can be measured in one high-throughput sequencing.
  • a distinguishable index sequence tag is added to one end of a DNA library that can be used for the establishment of standard library samples for Illumina high throughput sequencing.
  • tag sequence primer means that a sequence tag is embedded in a PCR primer sequence, whereby a tag sequence primer can be introduced into a target fragment during amplification reaction using a tag sequence primer.
  • the nucleic acid tag can be introduced to the 5' end, or the tag can be introduced to the 3' end.
  • a tag PCR primer when used as an upstream primer, that is, when a sequence tag is introduced at the 5' end of the target fragment, the tag sequence primer specifically recognizes the sequence of the 5' linker, and the downstream primer can be specifically identified.
  • the sequence of the 3' linker when a tag PCR primer is used as an upstream primer, that is, when a sequence tag is introduced at the 5' end of the target fragment, the tag sequence primer specifically recognizes the sequence of the 5' linker, and the downstream primer can be specifically identified.
  • the sequence of the 3' linker when a tag PCR primer is used as an upstream primer, that is, when a sequence tag is introduced at the 5' end of the target fragment, the tag sequence
  • a DNA library for sequencing of a plurality of DNA molecules can be simultaneously constructed, thereby making it possible to
  • the DNA library of the product is mixed and sequenced, and the DNA sequence is classified based on the tag sequence to obtain sequence information of various DNA molecules.
  • This allows for the full use of high-throughput sequencing technologies, such as the use of Solexa sequencing technology, while sequencing multiple DNA molecules to improve the efficiency and throughput of DNA sequencing.
  • the specific sequence of the sequence tag is not particularly limited, and can be designed according to its own needs, as long as the source of the sequence can be distinguished.
  • PCR primer consisting of the nucleotides shown in any one of SEQ ID NOs: 8 to 19 in Table 1 is used as a PCR primer, whereby the accuracy and accuracy of sequencing are further improved.
  • the sequence platform after data analysis of the sequencing results, can accurately distinguish the sequence information of the gene sequencing library of multiple samples based on the sequence information of the tag, thereby fully utilizing the high-throughput sequencing platform and saving time, cut costs.
  • the invention proposes an apparatus for constructing a DNA sequencing library of a genome to be tested.
  • the apparatus includes: a first digestion processing device 100 for digesting a genome to be measured by a microsphere nuclease to obtain a first digestion treatment product; an immunoprecipitation treatment device 200, the co-immunoprecipitation treatment device 200 is connected to the first digestion treatment device 100, and is used for co-immunoprecipitation treatment of the first digestion treatment product to obtain an immunoprecipitation treatment product; second digestion treatment a device 300, the second digestion treatment device 300 is connected to the co-precipitation treatment device 200, and is configured to perform a second digestion treatment on the co-co-precipitation treatment product with proteinase K to obtain a second digestion treatment product; Dephosphorylation treatment device 400, the dephosphorization treatment device 400 is connected to the second digestion treatment device 300, and the dephosphorylation treatment device 400 is used to treat the second digestion treatment product by using shrimp al
  • a DNA sequencing library for constructing a genome to be tested it is possible to establish a genome having a starting cell volume as low as about 500 cells or a starting DNA amount as low as 2.5 nanoliters.
  • a DNA sequencing library of a genome having a starting cell amount as low as 200 cells or a DNA starting amount of even 1 ng or less can be efficiently established.
  • the device proposed in the embodiment of the invention retains the specificity of the single-stranded DNA, has less DNA loss, and maintains the complete genetic information, and is particularly suitable for the construction of a DNA sequencing library of the genome of the primary cell or the exfoliated tissue.
  • the apparatus for constructing a DNA sequencing library of a genome to be tested may further include: a lysis apparatus 700 connected to the first digestion processing apparatus 100, and the lysis apparatus 700 is used for cells or tissues A lysis treatment is performed to obtain a genome to be tested.
  • the first digestion processing apparatus 100 further includes: a pre-contact unit 110, the pre-contact unit 110 is connected to the cracking apparatus 700, and the pre-contact unit 110 is used for
  • the test genome is contacted with the micrococcal nuclease working solution to obtain a precontacted mixture, and the genome is precontacted with the micrococcal nuclease working solution in the precontacting unit 110, so that the chromatin can be adapted to the enzyme digestion system in advance, and further Conducive to the improvement of the enzyme digestion efficiency;
  • the first digestion processing unit 120, the first digestion processing unit 120 is connected to the pre-contact unit 110, and the first digestion processing unit 120 is configured to utilize the micrococcal nuclease pair After the contact mixture
  • the first digestion process to obtain the first digestion treatment product; and terminating the digestion unit 130, the termination digestion unit 130 is connected to the first digestion processing unit 120, and the termination digestion unit 130 is for utilizing micro The cocci nuclease stop solution terminates the digestion process.
  • the efficiency of micrococcal nuclease digestion of the chromatin of the test genome is significantly improved, and the chromatin can be more completely broken into mononuclear or dinuclear bodies, further greatly improving the subsequent antigen-antibody.
  • the efficiency of the combination is significantly improved.
  • the immunoprecipitation device 200 comprises: a histone antibody incubation unit 210, the histone antibody incubation unit 210 is connected to the first digestion treatment device 100, and the histone antibody is incubated.
  • the unit 210 is configured to incubate the first digestion treatment product with a histone antibody to obtain an immunoprecipitation treatment product.
  • the immunoprecipitation device 200 further comprises: a protein A magnetic bead contact unit 220, The protein A magnetic bead contact unit 220 is connected to the histone antibody incubation unit 210, and the pr-otein A magnetic bead contact unit 220 is used to contact the immunocoprecipitation treatment product with the magnetic beads carrying the protein A,
  • the co-immunoprecipitation device 200 further comprises: a cleaning unit 230, the cleaning unit 230, the cleaning unit 230 is connected to the protein A magnetic bead contact unit 220, and is used for carrying and carrying a protein A
  • the immunoprecipitation treated product contacted by the magnetic beads is subjected to a washing treatment.
  • the first digestion treatment product is sequentially passed through the action of the histone antibody incubation unit 210 and the protein A magnetic bead contact unit 220, the chromatin is enriched, and the purity of the target product (chromatin) is improved, further in the cleaning unit 230. Under the action, unintentional binding can be removed to further improve the purity of the enriched target product (chromatin).
  • the second digestion processing device 300 further includes: a second digestion processing unit 310, the second digestion processing unit 310 is connected to the immunoprecipitation processing device 200, The second digestion processing unit 310 is configured to perform a second digestion treatment on the co-co-precipitation treatment product with an elution buffer and a proteinase K to obtain a second digestion treatment product.
  • the second digestion apparatus 300 further comprises: The proteinase K inactivation unit 320 is connected to the second digestion processing unit 310, and the proteinase K inactivation unit 320 is used to inactivate the protease K.
  • the second digestion processing unit 310 elutes the immunoprecipitated product from the magnetic beads by using an elution buffer, and further, the histone-free is performed under the digestion of the histone antibody by the proteinase K.
  • the digested product further enters the proteinase K inactivation unit 320, in which the proteinase K is inactivated, and according to a specific example, the proteinase K inactivation unit 320 is subjected to heat inactivation treatment so that the proteinase K terminates the reaction in a timely manner.
  • the TELP device 600 includes a tail connecting unit 610 connected to the denaturation processing device 400 and configured to use the single-stranded DNA molecule
  • the denatured treatment product is directly subjected to a 3' end modification treatment to form a poly(C)n tail at the 3' end of the single-stranded DNA molecule, thereby obtaining a single-stranded DNA molecule having a Poly(C)n tail, wherein Representing the number of bases C, and n is an integer between 5 and 30; an extension unit 620, the extension unit 620 and the tail connection unit 610 Connected, and for obtaining a double-stranded DNA molecule based on the single-stranded DNA molecule having a Poly(C) tail, wherein the extension primer comprises a H(G)m unit at its 3' end, and H is a base A, a base Base T or base C, m is the number of bases G, and m is an integer between 5 and
  • the TELP device 600 further includes: a purification unit 650, wherein the purification unit purifies the double-stranded DNA molecule to which the linker is attached, wherein the purification utilizes specific recognition of biotin
  • the bead is, for example, the bead is a magnetic bead, wherein streptavidin is attached to the magnetic bead.
  • the purification unit 650 further comprises: an elution module that uses a super-pure distilled water to heat at 72 degrees Celsius to separate the double-stranded DNA molecules linked to the beads and which are linked.
  • the eluate containing the double-stranded DNA molecule to which the half-linker is ligated can be directly added to the amplification unit 640 for amplification, reducing intermediate steps and avoiding DNA loss.
  • the linker ligation unit 630 further comprises: a half linker forming module 631 for annealing a single-stranded nucleic acid molecule of the nucleotide sequence shown by SEQ ID NO: 2-3, respectively.
  • a ligation enzyme is provided in the ligation module 632 to obtain a double-stranded DNA molecule to which a half linker is ligated, and optionally, a nucleotide represented by SEQ ID NOS: 4-7 is used as a primer, and the ligation is half
  • the double-stranded DNA molecule of the adaptor is amplified.
  • the ligation product is amplified by using a primer comprising a tag sequence.
  • the primer containing the tag sequence is A set of primer sequence primers, wherein the set of tag sequence primers consists of the nucleotides set forth in SEQ ID NOS: 8-19.
  • the invention proposes a method of determining DNA sequence information of a genome to be tested.
  • the method package comprises: constructing a DNA sequencing library of a genome to be tested according to the method described above; sequencing the DNA sequencing library to obtain a sequencing result; and determining the site based on the sequencing result Describe the DNA sequence information of the genome.
  • the DNA sequencing library for constructing the genome to be tested has been described in detail above and will not be described herein.
  • the method and apparatus for sequencing a sequencing library are not particularly limited, and in view of the maturity of the technique, according to an embodiment of the present invention, second generation sequencing techniques such as SOLEXA, SOLID, and 454 sequencing may be employed. technology.
  • the inventors have surprisingly found that using a method for determining DNA sequence information of a genome to be tested according to an embodiment of the present invention, it is possible to determine a trace amount of cells sensitively, accurately, and efficiently (the initial amount of cells is as low as 500 or even as low as 200). Sequence information of a single-stranded DNA molecule of the genome or microgenome (DNA starting amount as low as 2.5 ng, or even as low as 1 ng).
  • the invention provides a system for determining DNA sequence information of a genome to be tested, characterized in that, with reference to FIG. 12, the system comprises: a sequencing library construction device 1000, the sequencing library construction device 1000 As described above; a sequencing device 2000, the sequencing device 2000 is coupled to the sequencing library construction device 1000 for sequencing a gene sequencing library of the genome to obtain a sequencing result of the DNA of the genome; The analysis device 3000 analyzes the sequencing result of the DNA of the genome to obtain the DNA sequence information.
  • any device known in the art suitable for performing the above operations can be employed as a component of each of the above units.
  • the term "connected” as used herein is used in a broad sense and may be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms may be understood by one of ordinary skill in the art.
  • the inventors have found that using a system for determining DNA sequence information of a genome to be tested according to an embodiment of the present invention, it is possible to determine a trace amount of cells (initial amount as low as 500 or even as low as 200) sensitively, accurately, and efficiently. Sequence information for single-stranded DNA molecules in the genome or microgenome (initial amounts of DNA as low as 2.5 ng, or even as low as 1 ng).
  • the invention proposes a sequence for determining a chromatin target region of a genome to be tested
  • the method of information comprises: constructing a target region sequencing library of a genome to be tested according to the method described above, wherein the histone antibody specifically recognizes the target region; The target region sequencing library of the genome is sequenced to obtain a sequencing result; and based on the sequencing result, sequence information of the chromatin target region of the test genome is determined.
  • the inventors have surprisingly found that using a method for determining sequence information of a chromatin target region of a test genome according to an embodiment of the present invention, it is possible to determine a trace amount of cells sensitively, accurately, and efficiently (the initial amount of cells is as low as 500 or even low). Sequence information of chromatin target regions of up to 200 genomes or microgenomes (DNA starting amounts as low as 2.5 ng, or even as low as 1 ng), thereby enabling detection of chromatin target regions of the genome of microscopic cells.
  • NP ⁇ 40 now known as Igepal CA ⁇ 630
  • the collected cells were centrifuged to remove the supernatant, stored on ice, fresh lysate was placed, and protease inhibitor (PI) was added.
  • PI protease inhibitor
  • MNase micrococcal nuclease
  • MNase is a very sensitive enzyme whose nuclease activity varies slightly from batch to batch, and the optimal MNase dilution concentration needs to be retested each time a new batch of MNase is used.
  • the sample is placed on a magnetic stand, and after the magnetic beads are all adsorbed to the magnetic frame solution, the supernatant is removed.
  • the beads were washed four times with RIPA buffer and each was spun for five minutes at 4 degrees Celsius. Finally, wash once with lithium chloride buffer.
  • the DNA is cleaved to produce a 5' terminal hydroxyl group and a 3' terminal phosphate group.
  • rSAP shrimp alkaline phosphatase
  • the double-stranded DNA obtained after the above steps is directly subjected to heat denaturation treatment to obtain single-stranded DNA, and the obtained single-stranded DNA is directly used for TELP establishment.
  • the inventors screened the optimal dephosphorylation enzymes from T4PNK, T4 DNA polymerase, Klenow three combinations or T4PNK or rSAP.
  • the specific experimental procedure is as follows Said:
  • the mouse embryonic stem cells grown on the six-well cell culture plate were taken out from the incubator, the medium was aspirated, and gently washed with 1 ml of PBS, and the residual medium was washed away. Thereafter, 0.5 ml of 0.05% trypsin was added, digested at 37 ° for three minutes, and the digestion reaction was terminated with 1 ml of fresh medium.
  • To completely separate the cells into a single suspended cell state repeatedly blow with a 1 ml large head until there is no cell mass visible to the naked eye. It was then centrifuged at 2000 rpm for 5 minutes at low speed. After the supernatant was aspirated, the cells were resuspended in PBS to perform cell counting. After calculating the concentration, seven groups of 10k cells were taken into a 250 ul low-adsorption EP tube. After centrifugation again, the supernatant was carefully removed, and the prepared cells were stored on ice.
  • the fresh MNase dilution was configured and the MNase was diluted to the appropriate concentration (0.01 U) to allow the chromatin to be cleaved to the mononuclear body.
  • the sample is placed on a magnetic stand, and after the magnetic beads are all adsorbed to the magnetic frame solution, the supernatant is removed.
  • the beads were washed four times with 150 ul of RIPA buffer and each was spun for five minutes at 4 °C. Finally, wash once with 150 ul of lithium chloride buffer.
  • the A-F group was purified using Ampure beads, and the G group was subjected to column purification. After obtaining the purified DNA, the TELP was built, and the experimental results are shown in FIG.
  • Figure 14 shows that the band size is in the range of 200 bp to 500 bp, which is the desired band of interest, with the band of the D group having the highest brightness, indicating that the final DNA yield is the highest, indicating that the PNK enzyme alone is sufficient for dephosphorylation. End repair. Can be included in the next consideration to optimize experimental conditions.
  • any purification process will be severely affected in consideration of further reducing the number of cells. Based on this, try to remove the purification step after the end repair.
  • the initial cell volume of the experiment was reduced to 500 for comparative experiments.
  • DAN eluted 27 microliters of water, 1 microliter of elution buffer 10xExTaq, and 1 microliter of proteinase K were added to each sample. Mix on a vortexer and place on a shaker at 55 degrees Celsius for one hour. The liquid in the test tube was sucked into a new centrifuge tube and heated at 72 ° C for 40 minutes to completely inactivate the proteinase K.
  • T4Polynucleotide Kinase (T4PNK) enzyme was added directly to the other set.
  • the reaction system is shown in Table 3:
  • the reaction was carried out at 37 ° C for one hour, and then the enzyme was inactivated by heating. Without any purification step, the TELP was directly constructed, and the experimental results are shown in FIG. 15 .
  • the results in Figure 15 show that the band size is in the range of 200 bp to 500 bp, which is the desired band of interest.
  • the amount of the target band in the rSAP group is significantly larger than that in the PNK group, and the amount of DNA visible to the naked eye can be obtained. , enough downstream sequencing experiments, indicating that the experiment is feasible, select rSAP as the end repair enzyme for subsequent experiments.
  • the inventors compared the map data and the ENCODE map data obtained by the inventors and loaded them into the UCSC browser, and then judged the peak distribution of H3K4me3 and combined with the positional relationship between the gene promoter and the gene promoter. Whether the method of building a database proposed by the present invention is successful.
  • the inventors also conducted more accurate big data analysis.
  • the inventors divided the entire genome into two consecutive 2000 bp bins, and calculated the FPKM values at each position by comparing different cell volumes with ENCODE data. The FPKM values at the same location, and then their correlation is calculated.
  • first and second are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated.
  • features defining “first” and “second” may include one or more of the features either explicitly or implicitly.
  • the meaning of "a plurality” is two or more unless specifically and specifically defined otherwise.

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Abstract

提供了一种构建待测基因组的DNA测序文库的方法，该方法包括：（1）利用微球菌核酸酶对待测基因组进行第一消化处理，以便获得第一消化处理产物；（2）将所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；（3）利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；（4）利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；（5）将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及（6）基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库。

Description

构建待测基因组的DNA测序文库的方法及其应用
优先权信息
本申请请求2016年04月19日向中国国家知识产权局提交的、专利申请号为201610245445.5的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物技术领域。具体地，本发明涉及构建待测基因组的DNA测序文库的方法及其应用。更具体地，本发明涉及构建待测基因组的DNA测序文库的方法、构建待测基因组的DNA测序文库的设备、确定待测基因组的DNA序列信息的方法、确定待测基因组的DNA序列信息的系统和用于确定待测基因组染色质目标区域的序列信息的方法。
背景技术
近年来在表观遗传学领域的研究表明，组蛋白修饰对维持细胞的生长发育有着至关重要的作用。因此，如何获得不同种类细胞在不同发育阶段的系统的全面的组蛋白修饰信息成了亟待解决的问题。而二代测序技术的普及，使得结合测序的染色质免疫共沉淀技术成为了研究DNA与组蛋白相互作用的重要技术手段，为揭示细胞的系统全面的DNA组蛋白修饰图谱提供了强有力的技术支撑。然而，染色质免疫共沉淀技术极为受限于细胞数目和抗体的质量等多方面的因素，如果不能够获得足够量的用于建库的DNA，是无法得到有效组蛋白修饰信息的。传统的染色质免疫共沉淀技术包括交联、打断DNA、抗体孵育、解交联和洗脱DNA等几个重要步骤，然而传统的染色质免疫共沉淀技术通常都用于研究容易获得的、数量级在106以上的常见类型的细胞，对于研究处于胚胎发育早期的这种极难获得的、少量数目的类型的细胞是无能为力的。
从而，如何建立不易获得的、少量数量类型细胞的DNA测序文库并有效用于DNA测序，是有待解决的问题。
发明内容
本申请是基于发明人对以下问题的发现而做出的：
传统的染色质免疫共沉淀技术中的交联和断裂DNA的过程会造成大量的DNA损失，并且后续的几次纯化DNA的过程又使得DNA的回收效率成为了影响获得的DNA量的关键因素。基于发明人对以上传统染色质免疫共沉淀技术中影响获得DNA量因素的发现，发明人对传统的免疫共沉淀技术进行了创造性的改进，提出了一种新的基于染色质免疫共沉淀的建库和测序技术。该建库技术和测序技术可对原代细胞或数目低至500的细胞的DNA进行建库和有效测序，为研究胚胎发育早期的表观遗传学的精细调控研究带来了希望。
在本发明的第一方面，本发明提出了一种构建待测基因组的DNA测序文库的方法。根据本发明的实施例，该方法包括：(1)利用微球核酸酶对待测基因组进行第一消化处理， 以便获得第一消化处理产物；(2)将所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；(3)利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；(4)利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；(5)将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及(6)基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库。
利用根据本发明实施例的构建待测基因组的DNA测序文库的方法方法采用微球核酸酶切断裂染色质，进而避免了超声破碎造成的DNA的局部损伤，大大提高了抗原-抗体的识别效率；利用根据本发明实施例的构建待测基因组的DNA测序文库的方法不需对第二消化处理产物进行纯化回收，直接进行利用虾碱性磷酸酶去磷酸化处理，并且经过虾碱性磷酸酶的去磷酸化处理产物也不需要纯化回收，而可直接进行变性和获得测序文库，进而显著提高了可用于建库的DNA的量。本发明实施例所提出的方法能够高效地建立起始细胞量低至500个左右细胞的基因组或起始DNA量低至2.5纳克的基因组的DNA测序文库，根据本发明的具体实施例，甚至可以高效建立起始细胞量低至200个细胞的基因组或DNA起始量甚至低至1纳克的基因组的DNA侧序文库。本发明实施例所提出的方法特别适用于原代细胞或剥离组织的等微量细胞基因组的DNA测序文库的构建，进而能够高效、灵敏地应用于高通量测序技术，基于对测序结果的数据分析，就能够有效地获得基因序列信息。
在本发明的第二方面，本发明提出了一种构建待测基因组的DNA测序文库的设备。根据本发明的实施例，所述设备包括：第一消化处理装置，所述第一消化处理装置用于利用微球核酸酶对待测基因组进行消化处理，以便获得第一消化处理产物；免疫共沉淀处理装置，所述免疫共沉淀处理装置与所述第一消化处理装置相连，并且用于对所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；第二消化处理装置，所述第二消化处理装置与所述免疫共沉淀处理装置相连，并且用于利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；去磷酸化处理装置，所述去磷酸化处理装置与所述第二消化处理装置相连，所述去磷酸化处理装置用于利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；变性处理装置，所述变性处理装置与所述去磷酸化处理装置相连，并且用于将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及TELP装置，所述TELP装置与所述变性处理装置相连，所述TELP装置基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库，可选地，所述变性处理装置适于热变性而获得所述单链DNA。
利用根据本发明实施例的利用根据本发明实施例的用于构建待测基因组的DNA测序文库的设备，能够建立起始细胞量低至500个左右细胞的基因组或起始DNA量低至2.5纳克的基因组的DNA测序文库，根据本发明的具体实施例，甚至可以高效建立起始细胞量低至200个细胞的基因组或DNA起始量甚至低至1纳克的基因组的DNA测序文库。并且本发明实施例所提出的设备保留了单链DNA链的特异性，DNA损失少，基因信息保持完整， 特别适用于原代细胞或剥离组织的等微量细胞基因组的DNA测序文库的构建。
在本发明的第三方面，本发明提出了一种确定待测基因组的DNA序列信息的方法。根据本发明的实施例，所述方法包括：根据前面所述的方法构建待测基因组的DNA测序文库；对所述DNA测序文库进行测序，以便获得测序结果；以及基于所述测序结果，确定所述待测基因组的DNA序列信息。
利用根据本发明实施例的待测基因组的DNA序列信息的方法，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)基因组或微量基因组(起始DNA量低至2.5纳克，甚至低至1纳克)的单链DNA分子的序列信息。
在本发明的第四方面，本发明提出了一种确定待测基因组的DNA序列信息的系统。根据本发明的实施例，所述系统包括：测序文库构建设备，所述测序文库构建设备为前面所述的；测序设备，所述测序设备与所述测序文库构建设备相连，以便用于对所述基因组的基因测序文库进行测序，获得所述基因组DNA的测序结果；以及分析设备，对所述基因组DNA的测序结果进行分析，以便获得所述DNA序列信息。
利用根据本发明实施例的待测基因组的DNA序列信息的系统，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)基因组的或微量基因组(起始DNA量低至2.5纳克，甚至低至1纳克)的单链DNA分子的序列信息。
在本发明的第五方面，本发明提出了一种用于确定待测基因组染色质目标区域的序列信息的方法。根据本发明的实施例，所述方法包括：根据前面所述的方法构建待测基因组的目标区域测序文库，其中，所述组蛋白抗体特异性识别所述目标区域；对所述待测基因组的目标区域测序文库进行测序，以便获得测序结果；以及基于所述测序结果，确定所述待测基因组染色质目标区域的序列信息。
利用根据本发明实施例的确定待测基因组染色质目标区域的序列信息的方法，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)的基因组或微量基因组(起始DNA量低至2.5纳克，甚至低至1纳克)的染色质目标区域的序列信息，从而实现对微量细胞的基因组的染色质目标区域的检测。
需要说明的是，本发明所提出的待测基因组是指细胞或组织的全基因组或部分基因组，并且基因组由染色质或染色体组成。本领域的技术人员可以理解，基因组的来源不受特别限制，可以从任何可能的途径获得，可以是通过市售直接获得，也可以是从其他实验室直接获取，还可以是直接从细胞或组织样本中提取的。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
图1是根据本发明实施例的构建待测基因组的DNA测序文库的方法的流程图；
图2是根据本发明又一实施例的构建待测基因组的DNA测序文库的方法的流程图；
图3是根据本发明实施例的利用微球菌核酸酶对待测基因组进行第一消化处理的流程 图；
图4是根据本发明实施例的构建待测基因组的DNA测序文库的设备的结构示意图；
图5是根据本发明再一实施例的构建待测基因组的DNA测序文库的设备的结构示意图；
图6是根据本发明实施例的第一消化处理装置的结构示意图；
图7是根据本发明实施例的免疫共沉淀装置的结构示意图；
图8是根据本发明实施例的第二消化处理装置的结构示意图；
图9是根据本发明实施例的TELP装置的结构示意图；
图10是根据本发明再一实施例的TELP装置的结构示意图；
图11是根据本发明实施例的接头连接单元的结构示意图；
图12是根据本发明实施例的确定待测基因组的DNA序列信息的系统的结构示意图；
图13是根据本发明实施例的确定待测基因组染色质目标区域的序列信息的方法的流程图；
图14是根据本发明实施例的去磷酸化酶筛选实验的DNA凝胶电泳图；
图15是根据本发明再一实施例的去磷酸化酶筛选实验的DNA凝胶电泳图；
图16是根据本发明实施例的验证本发明实施例所提出的建库方法有效性的结果图；以及
图17是根据本发明再一实施例的验证本发明实施例所提出的建库方法有效性的结果图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
构建待测基因组的DNA测序文库的方法
在本发明的第一方面，本发明提出了一种构建待测基因组的DNA测序文库的方法。根据本发明的实施例，参考图1，该方法包括：(1)利用微球核酸酶对待测基因组进行第一消化处理，以便获得第一消化处理产物；(2)将第一消化处理产物进行免疫共沉淀处理；以便获得免疫共沉淀处理产物；(3)利用蛋白酶K对免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物，其中，蛋白酶K用于消化免疫共沉淀处理产物中的相应组蛋白抗体，以便裸露DNA,因而此处的第二消化处理产物是裸露DNA；(4)利用虾碱性磷酸酶对第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；(5)将去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及(6)基于含有单链DNA分子的变性处理产物，按照TELP法获得测序文库。利用根据本发明实施例的构建待测基因组的DNA测序文库的方法采用微球核酸酶切断裂染色质，进而避免了超声破碎造成的DNA的局部损伤，大大提高了抗原-抗体的识别效率；利用根据本发明实 施例的构建待测基因组的DNA测序文库的方法不需对第二消化处理产物进行纯化回收，直接进行利用虾碱性磷酸酶去磷酸化处理，并且经过虾碱性磷酸酶的去磷酸化处理产物也不需要纯化回收，而可直接进行变性和获得测序文库，进而显著提高了可用于建库的DNA的量。本发明实施例所提出的方法能够高效地建立起始细胞量低至500个左右细胞的基因组或DNA的起始量低至2.5纳克的基因组的DNA测序文库，根据本发明的具体实施例，甚至可以高效建立起始细胞量低至200个细胞的基因组或DNA起始量甚至低至1纳克的基因组的DNA侧序文库。本发明实施例所提出的方法特别适用于原代细胞或剥离组织的等微量细胞的基因组的DNA测序文库的构建，进而能够高效、灵敏地应用于高通量测序技术，基于对测序结果的数据分析，就能够有效地获得基因序列信息。
根据本发明的实施例，参考图2，所述待测基因组是通过裂解细胞或组织而获得的，其中，所述细胞为细胞系或原代细胞，进而释放细胞或组织中的基因组，以利于微球菌核酸酶直接对染色质进行消化。本发明实施例所提出的方法直接对天然状态的细胞或组织进行裂解处理，而现有技术中是利用甲醛交联固定细胞，而出现掩盖抗原表位信息的问题，本发明实施例所提出的方法大大提高了后续抗原-抗体的识别效率。根据本发明实施例的构建待测基因组的DNA测序文库的方法，是利用微球菌核酸酶将染色质打断成单核小体或双核小体的状态，即上述的第一消化处理产物即为片段化的染色质，进而有效暴露了组蛋白相关抗原，进一步有效提高了后续抗原-抗体反应效率。
根据另一具体实施例，参考图3，发明人发现利用微球菌核酸酶对待测基因组进行第一消化处理可进一步包括:(1-1)使所述待测基因组与微球菌核酸酶工作液进行预接触，以便获得预接触后混合物。裂解处理后产物与微球菌核酸酶工作液进行预接触，可预先使得染色质适应酶切体系，进而有利于酶切效率的提高；(1-2)利用微球菌核酸酶对所述预接触后混合物进行第一消化处理，以便获得第一消化处理产物；(1-3)利用微球菌核酸酶终止液终止消化处理。通过上述操作，微球菌核酸酶消化待测细胞样品的染色质的效率显著提高，进而染色质可更为彻底地断裂为单核小体或双核小体。
根据本发明的另一具体实施例，通过以下操作实现免疫共沉淀处理：将所述第一消化处理产物与组蛋白抗体进行孵育，以便获得免疫共沉淀处理产物，任选地，进一步包括：将所述免疫共沉淀处理产物与携带protein A的磁珠接触，任选地，进一步包括：利用RIPA缓冲液和氯化锂缓冲液对所述免疫共沉淀处理产物进行清洗处理。组蛋白抗体特异性识别核小体上组蛋白的特异性抗原位点，进而通过抗原抗体的免疫共沉淀，对染色质进行富集，另外，免疫共沉淀产物与携带protein A的磁珠接触，可进一步富集染色质，提高染色质的纯度，进一步地，利用RIPA缓冲液和氯化锂缓冲液对所述免疫共沉淀产物进行清洗，可去除非特意结合，进一步提高富集目的产物(染色质)的纯度。
根据本发明的再一具体实施例，通过以下操作实现所述第二消化处理：利用洗脱缓冲液和蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物，任选地，所述第二消化处理是在55摄氏度的条件下进行1小时，任选地，进一步包括：对所述蛋白酶K进行失活处理。根据本发明的实施例，上述洗脱缓冲液用于将免疫共沉淀产 物从磁珠上洗脱下来，进而在蛋白酶K对组蛋白抗体的消化作用下，不含组蛋白的裸露DNA游离出来，蛋白酶K的最适浓度反应温度是55摄氏度，在55摄氏度反应1个小时，发明人发现，裸露DNA释放完全，短于1个小时，消化不充分，长于1个小时，会使DNA产生非特异性降解。蛋白酶K消化处理1后，进一步对蛋白酶K进行失活处理，根据具体示例，发明人采用热失活处理使得蛋白酶K适时终止反应。
根据本发明的具体实施例，利用虾碱性磷酸酶的去磷酸化处理是在37摄氏度的条件下进行1小时。在37摄氏度的条件下，虾碱性磷酸酶的酶活最高，进行1小时的去磷酸化处理，既可保证反应底物的充分反应，又可保证不过度反应而造成非特异性产物增加。进一步地，去磷酸化处理后可对虾碱性磷酸酶进行失活处理，失活处理是在65摄氏度下进行10分钟，失活处理可有效终止去磷酸化反应。
根据本发明的具体实施例，上述的变性处理可采用但不限于热变性处理。变性处理是为了将双链DNA转变为单链DNA,进而进一步根据TELP法获得测序文库，热变性处理具有变性效率高、对DNA损伤小、DNA突变几率低以及不会引入影响后续建库的因子的优势。
另外，申请人在专利201410466261.2中对TELP法进行了详细阐述。根据本发明的具体实施例，TELP法是通过如下方式实现的：
首先，将含有单链DNA分子的变性处理产物直接在3’末端形成poly(C)n尾部，以便获得具有Poly(C)n尾部的单链DNA分子，其中，n代表碱基C的数目，并且n为5～30之间的整数。根据具体示例，所述单链DNA分子的量为≥5pg。由此，本发明获得测序文库的起始量显著小于其他二代测序文库的起始量，适于微量样本构建测序文库，尤其适于珍贵稀缺的样品或者临床病人标本或原代细胞构建测序文库。根据本发明具体示例，所述单链DNA分子的量为5pg～10ng。由此，构建测序文库的效率高，准确度好。根据具体示例，Poly(C)n尾部的n可以为15～25之间的整数。根据具体示例，n可以为20。由此，与延伸引物结合的效果好。根据具体实施例，Poly(C)n尾部添加至单链DNA分子3’末端的方式不受特别限制。根据具体示例，所述poly(C)n尾部可以是利用末端转移酶形成的。单链DNA在末端转移酶(TdT)的作用下，DNA链的3’末端能连上若干个多聚胞嘧啶脱氧核苷酸Poly(C)n，反应过程为：预先混合28μl DNA溶液，1μl 10x EX缓冲液(Takara，supplied with RR006A)，1μl 1mM dCTP(NEB，N0446S)，高温使DNA变性，获得单链DNA分子。接着加入1μl末端转移酶(TdT；NEB，M0315S)，并在37℃条件下反应35min。反应结束后，加热至75℃保持20分钟，以使TdT失活，获得3’末端连接寡聚Poly(C)n的单链DNA分子。
然后，利用延伸引物，基于所述具有Poly(C)n尾部的单链DNA分子，获得双链DNA分子，其中，延伸引物在其3’末端包括H(G)m单元，H为碱基A、碱基T或碱基C，m为碱基G的数目，并且m为5～15之间的整数。由此，延伸引物能在Poly(C)n尾部上的合适位置起始退火配对。根据本发明实施例，H(G)m单元的m可以为9。由此，H(G)m单元与寡聚Poly(C)n起始退火配对的位置适宜。根据具体示例，延伸引物的序列不受特 别的限制，只要能保证DNA延伸能够进行即可。根据本发明具体示例，所述延伸引物可以由SEQ ID NO：1所示的核苷酸构成。由此，延伸引物易于与poly(C)n尾部配对，延伸反应效率高。其中，SEQ ID NO：1的具体序列如下：
GTGACTGGAGTTCAGACGTGTGCTGGGGGGGGGH(SEQ ID NO：1)。
根据具体示例，可以利用KAPA 2G Robust HS对单链DNA分子进行延伸，获得所述双链DNA分子。例如，延伸的反应体系为：上一步骤获得的3’末端连接寡聚Poly(C)n的单链DNA分子中，加入6.2μl水，0.8μl KAPA 2G Robust HS(KAPA，KK5515)，12μl 5x KAPA缓冲液A(KAPA，KK5515)，4.8μl 2.5mM dNTP(Takara，RR006A)，以及6μl 2μM延伸引物。根据具体实施例，延伸反应的程序为：(1)95℃3min；(2)47℃1min，68℃2min，16个循环；(3)72℃10min。反应结束后，再加入核酸外切酶I(Exo I)于37℃反应1小时，消化掉多余的延伸引物，获得延伸产物。其中说明的是，利用延伸反应获得的延伸链末端，即延伸链的3’端为碱基A，由此，便于与5’以碱基T为首端的半接头连接。
根据具体实施例，所述延伸引物可以具有筛选标记，其中，所述筛选标记形成于所述延伸引物的5’末端。由此，通过筛选标记高效地对延伸后的双链DNA进行筛选、纯化，获得目的基因。根据具体示例，所述筛选标记可以为生物素。由此，采用连接有生物素的DNA片段与磁珠结合的方法对延伸的双链DNA产物的纯化，显著减少纯化过程中DNA的损失。根据本发明具体示例，生物素与磁珠结合的过程如下：预先用1x Binding&Wash(B&W)缓冲液(10mM Tris-HCl pH 8.0、0.5mM EDTA，1M NaCl)清洗magnetic streptavidin C1磁珠(Invitrogen，650.01)，然后和延伸后的产物混合后在温控混匀仪上的孵育，条件为：23℃1400rpm震荡(震荡频率：震荡10s，停止10s)30min。反应结束后，结合了DNA的磁珠用100μl 1x B&W缓冲液洗一次，150μl EBT缓冲液(10mM Tris-HCl pH 8.0，0.02％Triton X-100)洗三次，最后用8.4μl elution缓冲液(EB；10mM Tris-HCl pH 8.0)重悬，用于之后的连接反应。
最后，在所述双链DNA分子远离H(G)m单元的一端连接接头，并对所得到的连接产物进行扩增，以便获得扩增产物，所述扩增产物构成所述测序文库。根据本发明实施例，，该步骤可以进一步包括：将分别具有由SEQ ID NO：2-3所示核苷酸序列的单链核酸分子进行退火，以便获得半接头，将所述半接头与所述双链DNA分子的一端进行连接，以便获得连接有半接头的双链DNA分子。需要说明的是，若珠子与双链DNA的一端相连，则产生阻碍作用，因此半接头与远离双链DNA珠子连接端的一端相连。其中，半接头SEQ ID NO：2-3的3’端都有磷酸基团修饰以防止其自连，半接头连接引物所示核苷酸序列具体如下：
Adp_A：GACGCTCTTCCGATCT(SEQ ID NO：2)；
Adp_B：GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT(SEQ ID NO：3)
根据具体示例，连接反应的条件为：将1μl快速连接酶(NEB，M2200L)，10μl 2x快速连接缓冲液，8.4μl重悬的结合有延伸产物的磁珠以及0.6μl 10mM接头，置于离心管中，充分混匀。将离心管置于旋转培养器上以防磁珠沉降，整个反应在4℃条件下过夜进行(约15小时)，获得连接接头的双链DNA分子。由此，连接的背景小，并且连接效率高。 根据具体实施例，在所述双链DNA分子连接接头之后，进行扩增之前，对所述连接有接头的双链DNA分子进行纯化。根据具体的实施例，纯化的方式不受特别的限制。根据本发明具体示例，纯化可以是利用特异性识别生物素的珠子进行的。根据另一具体示例，所述珠子可以为磁珠，其中，所述磁珠上连接有链霉亲和素。由此，采用磁珠与连接有生物素的DNA片段结合的方法对延伸的双链DNA产物的纯化，纯化效果好，并且能够显著减少纯化过程中DNA的损失。根据具体实施例，纯化后可以采用超纯蒸馏水72℃加热进行洗脱，连接有半接头的双链DNA分子即在洗脱液中，获得纯化的连接有半接头的双链DNA分子。由此，含有连接有半接头的双链DNA分子的洗脱液可直接用于下一步的扩增产物，减少中间操作步骤，避免DNA损失。
获得连接有半接头的双链DNA分子后，对其进行扩增，以便获得扩增产物，所述扩增产物构成所述测序文库。根据本发明实施例，扩增的方式不受特别的限制，可以根据需要采用一轮PCR或多轮PCR。例如，本方法采用两轮PCR，第一轮PCR，利用由SEQ ID NO：4-5所示的核苷酸作为引物，对所述连接有半接头的双链DNA分子进行扩增，由此，DNA分子的扩增效率高；第二轮PCR，根据本发明实施例，第二轮PCR利用由SEQ ID NO：4-5所示的核苷酸作为引物。由此，DNA分子的扩增效率高。其中，扩增引物的具体核苷酸序列如下：
扩增引物：
第一轮PCR：
MP24_G5：GTGACTGGAGTTCAGACGTGTGCTGGGGG(SEQ ID NO：4)，
P1_FL：AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC TTCCGATCT(SEQ ID NO 5)；
第二轮PCR：
P1_Sh：AATGATACGGCGACCACCGA(SEQ ID NO：6)
去除标签的Index序列：CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGG AGTTCAGACG(SEQ ID NO：7)
根据本发明实施例，所述扩增单元中，可以采用包含标签序列的引物，对所述连接产物进行扩增，由此，能在一次高通量测序中测多个不同的样品。例如，在DNA文库的一端被加上了可区别的索引序列标签，该文库可用于Illumina高通量测序的标准文库样本的建立。
在本发明中所使用的术语“标签序列引物”是指在PCR引物序列中嵌入了序列标签，由此，在利用标签序列引物进行扩增反应的过程中，能够将标签序列引物引入到目的片段的一端，既可以将核酸标签引入到5’端，也可以将标签引入到3’端。例如，参考图6，当采用标签PCR引物作为上游引物，即在目的片段的5’端引入序列标签时，标签序列引物特异性地识别5’接头的序列，进而下游引物可以采用特异性地识别3’接头的序列。通过标签序列引物与DNA分子相连，可以精确地表征DNA分子的样品来源。由此，利用上述核酸标签，可以同时构建多种DNA分子的用于测序的DNA文库，从而可以通过将来源于不同样 品的DNA文库进行混合，同时进行测序，基于标签序列对DNA序列进行分类，获得多种DNA分子的序列信息。从而可以充分利用高通量的测序技术，例如利用Solexa测序技术，同时对多种DNA分子进行测序，从而提高DNA分子测序的效率和通量。根据本发明实施例，序列标签的具体序列不受特别的限制，可以根据自己的需要进行设计，只要能区分序列来源即可。例如，采用如表1中SEQ ID NO:8～19任一项所示的核苷酸构成的PCR引物作为标签PCR引物，由此，测序的准确度和精确度进一步提高。在本说明书中，核酸标签分别被命名为IndexN，其中N＝1-12中的任意整数，其序列如下表1所示：
表1：核酸标签序列引物Index1-12的序列
Figure PCTCN2017080142-appb-000001
由此，可以方便地同时构建多种样本的基因测序文库，并能够有效地应用于高通量测 序平台，从而在对测序结果进行数据分析后，基于标签的序列信息，能够准确地区分多种样本的基因测序文库的序列信息，进而，能够充分地利用高通量测序平台，且节省时间、降低成本。
构建待测基因组的DNA测序文库的设备
在本发明的第二方面，本发明提出了一种构建待测基因组的DNA测序文库的设备。参考图4，该设备包括：第一消化处理装置100，所述第一消化处理装置100用于利用微球核酸酶对待测基因组进行消化处理，以便获得第一消化处理产物；免疫共沉淀处理装置200，所述免疫共沉淀处理装置200与所述第一消化处理装置100相连，并且用于对所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；第二消化处理装置300，所述第二消化处理装置300与所述免疫共沉淀处理装置200相连，并且用于利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；去磷酸化处理装置400，所述去磷酸化处理装置400与所述第二消化处理装置300相连，所述去磷酸化处理装置400用于利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；变性处理装置500，所述变性处理装置500与所述去磷酸化处理装置400相连，并且用于将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及TELP装置600，所述TELP装置600与所述变性处理装置500相连，所述TELP装置600基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库，可选地，所述变性处理装置500适于热变性而获得所述单链DNA。利用根据本发明实施例的利用根据本发明实施例的用于构建待测基因组的DNA测序文库的设备，能够建立起始细胞量低至500个左右细胞的基因组或起始DNA量低至2.5纳克的基因组的DNA测序文库，根据本发明的具体实施例，甚至可以高效建立起始细胞量低至200个细胞的基因组或DNA起始量甚至低至1纳克的基因组的DNA侧序文库。并且本发明实施例所提出的设备保留了单链DNA的特异性，DNA损失少，基因信息保持完整，特别适用于原代细胞或剥离组织的等微量细胞的基因组的DNA测序文库的构建。
参考图5，构建待测基因组的DNA测序文库的设备可进一步包括：裂解装置700，所述裂解装置700与所述第一消化处理装置相连100，并且所述裂解装置700用于对细胞或组织进行裂解处理，以便获得待测基因组。
根据再一具体实施例，参考图6，所述第一消化处理装置100进一步包括：预接触单元110，所述预接触单元110与所述裂解装置700相连，并且所述预接触单元110用于所述待测基因组与微球菌核酸酶工作液进行接触，以便获得预接触后混合物，基因组与微球菌核酸酶工作液在预接触单元110进行预接触，可预先使得染色质适应酶切体系，进而有利于酶切效率的提高；第一消化处理单元120，所述第一消化处理单元120与所述预接触单元110相连，所述第一消化处理单元120用于利用所述微球菌核酸酶对所述接触后混合物进行 所述第一消化处理，以便获得所述第一消化处理产物；以及终止消化单元130，所述终止消化单元130与所述第一消化处理单元120相连，所述终止消化单元130用于利用微球菌核酸酶终止液终止消化处理。在上述第一消化处理装置100中微球菌核酸酶消化待测基因组的染色质的效率显著提高，染色质可更为彻底地断裂为单核小体或双核小体，进一步大大提高后续抗原-抗体的结合效率。
根据具体实施例，参考图7，所述免疫共沉淀装置200包括：组蛋白抗体孵育单元210，所述组蛋白抗体孵育单元210与所述第一消化处理装置100相连，所述组蛋白抗体孵育单元210用于将所述第一消化处理产物与组蛋白抗体进行孵育，以便获得免疫共沉淀处理产物，任选地，所述免疫共沉淀装置200包括进一步包括：protein A磁珠接触单元220，所述protein A磁珠接触单元220与所述组蛋白抗体孵育单元210相连，并且所述pr-otein A磁珠接触单元220用于所述免疫共沉淀处理产物与携带protein A的磁珠接触，任选地，所述免疫共沉淀装置200进一步包括：清洗单元230，所述清洗单元230，所述清洗单元230与所述protein A磁珠接触单元220相连，并且用于对与携带protein A的磁珠接触的所述免疫共沉淀处理产物进行清洗处理。第一消化处理产物依次通过在上述组蛋白抗体孵育单元210和protein A磁珠接触单元220中的作用，染色质得到富集，目的产物(染色质)的纯度得到提高，进一步在清洗单元230的作用下，可去除非特意结合，进一步提高富集目的产物(染色质)的纯度。
根据再一具体实施例，参考图8，所述第二消化处理装置300进一步包括：第二消化处理单元310，所述第二消化处理单元310与所述免疫共沉淀处理装置200相连，所述第二消化处理单元310用于利用洗脱缓冲液和蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物，任选地，第二消化装置300进一步包括：蛋白酶K失活单元320，所述蛋白酶K失活单元320与所述第二消化处理单元310相连，所述蛋白酶K失活单元320用于对所述蛋白酶K进行失活处理。根据本发明的实施例，第二消化处理单元310利用洗脱缓冲液将免疫共沉淀产物从磁珠上洗脱下来，进而在蛋白酶K对组蛋白抗体的消化作用下，将不含组蛋白的裸露DNA游离出来时。蛋白酶K消化处理后，消化产物进一步进入蛋白酶K失活单元320，在其中，对蛋白酶K进行失活处理，根据具体示例，蛋白酶K失活单元320采用热失活处理使得蛋白酶K适时终止反应。
根据本发明的具体实施例，参考图9，所述TELP装置600包括：尾部连接单元610，所述尾部连接单元610与所述变性处理装置400相连，并且用于将所述含有单链DNA分子的变性处理产物直接进行3’末端修饰处理，以便在所述单链DNA分子的3’末端形成poly(C)n尾部，从而获得具有Poly(C)n尾部的单链DNA分子，其中，n代表碱基C的数目，并且n为5～30之间的整数；延伸单元620，所述延伸单元620与所述尾部连接单元610 相连，并且用于基于所述具有Poly(C)尾部的单链DNA分子，获得双链DNA分子，其中，延伸引物在其3’末端包括H(G)m单元，H为碱基A、碱基T或碱基C，m为碱基G的数目，并且m为5～15之间的整数；接头连接单元630，所述接头连接单元630与所述延伸单元620相连，并且用于在所述双链DNA分子远离H(G)m单元的一端连接接头；以及扩增单元640，所述扩增单元640与所述接头连接单元630相连，并且用于对所述接头连接单元630的连接产物进行扩增，以便获得扩增产物，所述扩增产物构成所述测序文库，可选地，在所述尾部连接单元610中设置有末端转移酶，可选地，所述延伸单元620中设置有KAPA 2G Robust HS，以便获得所述双链DNA分子，可选地，所述延伸引物由SEQ ID NO：1所示的核苷酸构成，可选地，所述延伸单元620中设置的所述延伸引物，具有筛选标记，其中，所述筛选标记形成于所述延伸引物的5’末端，可选地，所述筛选标记为生物素。利用根据本发明实施例的TELP装置，能够通过获得的基于单链DNA的测序文库，并且保留了DNA链的特异性，样品损失更少，基因信息保持更加完整。
可选地，参考图10，所述TELP装置600进一步包括：纯化单元650，所述纯化单元对所述连接有接头的双链DNA分子进行纯化，其中，所述纯化是利用特异性识别生物素的珠子进行的，可选地，所述珠子为磁珠，其中，所述磁珠上连接有链霉亲和素。可选地，所述纯化单元650进一步包括：洗脱模块，所述洗脱模块利用超纯蒸馏水72摄氏度加热，将与珠子结合的连接有接头的双链DNA分子进行分离。由此，含有连接有半接头的双链DNA分子的洗脱液可直接加入扩增单元640中，进行扩增，减少中间操作步骤，避免DNA损失。
根据本发明的具体实施例，参考图11，所述接头连接单元630进一步包括：半接头形成模块631，将分别由SEQ ID NO：2-3所示核苷酸序列的单链核酸分子进行退火，以便获得半接头；以及连接模块632，所述连接模块用于将所述半接头与所述双链DNA分子进行连接，以便获得连接有半接头的双链DNA分子，可选地，所述连接模块632内设置有快速连接酶，以便获得连接有半接头的双链DNA分子，可选地，利用由SEQ ID NO：4-7所示的核苷酸作为引物，对所述连接有半接头的双链DNA分子进行扩增，可选地，所述扩增单元640中，采用包含标签序列的引物，对所述连接产物进行扩增，可选地，所述含标签序列的引物为一组标签序列引物的一种，其中，所述一组标签序列引物由SEQ ID NO：8-19所示的核苷酸构成。
关于这些设备、装置、单元、模块所实施处理的优点，前面已经进行了详细描述，在此不再详述。本领域技术人员能够理解的是，可以采用本领域中已知的任何适于进行上述操作的装置作为上述各个单元的组成部件。在本文中所使用的术语“相连”应作广义理解，可以是直接相连，也可以通过中间媒介间接相连，对于本领域的普通技术人员而言，可以根据具体情况理解上述术语的具体含义。
确定待测基因组的DNA序列信息的方法
在本发明的第三方面，本发明提出了一种确定待测基因组的DNA序列信息的方法。根据本发明的实施例，该方法包包括：根据前面所述的方法构建待测基因组的DNA测序文库；对所述DNA测序文库进行测序，以便获得测序结果；以及基于所述测序结果，确定所述待测基因组的DNA序列信息。关于构建待测基因组的DNA测序文库，前面已经进行了详细描述，在此不再赘述。根据本发明的实施例，对测序文库进行测序的方法和装置不受特别限制，考虑到技术的成熟度，根据本发明的实施例，可以采用第二代测序技术，诸如SOLEXA、SOLID和454测序技术。当然，也可以采用正在开发或者尚未开发的新型测序技术，例如单分子测序技术，诸如：Helicos公司的True Single Molecule DNA sequencing技术，Pacific Biosciences公司的the single molecule，real-time(SMRT.TM.)技术，以及Oxford Nanopore Technologies公司的纳米孔测序技术等(Rusk,Nicole(2009-04-01).Cheap Third-Generation Sequencing.Nature Methods 6(4):244–245)。
发明人惊奇地发现，利用根据本发明实施例的确定待测基因组的DNA序列信息的方法，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)的基因组或微量基因组(DNA起始量低至2.5纳克，甚至低至1纳克)的单链DNA分子的序列信息。
确定待测基因组的DNA序列信息的系统
在本发明的第四方面，本发明提出了一种确定待测基因组的DNA序列信息的系统，其特征在于，参考图12，该系统包括：测序文库构建设备1000，所述测序文库构建设备1000为前面所描述的；测序设备2000，所述测序设备2000与所述测序文库构建设备1000相连，以便用于对所述基因组的基因测序文库进行测序，获得所述基因组的DNA的测序结果；以及分析设备3000，对所述基因组的DNA的测序结果进行分析，以便获得所述DNA序列信息。本本领域技术人员能够理解的是，可以采用本领域中已知的任何适于进行上述操作的设备作为上述各个单元的组成部件。在本文中所使用的术语“相连”应作广义理解，可以是直接相连，也可以通过中间媒介间接相连，对于本领域的普通技术人员而言，可以根据具体情况理解上述术语的具体含义。
发明人发现，利用根据本发明实施例的确定待测基因组的DNA序列信息的系统，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)的基因组或微量基因组(DNA起始量低至2.5纳克，甚至低至1纳克)的单链DNA分子的序列信息。
确定待测基因组染色质目标区域的序列信息的方法
在本发明的第五方面，本发明提出了一种用于确定待测基因组染色质目标区域的序列 信息的方法。根据本发明的实施例，参考图13，该方法包括：根据前面所述的方法构建待测基因组的目标区域测序文库，其中，所述组蛋白抗体特异性识别所述目标区域；对所述待测基因组的目标区域测序文库进行测序，以便获得测序结果；以及基于所述测序结果，确定所述待测基因组染色质目标区域的序列信息。
发明人惊奇地发现，利用根据本发明实施例的确定待测基因组染色质目标区域的序列信息的方法，能够灵敏、准确、高效地确定微量细胞(细胞起始量低至500个左右，甚至低至200个)基因组或微量基因组(DNA起始量低至2.5纳克，甚至低至1纳克)的染色质目标区域的序列信息，从而实现对微量细胞的基因组的染色质目标区域的检测。
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅是说明性的，而不能理解为对本发明的限制。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品，例如可以采购自Illumina公司。
实施例1构建细胞基因组的DNA测序文库
1.1试剂准备
裂解液(Lysis buffer)
·0.5％NP-40
·0.5％Tween
·0.1％SDS
MNase工作液(MNase working buffer)
·100mM Tris-HCl ph8.0
·2mM CaCl2
MNase稀释液(MNase Dilute buffer)
·50mM Tris‐HCl,pH 8.0
·1mM CaCl2
·0.2％Triton X‐100
10x MNase终止液(10xMNase Stop buffer)
·110mM Tris‐HCl pH 8.0
·55mM EDTA
2x RIPA缓冲液2xRIPA buffer(on ice)
·280mM NaCl
·1％Triton X‐100
·0.1％SDS
·0.2％Na‐Deoxycholate
·5mM EGTA
RIPA缓冲液RIPA buffer
·10mM Tris pH 8.0
·1mM EDTA
·140mM NaCl
·1％Triton X‐100
·0.1％SDS
·0.1％Na‐Deoxycholate
氯化锂缓冲液(LiCl wash buffer)
·250mM LiCl
·10mM Tris pH 8.0
·1mM EDTA
·0.5％NP‐40(now known as Igepal CA‐630)
·0.5％Na‐deoxycholate
1.2裂解细胞样品
将收集的细胞离心去掉上清以后置于冰上保存，配置新鲜的裂解液，加入蛋白酶抑制剂(PI)。每个样品中加入19微升裂解液，用低吸附的枪头下上反复吹打数次，直至细胞完全裂解，注意过程中避免吹出气泡。待细胞完全裂解后，向样品中加入19微升微球菌核酸酶(MNase)工作液，充分混匀。
配置新鲜的MNase稀释液，将MNase稀释到合适的浓度。使得染色质被酶切断裂到单核小体的状态。MNase是一种非常敏感的酶，其核酸酶的活性会随批次的不同略有变化，在每次使用新批次的MNase之前，需要重新检测最佳MNase稀释浓度。向样品中加入2微升稀释后的MNase,充分混匀后放到37摄氏度水浴锅静置5分钟，之后加入5微升10xMNase终止液，充分混匀以防止过度打断。
向样品中加入50微升2xRIPA缓冲液，补充蛋白酶抑制剂，充分混匀后于4摄氏度离心机内高速离心15分钟。取上清溶液到一个新的150微升管中，置于冰上保存。
1.3抗体孵育
向样品中加入40微升RIPA缓冲液，混匀后加入1-2微克相应的组蛋白修饰抗体。上 下颠倒轻柔混匀，在4摄氏度试管混悬设备上孵育过夜。
1.4DNA洗脱
第二天，准备带有proteinA的磁珠。每一个免疫共沉淀样品(IP样品)需要10微克磁珠。用RIPA缓冲液清洗磁珠两次，然后用RIPA缓冲液重悬。向每个样品中加入10微克磁珠，上下颠倒混匀。放回到试管混悬仪上，4摄氏度结合2个小时。
将样品放置在磁力架上，待磁珠全部吸附到磁力架溶液变澄清后，吸掉上清。用RIPA缓冲液清洗磁珠四次，每次在4摄氏度试管混悬疑上旋转五分钟。最后用氯化锂缓冲液清洗一次。
将磁珠迅速短暂离心，置于磁力架上去掉磁珠上沾附的多余液体。向每个样品中加入27微升水，一微升10xExTaq缓冲液，一微升蛋白酶K。涡旋仪上混匀后置于55摄氏度的摇床上一个小时。将试管内的液体吸到一个新的离心管中，72摄氏度条件下加热40分钟，使蛋白酶K完全失去活性。
1.5去磷酸化处理
因为MNase核酸酶自身的特殊性，DNA被其切割后产生的是5’末端羟基，3’末端磷酸基团。为了下游的建库，需要对其进行3末端的修复。向洗脱后的DNA样品中加入1微升的虾碱性磷酸酶(rSAP)，37摄氏度条件下反应一个小时，保证3’末端被去磷酸化变为羟基。随后升温到65摄氏度，继续加热10分钟使酶失活。
1.6变性处理和TELP建库
经过上述步骤后得到的双链DNA直接经过热变性处理后得到单链DNA,所获得的单链DNA直接用于TELP建库。
实施例2去磷酸化处理酶的筛选
为了获得最好的去磷酸化末端修复结果并且保证最终DNA的产量，发明人从T4PNK，T4DNA聚合酶，Klenow三个组合或者T4PNK或者rSAP中筛选最适的去磷酸化处理酶，具体实验过程如下所述：
将生长在六孔细胞培养板上的小鼠胚胎干细胞从孵箱中取出，吸掉培养基后用1ml PBS轻柔清洗一遍，洗掉残余的培养基。之后加入0.5ml浓度为0.05％的胰酶，在37°消化三分钟，用1ml的新鲜培养基终止消化反应。为使细胞完全成为单个悬浮细胞状态，用1ml的大枪头反复吹打，直到无肉眼可见的细胞团块。之后以2000rmp的转速低速离心5分钟。吸掉上清后用PBS重悬细胞，进行细胞计数。计算浓度后取七组10k个细胞到250ul低吸附EP管中，再次离心后小心去除上清，将准备好的细胞放在冰上保存。
配置新的裂解液，加入50x蛋白酶抑制剂(PI)到1x的浓度。向每组样品中加入19微 升裂解液，用低吸附的枪头上下反复吹打数次，直至细胞完全裂解，注意过程中避免吹出气泡。待细胞完全裂解后，向每组样品中加入19微升MNase工作液，充分混匀。
配置新鲜的MNase稀释液，将MNase稀释到合适的浓度(0.01U)，使得染色质被酶切断裂到单核小体的状态。向每组样品中加入2微升稀释后的MNase,充分混匀后放到37摄氏度水浴锅静置5分钟，之后加入5微升10xMNase终止液，充分混匀以防止过度打断。
向每组样品中加入50微升2xRIPA缓冲液，补充蛋白酶抑制剂，充分混匀后于4摄氏度离心机内高速离心15分钟。取上清溶液到一个新的150微升管中，置于冰上保存。
向每组样品中加入40微升RIPA缓冲液，混匀后加入1.5微克H3K4me3组蛋白修饰抗体。上下颠倒轻柔混匀，在4摄氏度试管混悬设备上孵育过夜。
第二天，准备带有proteinA的磁珠(共700ug)。向每组样品中加入100ug磁珠。用500ulRIPA缓冲液清洗磁珠两次，然后将磁珠用10ul RIPA缓冲液重悬。向每个样品中加入10ul磁珠，上下颠倒混匀。放回到试管混悬仪上，4摄氏度结合2个小时。
将样品放置在磁力架上，待磁珠全部吸附到磁力架溶液变澄清后，吸掉上清。用150ulRIPA缓冲液清洗磁珠四次，每次在4摄氏度试管混悬疑上旋转五分钟。最后用150ul氯化锂缓冲液清洗一次。
将磁珠迅速短暂离心，置于磁力架上去掉磁珠上沾附的多余液体。向每个样品中加入20微升Tris-EDTA缓冲液(10mM Tris,1mM EDTA)，一微升蛋白酶K进行洗脱反应。涡旋仪上混匀后置于55摄氏度的摇床上一个小时。将试管内的液体吸到一个新的离心管中，72摄氏度条件下加热40分钟，使蛋白酶K完全失去活性。
按照表2所示的实验条件配置末端修复的反应体系(单位：ul)，
表2：
Figure PCTCN2017080142-appb-000002
23摄氏度条件下反应一个小时之后，A-F组使用Ampure beads进行纯化，G组使用过柱纯化的方法。获得纯化后的DNA后，进行TELP建库，实验结果如图15所示。
图14显示，条带大小在200bp-500bp的范围内是想要的目的条带，其中D组的条带亮度最高，表明最终获得的DNA产量最高，说明单独使用PNK酶足以进行去磷酸化的末端修复。可以纳入下一步优化实验条件的考虑范围内。
在接下来的实验优化过程中，考虑到进一步降低细胞数量时，任何纯化过程都会严重影 响最终的DNA产量，基于此，尝试去掉末端修复后的纯化步骤。为了获得真实可靠的实验结果，实验的起始细胞量降低到500进行对比实验。DAN洗脱时，向每个样品中加入27微升水，1微升洗脱缓冲液10xExTaq，1微升蛋白酶K。涡旋仪上混匀后置于55摄氏度的摇床上一个小时。将试管内的液体吸到一个新的离心管中，72摄氏度条件下加热40分钟，使蛋白酶K完全失去活性。
在进行末端修复时，向一组样品中直接加入1微升rSAP酶，向另外一组中直接加入一微升T4Polynucleotide Kinase(T4PNK)酶。反应体系如表3所示：
表3：
  rSAP PNK
10xEx Taq 1 1
蛋白酶K 1 1
ddH2O 27 27
T4多聚核苷酸激酶(T4PNK) 0 1
rSAP 1 0
37摄氏度条件下反应一个小时，之后加热使酶失去活性，不进行任何纯化步骤，直接TELP建库，实验结果如图15所示。
图15结果显示，条带大小在200bp-500bp的范围内是想要的目的条带，其中rSAP组的目的条带的量要明显多于PNK实验组，能够获得在胶上肉眼可见的DNA量，足够下游的测序实验，说明实验可行，选择rSAP作为后续实验的末端修复酶。
实施例3
为了验证本申请所提建库方法的有效性，发明人采取了最为严格的检验手段：通过与ENCODE数据库中被当成黄金标准的大量(1x10e7)小鼠胚胎干细胞H3K4me3基于免疫共沉淀的DNA测序技术(ChIP-sequence)的结果相比较，看两者之间的相关性。
具体地，发明人一方面比较了发明人所得到的图谱数据和ENCODE图谱数据，并将其载入到UCSC browser中，进而通过观察H3K4me3的峰值分布，结合其与基因启动子的位置关系，判断本发明所提出的建库方法是否成功。
结果如图16所示，从结果(基因组位置：chr6:51,271,690-52,394,589)中可以看出，无论是从上百万个细胞中获得的结果(ENCODE)还是发明人低至200个细胞的ChIP-sequence结果，在转录活跃的基因的启动子区域，都有H3K4me3的分布。其信噪比也令人满意，直观上看，五条轨迹的相似性极高，进而有效验证了本发明所提出的建库方法的有效性。
另外一方面，发明人也进行了更为精确的大数据分析，发明人将整个基因组按照位置分为连续的2000bp的bin,在每一个位置计算其FPKM的数值，通过比较不同细胞量与ENCODE数据在相同位置处的FPKM值，进而计算其相关性。
结果如图17所示，从图17结果中可以看到，5000个细胞数目数据与ENCODE数据的相关性高达0.8，而细胞数量最少的(200)也有0.75。相关性随着细胞数量的减少而降低，也是合理的。因为是全基因组的比较，有这样的相关性足以说明即使在数百数量级细胞的实验条件下，发明人仍然能够得到可信的ChIP-sequence结果。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种构建待测基因组的DNA测序文库的方法，其特征在于，包括：
    (1)利用微球菌核酸酶对待测基因组进行第一消化处理，以便获得第一消化处理产物；
    (2)将所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；
    (3)利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；
    (4)利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；
    (5)将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及
    (6)基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库。
  2. 根据权利要求1所述的方法，其特征在于，所述去磷酸化处理是在37摄氏度的条件下进行1小时，
    任选地，所述去磷酸化处理后进一步包括：对所述虾碱性磷酸酶进行失活处理，所述失活处理是在65摄氏度下进行10分钟，
    任选地，所述变性处理是热变性处理。
  3. 根据权利要求1所述的方法，其特征在于，所述TELP法是通过如下方式实现的：
    (6-1)将所述含有单链DNA分子的变性处理产物直接进行3’末端修饰处理，以便在所述单链DNA分子的3’末端形成poly(C)n尾部，从而获得具有Poly(C)n尾部的单链DNA分子，其中，n代表碱基C的数目，并且n为5～30之间的任意整数；
    (6-2)利用延伸引物，基于所述具有Poly(C)n尾部的单链DNA分子，获得双链DNA分子，其中，所述延伸引物在3’末端包括H(G)m单元，H为碱基A、碱基T或碱基C，m为碱基G的数目，并且m为5～15之间的整数；以及
    (6-3)在所述双链DNA分子远离H(G)m单元的一端连接接头，并对所得到的连接产物进行扩增，以便获得扩增产物，所述扩增产物构成所述DNA测序文库，
    任选地，所述单链DNA分子的量为≥25pg，
    任选地，所述单链DNA分子的量为25pg～1000pg，
    任选地，所述单链DNA分子的长度为200～500nt，
    任选地，n为15～25之间的整数，优选地n为20，
    任选地，所述poly(C)n尾部是利用末端转移酶形成的，
    任选地，在步骤(6-2)中，利用KAPA 2G Robust HS获得所述双链DNA分子，
    任选地，m为9，
    任选地，所述延伸引物由SEQ ID NO：1所示的核苷酸构成，
    任选地，所述延伸引物具有筛选标记，其中，所述筛选标记形成于所述延伸引物的5’末端，
    任选地，所述筛选标记为生物素，
    任选地，在步骤(6-3)中，在所述双链DNA分子连接接头之后，进行扩增之前，对所述连接有接头的双链DNA分子进行纯化，其中，所述纯化是利用特异性识别生物素的珠子进行的，
    任选地，所述珠子为磁珠，其中，所述磁珠上连接有链霉亲和素，
    任选的，通过超纯蒸馏水72摄氏度加热对所述纯化产物进行洗脱，以便获得纯化的接有接头的双链DNA分子。
  4. 根据权利要求1所述的方法，其特征在于，所述待测基因组是通过裂解细胞或组织而获得的全基因组或部分基因组。
  5. 根据权利要求4所述的方法，其特征在于，步骤(1)进一步包括：
    (1-1)使所述待测基因组与微球菌核酸酶工作液进行预接触，以便获得预接触后混合物；
    (1-2)利用所述微球菌核酸酶对所述接触后混合物进行所述第一消化处理，以便获得所述第一消化处理产物；以及
    (1-3)利用微球菌核酸酶终止液终止消化处理。
  6. 根据权利要求1所述的方法，其特征在于，通过以下操作实现免疫共沉淀处理：
    将所述第一消化处理产物与组蛋白抗体进行孵育，以便获得免疫共沉淀处理产物，
    任选地，进一步包括：将所述免疫共沉淀处理产物与携带protein A的磁珠接触，
    任选地，进一步包括：利用RIPA缓冲液和氯化锂缓冲液对所述免疫共沉淀处理产物进行清洗处理。
  7. 根据权利要求1所述的方法，其特征在于，通过以下操作实现所述第二消化处理：
    利用洗脱缓冲液和蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物，
    任选地，所述第二消化处理是在55摄氏度的条件下进行1小时，
    任选地，进一步包括：对所述蛋白酶K进行失活处理。
  8. 根据权利要求3所述的方法，其特征在于，步骤(6-3)进一步包括：
    (6-3-1)将分别具有由SEQ ID NO：2-3所示核苷酸序列的单链核酸分子进行退火，以便获得半接头；
    (6-3-2)将所述半接头与所述双链DNA分子的一端进行连接，以便获得连接有半接头 的双链DNA分子；
    (6-3-3)利用由SEQ ID NO：4-7所示的核苷酸作为引物，对所述连接有半接头的双链DNA分子进行扩增，
    任选地，通过快速连接酶将所述半接头连接至所述双链DNA分子而获得连接有半接头的双链DNA分子，
    任选地，在步骤(6-3)中，采用包含标签序列的引物，对所述连接产物进行扩增，
    任选地，所述含标签序列的引物为一组标签序列引物的一种，其中，所述一组标签序列引物由SEQ ID NO：8-19所示的核苷酸构成。
  9. 一种构建待测基因组的DNA测序文库的设备，其特征在于，包括：
    第一消化处理装置，所述第一消化处理装置用于利用微球核酸酶对待测基因组进行消化处理，以便获得第一消化处理产物；
    免疫共沉淀处理装置，所述免疫共沉淀处理装置与所述第一消化处理装置相连，并且用于对所述第一消化处理产物进行免疫共沉淀处理，以便获得免疫共沉淀处理产物；
    第二消化处理装置，所述第二消化处理装置与所述免疫共沉淀处理装置相连，并且用于利用蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物；
    去磷酸化处理装置，所述去磷酸化处理装置与所述第二消化处理装置相连，所述去磷酸化处理装置用于利用虾碱性磷酸酶对所述第二消化处理产物直接进行去磷酸化处理，以便获得去磷酸化处理产物；
    变性处理装置，所述变性处理装置与所述去磷酸化处理装置相连，并且用于将所述去磷酸化处理产物直接进行变性处理，以便获得含有单链DNA分子的变性处理产物；以及
    TELP装置，所述TELP装置与所述变性处理装置相连，所述TELP装置基于所述含有单链DNA分子的变性处理产物，按照TELP法获得测序文库，
    可选地，所述变性处理装置适于热变性而获得所述单链DNA。
  10. 根据权利要求9所述的设备，其特征在于，所述TELP文库构建装置包括：
    尾部连接单元，所述尾部连接单元与所述变性处理装置相连，并且用于将所述含有单链DNA分子的变性处理产物直接进行3’末端修饰处理，以便在所述单链DNA分子的3’末端形成poly(C)n尾部，从而获得具有Poly(C)n尾部的单链DNA分子，其中，n代表碱基C的数目，并且n为5～30之间的整数；
    延伸单元，所述延伸单元与所述尾部连接单元相连，并且用于基于所述具有Poly(C)尾部的单链DNA分子，获得双链DNA分子，其中，延伸引物在其3’末端包括H(G)m单元，H为碱基A、碱基T或碱基C，m为碱基G的数目，并且m为5～15之间的整数；
    接头连接单元，所述接头连接单元与所述延伸单元相连，并且用于在所述双链DNA分 子远离H(G)m单元的一端连接接头；以及
    扩增单元，所述扩增单元与所述接头连接单元相连，并且用于对所述接头连接单元的连接产物进行扩增，以便获得扩增产物，所述扩增产物构成所述测序文库，
    可选地，在所述尾部连接单元中设置有末端转移酶，
    可选地，所述延伸单元中设置有KAPA 2G Robust HS，以便获得所述双链DNA分子，
    可选地，所述延伸引物由SEQ ID NO：1所示的核苷酸构成，
    可选地，所述延伸单元中设置的所述延伸引物，具有筛选标记，其中，所述筛选标记形成于所述延伸引物的5’末端，
    可选地，所述筛选标记为生物素，
    可选地，进一步包括：纯化单元，所述纯化对所述连接有接头的双链DNA分子进行纯化，其中，所述纯化是利用特异性识别生物素的珠子进行的，
    可选地，所述珠子为磁珠，其中，所述磁珠上连接有链霉亲和素，
    可选地，所述纯化单元进一步包括：洗脱模块，所述洗脱模块利用超纯蒸馏水72摄氏度加热，将与珠子结合的连接有接头的双链DNA分子进行分离。
  11. 根据权利要求9所述的设备，其特征在于，进一步包括：裂解装置，所述裂解装置与所述第一消化处理装置相连，并且用于对细胞或组织进行裂解处理，以便获得待测基因组。
  12. 根据权利要求11所述的设备，其特征在于，所述第一消化处理装置进一步包括：
    预接触单元，所述预接触单元与所述裂解装置相连，并且用于所述待测基因组与微球菌核酸酶工作液进行预接触，以便获得预接触后混合物；
    第一消化处理单元，所述第一消化处理单元与所述预接触单元相连，用于利用所述微球菌核酸酶对所述接触后混合物进行所述第一消化处理，以便获得所述第一消化处理产物；以及
    终止消化单元，所述终止消化单元与所述第一消化处理单元相连，并且用于利用微球菌核酸酶终止液终止消化处理。
  13. 根据权利要求9所述的设备，其特征在于，所述免疫共沉淀装置包括：
    组蛋白抗体孵育单元，所述组蛋白抗体孵育单元与所述第一消化处理装置相连，并且用于将所述第一消化处理产物与组蛋白抗体进行孵育，以便获得免疫共沉淀处理产物，
    任选地，进一步包括：protein A磁珠接触单元，所述protein A磁珠接触单元与所述组蛋白抗体孵育单元相连，并且用于所述免疫共沉淀处理产物与携带protein A的磁珠接触，
    任选地，进一步包括：清洗单元，所述清洗单元与所述protein A磁珠接触单元相连，并且用于对与携带protein A的磁珠接触的所述免疫共沉淀处理产物进行清洗处理。
  14. 根据权利要求9所述的设备，其特征在于，所述第二消化装置进一步包括：
    第二消化处理单元，所述第二消化处理单元与所述免疫共沉淀处理装置相连，并且用于利用洗脱缓冲液和蛋白酶K对所述免疫共沉淀处理产物进行第二消化处理，以便获得第二消化处理产物，
    任选地，进一步包括：蛋白酶K失活单元，所述蛋白酶K失活单元与所述第二消化处理单元相连，并且用于对所述蛋白酶K进行失活处理。
  15. 根据权利要求10所述的设备，其特征在于，所述接头连接单元进一步包括：
    半接头形成模块，将分别由SEQ ID NO：2-3所示核苷酸序列的单链核酸分子进行退火，以便获得半接头；以及
    连接模块，所述连接模块用于将所述半接头与所述双链DNA分子进行连接，以便获得连接有半接头的双链DNA分子，
    可选地，所述连接模块内设置有快速连接酶，以便获得连接有半接头的双链DNA分子，
    可选地，利用由SEQ ID NO：4-7所示的核苷酸作为引物，对所述连接有半接头的双链DNA分子进行扩增，
    可选地，所述扩增单元中，采用包含标签序列的引物，对所述连接产物进行扩增，
    可选地，所述含标签序列的引物为一组标签序列引物的一种，其中，所述一组标签序列引物由SEQ ID NO：8-19所示的核苷酸构成。
  16. 一种确定待测基因组的DNA序列信息的方法，其特征在于，包括：
    根据权利要求1～8任一项所述的方法构建待测基因组的DNA测序文库；
    对所述DNA测序文库进行测序，以便获得测序结果；以及
    基于所述测序结果，确定所述待测基因组的DNA序列信息。
  17. 一种确定待测基因组的DNA序列信息的系统，其特征在于，包括：
    测序文库构建设备，所述测序文库构建设备为权利要求9～15任一项所述的；
    测序设备，所述测序设备与所述测序文库构建设备相连，以便用于对所述基因组的基因测序文库进行测序，获得所述基因组的DNA测序结果；以及
    分析设备，对所述基因组的DNA的测序结果进行分析，以便获得所述DNA序列信息。
  18. 一种用于确定待测基因组染色质目标区域的序列信息的方法，其特征在于，包括：
    根据权利要求1～8任一项所述的方法构建待测基因组的染色质目标区域测序文库，其中，所述组蛋白抗体特异性识别所述目标区域；
    对所述待测基因组的目标区域测序文库进行测序，以便获得测序结果；以及
    基于所述测序结果，确定所述待测基因组染色质目标区域的序列信息。
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