WO2017181880A1 - Procédé de construction d'une bibliothèque de séquençage d'adn destiné à un génome à détecter, et ses applications - Google Patents

Procédé de construction d'une bibliothèque de séquençage d'adn destiné à un génome à détecter, et ses applications 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
product
optionally
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颉伟
张冰洁
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清华大学
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    • 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
    • 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

La présente invention décrit un procédé de construction d'une bibliothèque de séquençage d'ADN destiné à un génome à détecter. Le procédé comprend : (1), la conduite d'un premier traitement par digestion sur un génome à détecter à l'aide d'une nucléase micrococcique, de façon à obtenir un premier produit de traitement par digestion; (2), la conduite d'un traitement par co-immunoprécipitation sur le premier produit de traitement par digestion, de façon à obtenir un produit de traitement par co-immunoprécipitation; (3), la conduite d'un second traitement par digestion sur le produit de traitement par co-immunoprécipitation à l'aide d'une protéase K, de façon à obtenir un second produit de traitement par digestion; (4), la conduite directe d'un traitement par déphosphorylation sur le second produit de traitement par digestion à l'aide d'une phosphatase alcaline de crevette, de façon à obtenir un produit de traitement par déphosphorylation; (5), la conduite directe d'un procédé de dénaturalisation sur le produit de traitement par déphosphorylation, de façon à obtenir un produit de traitement de dénaturalisation comportant des molécules d'ADN simple brin; et (6), sur la base du produit de traitement de dénaturalisation comportant des molécules d'ADN simple brin, l'obtention d'une bibliothèque de séquençage selon un procédé TELP.
PCT/CN2017/080142 2016-04-19 2017-04-11 Procédé de construction d'une bibliothèque de séquençage d'adn destiné à un génome à détecter, et ses applications WO2017181880A1 (fr)

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CN105754995B (zh) * 2016-04-19 2019-03-29 清华大学 构建待测基因组的dna测序文库的方法及其应用
CN106192022B (zh) * 2016-08-08 2018-07-03 中国科学院北京基因组研究所 16SrRNA多重测序文库的构建方法
CN106191037B (zh) * 2016-08-08 2018-10-30 中国科学院北京基因组研究所 一种多重测序文库的构建方法
CN107217309A (zh) * 2017-07-07 2017-09-29 清华大学 构建待测基因组的dna测序文库的方法及其应用
CN109517889B (zh) * 2017-09-18 2022-04-05 苏州吉赛基因测序科技有限公司 一种基于高通量测序分析寡核苷酸序列杂质的方法及应用
CN108181461A (zh) * 2017-12-29 2018-06-19 上海嘉因生物科技有限公司 应用于组织样本中组蛋白修饰染色体免疫共沉淀优化方法
CN108285494B (zh) * 2018-02-11 2021-09-07 北京大学 一种融合蛋白、试剂盒以及CHIP-seq检测方法
CN108330181A (zh) * 2018-02-27 2018-07-27 苏州睿迈英基因检测科技有限公司 一种适用于少细胞的染色质免疫共沉淀测序方法及其试剂盒和应用
CN108753939B (zh) * 2018-06-01 2021-08-03 华侨大学 一种检测全基因组的单链dna损伤的方法
CN110628886B (zh) * 2019-09-05 2022-09-30 华侨大学 一种检测dna中的单链断裂的方法
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