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WO2019006975A1 - Méthode de capture in situ de conformation de chromatine de génome entier pour cellules infinitésimales - Google Patents

Méthode de capture in situ de conformation de chromatine de génome entier pour cellules infinitésimales Download PDF

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Publication number
WO2019006975A1
WO2019006975A1 PCT/CN2017/114475 CN2017114475W WO2019006975A1 WO 2019006975 A1 WO2019006975 A1 WO 2019006975A1 CN 2017114475 W CN2017114475 W CN 2017114475W WO 2019006975 A1 WO2019006975 A1 WO 2019006975A1
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treatment
dna
genome
sequencing
product
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Chinese (zh)
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颉伟
杜振海
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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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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

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, a method of determining DNA sequence information of a genome to be tested, and a method of determining a three-dimensional spatial structure of a genome to be tested.
  • genome-wide chromatin conformation capture technology is extremely limited by the number of cells. If a sufficient amount of DNA for building a database containing enough effective ligation events is not available, it is impossible to obtain efficient and sufficiently high-resolution genomic three-dimensional structural information. of.
  • the traditional Hi-C technology has a large reaction system, and there are many steps of centrifugation, washing, and tube exchange.
  • the selection of fragment size before DNA amplification causes a large amount of DNA loss.
  • the selection range of DNA fragments is narrower than the degree of enrichment of DNA fragments by ultrasonic interruption, further reducing the amount of DNA used for second-generation sequencing.
  • Hi-C technology is commonly used to study the commonly available cells of the order of magnitude above 107, which is extremely incapable of obtaining a large number of cell types in the early stages of embryonic development. Based on the discovery of the above traditional technical problems by the inventors, the inventors selected by narrowing the reaction system, changing the mode of cell transfer, and changing the size of the DNA fragment.
  • sisHi-C small scale in situ Hi-C
  • the invention proposes a method of constructing a DNA sequencing library of a genome to be tested.
  • the method comprises: (1) digesting the genome to be tested with a restriction endonuclease to obtain a digestion treatment product; and (2) subjecting the digestion treatment product to biotin labeling treatment so that Obtaining a biotin labeling treatment product; (3) linking the biotin labeling treatment product with DNA ligase to obtain a ligation product; (4) de-crosslinking the ligation product; (5) decrosslinking the solution Treating the product for purification treatment; (6) subjecting the purified product to ultrasonication and precipitation treatment by contacting the sonicated product with a streptavidin magnetic bead to obtain a binding enzyme affinity a target DNA fragment of the magnetic beads; and (7) a target DNA fragment based on the magnetic chain bound to the streptavidin magnetic beads.
  • a method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention can be used to construct a DNA sequencing library having a cell number as low as 10 cells or a genome amount as low as 50 pg, thereby realizing a non-obtainable, small number of types of cells. Capture of genomic chromatin conformation.
  • the invention proposes a sequencing library.
  • the sequencing library is obtained by the method of constructing a DNA sequencing library of the genome to be tested as described above.
  • sequencing libraries according to embodiments of the present invention for sequencing it is possible to obtain genomic sequence information with a cell number as low as 10 or a genome amount as low as 50 pg and genomic three-dimensional structure information of sufficiently high resolution.
  • 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.
  • the method of determining the DNA sequence information of the genome to be tested according to an embodiment of the present invention it is possible to obtain genomic sequence information of cells having a cell number as low as 10 or a genome amount as low as 50 pg.
  • the invention proposes a method for determining a three-dimensional spatial structure 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
  • the three-dimensional spatial structure information of the detected genome is described.
  • 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 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. 5 is a flowchart of a TruSeq database according to still another embodiment of the present invention.
  • FIG. 6 is a flow chart of a TruSeq library according to still another embodiment of the present invention.
  • FIG. 7 is a flow chart of a TruSeq library according to still another embodiment of the present invention.
  • FIG. 8 is a diagram showing the results of verifying that the method of building a database according to an embodiment of the present invention has significant advantages, according to an embodiment of the present invention.
  • the invention proposes a method of constructing a DNA sequencing library of a genome to be tested.
  • the method includes: S100: digesting a genome to be tested with a restriction endonuclease to obtain a digestion treatment product; S200: performing biotin labeling treatment on the digestion treatment product, In order to obtain a biotin labeling treatment product; S300: ligating the biotin labeling treatment product with DNA ligase to obtain a ligation product; S400: de-crosslinking the ligation product; S500: decrosslinking the treated product Purification treatment; S600: subjecting the purified treatment product to ultrasonication and precipitation treatment by contacting the sonicated product with a streptavidin magnetic bead to obtain a streptavidin-coupled magnetic bead Target DNA fragment; and S700: based on a target DNA fragment bound with a streptavidin magnetic beads.
  • a method of constructing a DNA sequencing library of a genome to be tested according to an embodiment of the present invention can be used to construct a DNA sequencing library having a cell number as low as 10 or a genome amount as low as 50 pg, thereby realizing a readily available, small number of types of cells. Capture of the genomic chromatin conformation.
  • the genome to be tested is obtained by lysing cells or tissues, optionally the cells are cell lines or primary cells.
  • the cells or tissues are lysed to release the genome in the cells or tissues.
  • the cells or tissue are previously subjected to a formaldehyde cross-linking treatment.
  • formaldehyde temporarily immobilizes DNA-protein and protein-protein complexes that are spatially close in the natural state of cells or tissues.
  • the cell or genome is subjected to a transfer treatment by a mouth pipette.
  • a transfer treatment by a mouth pipette.
  • the inventor found that using the mouth The precise transfer of cells or tissue by the pipette can effectively avoid cell loss caused by centrifugation of the replacement solution.
  • the restriction enzyme is MboI.
  • the inventors found that the recognition site of MboI is GATC, the base distribution is uniform, and there is no obvious preference when cutting on the genome; at the same time, the recognition site of MboI is four bases, compared with the commonly used six The base restriction endonuclease has a higher cutting frequency and a higher resolution of the theoretically obtained data. Finally, MboI is highly commercialized and low in cost, and can be easily obtained, thereby effectively controlling the entire DNA sequencing library. cost.
  • the biotin labeling treatment is carried out by treating the digestion treatment product with adenine triphosphate deoxynucleotide, guanine deoxynucleotide triphosphate, thymidine triphosphate deoxynucleotide
  • the biotin-labeled triphosphate cytosine deoxynucleotide and the DNA polymerase large fragment were contacted, and the contact was carried out at 37 ° C for 1.5 hours. Biotin can be efficiently labeled to the end of the MboI fragment by the above method.
  • the ligation process is a T4 linkage, which is carried out by contacting the ligation product with proteinase K, SDS and sodium chloride.
  • T4 DNA ligase By ligation with T4 DNA ligase, different nick ends of DNA can be ligated into a circular chimeric molecule, and the DNA can be efficiently released by separating the DNA from the bound protein by the above-described decrosslinking treatment.
  • the purification treatment is carried out by contacting the decrosslinked treatment product with pre-cooled anhydrous ethanol, which is carried out at -80 ° C for 15 minutes.
  • DNA is efficiently precipitated at the bottom of the tube to further purify the DNA.
  • the decrosslinking treatment product is contacted with hepatic glycogen and sodium acetate during the purification treatment.
  • the inventors have found that in the process of purifying DNA, since the number of cells is small, the amount of DNA is extremely small, and co-precipitation with hepatic glycogen and DNA can indicate the position of DNA precipitation in the EP tube, thereby effectively preventing DNA loss during washing. .
  • the ultrasound is performed for 134 s with a Peak Power of 50, a Duty Factor of 20, and a Cycles/Burst of 200.
  • Peak Power indicates the highest incident power, which is the instantaneous ultrasonic power acting on the sample
  • Duty Factor indicates the working coefficient, that is, the time when the ultrasonic wave acts on the sample as a percentage of the total time period
  • Cycles/Burst indicates that the ultrasonic wave acts on the sample during the ultrasonic wave The number of energy transfers.
  • the inventors have found that under the above ultrasonic conditions, DNA loss during DNA fragment size selection can be effectively reduced.
  • the database in S700 is built by TruSeq, and S700 includes S710: end-repairing, S720: end-addition of target DNA fragment bound with streptavidin magnetic beads. Adenine phosphate deoxyribonucleotides and S730: ligation sequencing linker sequences were processed.
  • the S720 end plus adenosine triphosphate deoxyribonucleotide treatment further comprises a Tween wash of the magnetic bead treatment; preferably, the S720 end plus adenosine triphosphate deoxyribose
  • the S730 is connected to the sequencing linker sequence to further include a Tween wash to treat the magnetic beads;
  • the S730-linked sequencing linker sequence further comprises a Tween wash to treat the magnetic beads.
  • the magnetic beads are directly washed twice with the Tween washing solution, and the solution can be changed simply and quickly, thereby avoiding the loss of DNA during the purification process.
  • the ligation sequencing linker sequence processing product is subjected to S740: DNA first elution treatment and S750: PCR amplification.
  • S740 DNA first elution treatment
  • S750 PCR amplification.
  • the target DNA fragment can be efficiently eluted from the streptavidin magnetic beads, and the target DNA can be efficiently enriched by PCR amplification.
  • a sequencing library obtained by the above method according to an embodiment of the present invention can be used as a sisHi-C library for second generation sequencing.
  • the above method according to an embodiment of the present invention uses a small reaction system to increase the efficiency of enzymatic cleavage and ligation reaction while greatly reducing the cost; accurately transferring cells using a mouth pipette before cell lysis avoids the replacement of the solution by centrifugation Cell loss; binding of DNA to streptavidin magnetic beads for TruSeq library construction, use of milder magnetic bead wash conditions, and controlled library construction in the same EP tube, these measures successfully bypass the purification and recovery steps And effectively reduce the DNA loss during the construction process; optimize the condition of ultrasonic to break the DNA fragment, elute the DNA in two steps, and retain all the DNA in the range of 200bp to 1000bp after PCR, further improve The amount of DNA that is finally available.
  • the whole method not only significantly reduces the amount of starting cells, but also costs one-tenth of the cost of traditional methods, thus efficiently achieving genomic chromatin capture.
  • the invention proposes a sequencing library.
  • the sequencing library is obtained by the method of constructing a DNA sequencing library of the genome to be tested as described above.
  • sequencing libraries according to embodiments of the present invention for sequencing it is possible to obtain genomic sequence information with a cell number as low as 10 or a genome amount as low as 50 pg and genomic three-dimensional structure information of sufficiently high resolution.
  • the invention proposes a method of determining DNA sequence information of a genome to be tested.
  • Root According to an embodiment of the present invention, 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 result based on the sequencing result DNA sequence information of the genome to be tested.
  • the method for constructing a DNA sequencing library of 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.
  • second generation sequencing techniques such as SOLEXA, SOLID, and 454 sequencing
  • technology such as single-molecule sequencing technologies such as Helicos' True Single Molecule DNA sequencing technology, Pacific Biosciences' single single molecule, real-time (SMRT.TM.), can also be used.
  • Single-molecule sequencing technologies such as Helicos' True Single Molecule DNA sequencing technology, Pacific Biosciences' single single molecule, real-time (SMRT.TM.
  • SMRT.TM. real-time
  • Technology, and nanopore sequencing technology from Oxford Nanopore Technologies, Inc. (Rusk, Nicole (2009-04-01). Cheap Third-Generation Sequencing. Nature Methods 6(4): 244-245).
  • 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 sensitively, accurately and efficiently determine a genomic or microgenome of a trace amount of cells (starting up to 10 cells). Genomic sequence information with a starting amount of DNA as low as 50 pg).
  • the invention proposes a method for determining a three-dimensional spatial structure 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 The three-dimensional spatial structure information of the detected genome is described.
  • the method comprises constructing a DNA sequencing library of the genome to be tested according to the method described above; performing double-end sequencing on the obtained library to obtain sequence information at both ends of each DNA fragment in the library; The sequence information at both ends is compared to the genome, so that the spatial proximity information between each two segments with different linear distances in the genome can be obtained, and the three-dimensional structure of the genome can be inferred by mathematical methods (Aiden et al.Comprehensive Mapping of long-range interactions reveals folding principle of the human genome. Science. 2009).
  • the inventors have surprisingly found that the method for determining the sequence information of the chromatin target region of the genome to be tested according to an embodiment of the present invention enables sensitive, accurate and efficient determination of trace cells (up to 10 cells starting from the genome) or trace amounts.
  • a sufficiently high resolution genomic three-dimensional structural information of the genome initial amount of DNA as low as 50 pg).
  • the collected samples were transferred to a freshly prepared PBS solution containing 1% formaldehyde by a mouthpiece under a stereoscopic microscope, fixed at room temperature for 10 minutes, and added with a 2.5 M glycine solution to a final concentration of 0.2 M, and allowed to stand at room temperature for 10 minutes.
  • the sample was transferred to a PBS solution by a mouth pipette and washed once, and then transferred to a PCR tube.
  • the solution containing the sample was transferred from the PCR tube to a low adsorption 1.5 ml EP tube.
  • the sample was placed on a Thermo mixer, 500 rpm, and treated at 24 ° C for 5.5 hours, and the different nick ends were ligated into circular chimeric molecules by DNA ligase.
  • the sample was taken out of the oven.
  • 1 ⁇ l of hepatic glycogen and 15 ⁇ l of 3 M sodium acetate were added to each sample, mixed on a vortex, and then 240 ⁇ m was added.
  • the pre-cooled anhydrous ethanol was stirred upside down and allowed to stand at -80 ° C for 15 minutes.
  • the precipitate was washed twice with high speed centrifugation with 75% absolute ethanol.
  • the purified DNA was dissolved in 50 ⁇ l of water, and the metal bath was allowed to stand at 37 ° C for 15 minutes.
  • the process of purifying DNA since the number of cells is small, the amount of DNA is extremely small, and hepatic glycogen is co-precipitated with DNA to indicate the position of DNA precipitation in the EP tube, preventing DNA loss during washing.
  • the sample DNA was shredded into fragments of 300-500 base pairs (bp) using a Covaris M220 ultrasonic DNA disruptor, and the ultrasonic conditions were: Peak Power 50, Duty Factor 20, Cycles/Burst 200, time 134s.
  • the Covaris sonic tube was washed with 20 microliters of water to reduce DNA loss. The inventors have found that DNA loss during DNA fragment size selection can be reduced under the above-described ultrasonic conditions.
  • the target DNA fragment bearing the biotin label is bound to the streptavidin magnetic beads. Because the binding of biotin-labeled DNA to streptavidin magnetic beads is very stable, these magnetic beads bound to the target DNA can be used directly for TruSeq library construction. Terminal repair was performed in sequence, adenine triphosphate deoxyribonucleotide (dATP) was added to the end of the DNA fragment, and the sequencing sequence was ligated. After each step of the reaction, the magnetic beads are directly washed twice with the Tween washing solution, and the solution can be changed simply and quickly, thereby avoiding the loss of DNA during the purification process.
  • dATP adenine triphosphate deoxyribonucleotide
  • the sample On the fourth day, after the sequence of the sequencing linker is connected, the sample is placed on a magnetic stand. After the magnetic beads are all adsorbed to the magnetic frame, the solution becomes clear, the supernatant is discarded, and the magnetic beads are washed twice with the Tween washing solution, each time in the Rotate the mixer for 2 minutes at room temperature. Add 20 ⁇ l of water to the sample, mix by pipetting, and place the sample on a constant temperature mixer at 66 ° C, 1400 rpm for 20 minutes, and elute the DNA twice. PCR amplification was performed.
  • AMPure XP magnetic beads were added to 150 ⁇ l of the sample, pipetted and mixed, and then incubated on a rotary mixer for 5 minutes at room temperature. Transfer the supernatant to a new low-adsorption EP tube, add 78 ⁇ l (1:1) AMPure XP magnetic beads, mix and mix for 5 minutes at room temperature on a rotary mixer and discard the supernatant.
  • the magnetic beads were washed twice with 75% absolute ethanol, and after air drying, 50 ⁇ l of water was added to the EP tube to elute, thereby obtaining a DNA fragment having a size ranging from 200 base pairs to 1000 base pairs.
  • the library obtained by the above procedure (in the present application, the inventors obtained the library obtained by the above method as a sisHi-C library) can be used for second generation sequencing.
  • 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

L'invention concerne une méthode de construction d'une bibliothèque de séquençage d'ADN d'un génome à tester, la méthode comprenant les étapes consistant à : (1) digérer un génome à tester au moyen d'une enzyme de restriction ; (2) effectuer un marquage à la biotine sur les produits digérés ; (3) lier les produits marqués à la biotine à l'aide d'une ADN ligase ; (4) déréticuler les produits liés ; (5) purifier les produits déréticulés ; (6) effectuer des traitements par ultrasons et par précipitation sur les produits purifiés, le traitement de précipitation étant pour amener les produits soniqués en contact avec des billes magnétiques de streptavidine pour obtenir des fragments d'ADN cibles liés à des billes magnétiques de streptavidine ; et (7) construire une bibliothèque sur la base des fragments d'ADN cibles liés à des billes magnétiques de streptavidine.
PCT/CN2017/114475 2017-07-07 2017-12-04 Méthode de capture in situ de conformation de chromatine de génome entier pour cellules infinitésimales Ceased WO2019006975A1 (fr)

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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN108265104B (zh) * 2018-01-02 2021-07-30 北京诺禾致源科技股份有限公司 染色体构型捕捉文库及其构建方法
CN109797436B (zh) * 2018-12-29 2021-10-08 阅尔基因技术（苏州）有限公司 一种测序文库构建方法
CN109735900A (zh) * 2019-03-20 2019-05-10 嘉兴菲沙基因信息有限公司 一种适用于Hi-C的小片段DNA文库构建方法
CN110396534A (zh) * 2019-08-12 2019-11-01 华大生物科技（武汉）有限公司 基因文库的构建方法、待测核酸样本基因突变的检测方法及试剂盒
CN111364105B (zh) * 2020-04-30 2021-09-07 华中农业大学 一种简便有效的植物长片段in situ DLO Hi-C测序文库的构建方法
CN112522251A (zh) * 2020-12-29 2021-03-19 上海派森诺生物科技股份有限公司 一种用于Hi-C的动物组织提取方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754995A (zh) * 2016-04-19 2016-07-13 清华大学 构建待测基因组的dna测序文库的方法及其应用
CN106566828A (zh) * 2016-11-11 2017-04-19 中国农业科学院农业基因组研究所 一种高效的全基因组染色质构象技术eHi‑C
CN106637422A (zh) * 2016-12-16 2017-05-10 中国人民解放军军事医学科学院生物工程研究所 一种构建Hi‑C高通量测序文库的方法
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

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030196214A1 (en) * 2002-03-27 2003-10-16 Priti Sharma Novel genes from drought stress tolerant tea plant and a method of introducing water-stress tolerance
EP1597395A2 (fr) * 2003-02-21 2005-11-23 Nuevolution A/S Procede de production d'une banque de deuxieme generation
CN106591955B (zh) * 2015-10-19 2019-10-29 浙江安诺优达生物科技有限公司 构建高分辨率、大信息量单细胞Hi-C文库的方法
CN106591285B (zh) * 2015-10-19 2019-11-29 浙江安诺优达生物科技有限公司 一种构建高可利用数据率的Hi-C文库的方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105754995A (zh) * 2016-04-19 2016-07-13 清华大学 构建待测基因组的dna测序文库的方法及其应用
CN106566828A (zh) * 2016-11-11 2017-04-19 中国农业科学院农业基因组研究所 一种高效的全基因组染色质构象技术eHi‑C
CN106637422A (zh) * 2016-12-16 2017-05-10 中国人民解放军军事医学科学院生物工程研究所 一种构建Hi‑C高通量测序文库的方法
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

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