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WO2017066909A1 - Procédé simple, rapide et à faible coût pour construire une banque hi-c - Google Patents

Procédé simple, rapide et à faible coût pour construire une banque hi-c Download PDF

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Publication number
WO2017066909A1
WO2017066909A1 PCT/CN2015/092182 CN2015092182W WO2017066909A1 WO 2017066909 A1 WO2017066909 A1 WO 2017066909A1 CN 2015092182 W CN2015092182 W CN 2015092182W WO 2017066909 A1 WO2017066909 A1 WO 2017066909A1
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WIPO (PCT)
Prior art keywords
chromatin
dna
immobilized
library
dna fragment
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PCT/CN2015/092182
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English (en)
Chinese (zh)
Inventor
李小林
张介中
赵红梅
裴志华
玄兆伶
李大为
梁峻彬
陈重建
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Annoroad Gene Technology (beijing) Co Ltd
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Annoroad Gene Technology (beijing) Co Ltd
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Priority to PCT/CN2015/092182 priority Critical patent/WO2017066909A1/fr
Publication of WO2017066909A1 publication Critical patent/WO2017066909A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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 a library construction method capable of capturing chromatin three-dimensional conformation in a genome-wide range, and belongs to the technical field of gene sequencing.
  • DNA is the carrier of cellular genetic information, which exists in the body in the form of chromatin in each cell and controls the progress of life activities.
  • most of the research on DNA information is carried out by studying the sequence of bases in DNA molecules (one-dimensional information of DNA), and analyzing the law of life activities by analyzing the base arrangement information.
  • the nucleus in the real state is a narrow three-dimensional space.
  • the DNA of the linear molecular structure is located in the nucleus in a complex crimping manner.
  • the original one-dimensional DNA sequence is assigned a three-dimensional conformation and leads to a large number of complex gene regulation modes. .
  • simple one-dimensional DNA sequence information cannot provide information related to the spatial distribution of real DNA, and therefore cannot explain a series of gene regulation phenomena caused by spatial conformation.
  • Hi-C technology is a technique that combines high-throughput sequencing methods to detect chromatin information throughout the nucleus.
  • Hi-C technology is a derivative technology of Chromosome conformation capture (3C), which refers to the capture of chromosome conformation based on high-throughput, which can capture the space between different gene loci in the whole genome. Interacting to study DNA elements that regulate genes in three dimensions.
  • Patent Document 1 and Non-Patent Document 1 report a Hi-C method which immobilizes a chromatin structure by formaldehyde, then interrupts the original genome sequence by restriction endonuclease, and performs biotin labeling, and then re- The ligation forms a new DNA molecule with structural information.
  • This process if two DNA fragments of different genomic locations are joined to form a hybrid molecule, this will be considered as evidence that the two DNA molecules are spatially adjacent to each other.
  • the DNA is then purified and disrupted, and then the labeled biotin molecules are harvested and enriched to obtain the desired spatially interacting DNA hybrid molecules.
  • the method mainly comprises the following steps: 1) firstly performing formaldehyde cross-linking immobilization on the sample cells, and the DNA having a relatively close internal space is cross-linked by the protein and collecting the cells; 2) using the lysis system and performing the grinding with the cells.
  • the Hi-C method has a high cost of building a database. Due to the need to use biotin-labeled bases and a large number of streptavidin magnetic beads for affinity purification, the cost of building the method is several times higher than that of genome building, expression profiling, and apparent group building. The cost of building a high-throughput sequencing library.
  • the Hi-C method is complicated in operation and long in operation time. Because of the need for biotin labeling and end-repair filling, blunt-end ligation, removal of unligated end biotin labeling, streptavidin magnetic bead fishing and other cumbersome steps, this method is very waste time.
  • the Hi-C method can only perform chromatin digestion using a type II restriction endonuclease which forms a 5'-end protruding sticky end after enzymatic cleavage, so that the chromatin digestion mode is relatively simple.
  • Patent Document 1
  • the inventors have conducted intensive studies to solve the above technical problems, and have made ingenious improvements based on the conventional method of constructing a Hi-C library, which makes the operation simple, time-consuming, and low in cost, thereby completing the present invention.
  • the present invention includes:
  • a method of constructing a Hi-C library comprising the steps of:
  • Step B obtaining immobilized chromatin
  • Step C digesting the immobilized chromatin obtained in the step B to obtain an immobilized chromatin fragment
  • Step D directly re-ligating the immobilized chromatin fragments obtained in the step C to obtain re-ligated immobilized chromatin fragments;
  • Step E de-immobilizing the religated immobilized chromatin fragment obtained in the step D to release the DNA fragment;
  • Step G Using the DNA fragment released in the step E as a DNA fragment to be sequenced, a DNA library for sequencing is constructed.
  • Step F performed after the step E: further fragmenting the DNA fragment released in the step E to obtain a smaller DNA fragment;
  • the smaller DNA fragment is used as the DNA fragment to be sequenced, and a DNA library for sequencing is constructed.
  • deoxyribonuclease is a type I restriction endonuclease, a type II restriction endonuclease or a type III restriction endonuclease.
  • Step A obtaining cells in which chromatin is immobilized
  • the cells obtained in the step A are cleaved to obtain immobilized chromatin.
  • a method of determining a chromatin region that may interact spatially comprising:
  • the Hi-C library was sequenced and the obtained information was aligned with chromatin DNA primary sequence information.
  • the advantages of the present invention are at least:
  • the invention provides a method of constructing a Hi-C library (Hi-C library construction method of the invention), the method comprising the steps of:
  • Step B obtaining immobilized chromatin
  • Step C digesting the immobilized chromatin obtained in the step B to obtain an immobilized chromatin fragment
  • Step D directly re-ligating the immobilized chromatin fragments obtained in the step C to obtain re-ligated immobilized chromatin fragments;
  • Step E de-immobilizing the religated immobilized chromatin fragment obtained in the step D to release the DNA fragment;
  • Step G Using the DNA fragment released in the step E as a DNA fragment to be sequenced, a DNA library for sequencing is constructed.
  • the Hi-C library construction method of the present invention may further comprise:
  • Step F performed after the step E: further fragmenting the DNA fragment released in the step E to obtain a smaller DNA fragment, and in the step G, the smaller DNA fragment is to be sequenced
  • a DNA fragment was constructed to construct a DNA library for sequencing.
  • the Hi-C library construction method of the present invention may further comprise:
  • Step A Obtaining cells in which chromatin is immobilized; and in the step B, lysing the cells obtained in the step A to obtain immobilized chromatin.
  • Hi-C refers to the three-dimensional interaction group of chromatin, which is a kind of chromatin spatial conformation capture in the whole genome, and studies the three-dimensional structure of chromatin and the spatial relationship of different DNA regions.
  • Hi-C library refers to: High-throughput sequencing in Hi-C method to obtain possible chromatin interaction information, and the DNA library used for such high-throughput sequencing is Hi-C library.
  • the amount of chromatin to be immobilized is not particularly limited, and from the viewpoint of ease of handling, it may be a chromatin of 10,000 to 1 billion cells, preferably a chromatin of 25 to 100 million cells. More preferably, the chromatin of 50 million to 100 million cells is used.
  • the amount of chromatin immobilized may be about 1 ng to 1,000,000 ng in terms of mass, based on naked DNA.
  • the amount of cells to which chromatin is immobilized is not particularly limited, and may be 10,000 to 1 billion cells, preferably 25 to 100 million cells, more preferably 50 million cells, from the viewpoint of ease of handling. 100 million cells.
  • immobilized means that a portion of a cell in which a chromatin is close to each other in three dimensions is fixed in a state close to a natural conformation.
  • chromatin also includes chromosome morphology.
  • the immobilization can usually be carried out by crosslinking the proteins on the chromatin.
  • Crosslinking methods for proteins on chromatin are known to those skilled in the art, for example, ultraviolet rays may be used alone, or tetranitromethane, carbodiimide, formaldehyde, methanol, ethanol, valeraldehyde, or the like may be used alone.
  • Chemical reagents such as nitrogen mustard, dimethyl sulfate, formaldehyde release agent, imide ester, mitogen C, mustard gas, and chlorhexidine may also be crosslinked by the above chemical reagent in combination with ultraviolet irradiation.
  • the cells may be placed in an appropriate amount (for example, 1 to 10,000,000 ⁇ L) of water, TE buffer, physiological saline, PBS or cell culture medium to prepare a cell suspension.
  • the droplets are further added with an appropriate amount (for example, 1-10,000,000 ⁇ L) of a formaldehyde solution (the concentration thereof is not limited, for example, 1 to 20% by weight), and the mixture is allowed to stand at room temperature for a certain period of time (for example, 1-100 min) to carry out crosslinking. Then, a certain amount of an amino acid (a mixture of one amino acid or a plurality of amino acids) or a protein (for example, BSA or the like) is added to the above reaction droplets to terminate the crosslinking reaction.
  • an appropriate amount for example, 1-10,000,000 ⁇ L
  • a formaldehyde solution the concentration thereof is not limited, for example, 1 to 20% by weight
  • a certain period of time for example, 1-100 min
  • the cells obtained in the step A are lysed to obtain immobilized chromatin.
  • Cell lysis can usually be carried out by placing the cells in an appropriate cell lysate.
  • the formulation and amount of the cell lysate can be suitably determined by those skilled in the art according to the type and amount of the cells.
  • the immobilized chromatin obtained in the step B is digested to obtain an immobilized chromatin fragment.
  • the digestion can be carried out using deoxyribonuclease.
  • deoxyribonuclease a type I restriction endonuclease, a type II restriction endonuclease or a type III restriction endonuclease is preferred.
  • the type II restriction endonuclease which forms a 5'-end protruding sticky end after digestion can be used for chromatin digestion, which results in a single chromatin digestion mode, but The Hi-C library construction method of the present invention may not be limited thereto.
  • deoxyribonuclease can be used to digest chromatin, enriching the chromatin digestion mode, and improving the resolution of the Hi-C method.
  • the immobilized chromatin digestion of the method can also be carried out using an enzyme other than deoxyribonuclease, for example, a non-specific nuclease (such as a broad-spectrum nuclease).
  • the immobilized chromatin fragments obtained in the step C are directly religated to obtain religated immobilized chromatin fragments.
  • directly reconnecting means that the immobilized chromatin fragments are not biotinylated, and the fragments are rejoined.
  • the obtained immobilized chromatin fragment has a sticky end, preferably, in the Hi-C library construction method of the present invention Instead of end-repairing these cohesive ends to blunt ends as in the prior art, these chromatin fragments are rejoined by cohesive end joining, which is more efficient than blunt-end ligation.
  • the method can also be carried out by blunt-end ligation after repair (but without the attachment point labeling such as adding a biotin-labeled nucleotide to repair the sticky end) or by restriction endonuclease producing a blunt end, and then performing Flat end connection.
  • the ligation can be carried out, for example, by using a DNA ligase having a terminal ligation activity such as T4 DNA ligase, T3 DNA ligase, E. coli DNA ligase, thermostable DNA ligase Enzyme and so on.
  • the amount of the enzyme and the substrate used in the ligation reaction, and the reaction conditions can be appropriately selected by those skilled in the art as needed. For example, it can be usually carried out at 0 to 80 ° C (preferably 10 to 40 ° C) in 0.1 to 10 ⁇ ligase buffer for about 1 minute to 200 hours (preferably 1 to 30 hours).
  • the religated immobilized chromatin fragment obtained in the step D is deactivated to release the DNA fragment.
  • de-immobilization means that the fixed state of the portion adjacent to each other in three-dimensional space among the immobilized chromatin fragments is released.
  • the immobilization is achieved by crosslinking a protein on chromatin
  • the "un-immobilization” means that the protein is decrosslinked.
  • the protein solution can be carried out by placing the system after the above-mentioned ligation reaction at 50 to 100 ° C (preferably 60 to 80 ° C) for 1 minute to 200 hours (preferably 1 to 30 hours).
  • Cross-linking As a method for biologically and chemically treating decrosslinking, endopeptide, serine protease, thiol protease, metalloproteinase, aspartic protease, pepsin, trypsin, cathepsin, papain, It is carried out with subtilisin, proteinase K, DTT, NaCl, KCl or a combination thereof.
  • the DNA fragment released in the step E is further fragmented to obtain a smaller DNA fragment.
  • "smaller DNA fragment” refers to a DNA library, such as an Illumina DNA sequencing library, that is sized to construct a sequencing (eg, second generation sequencing, third generation sequencing, or fourth generation sequencing).
  • the specific size of the "smaller DNA fragment” may be, for example, 10 to 50000 bp, preferably 50 to 1000 bp.
  • the method of fragmentation is not particularly limited, and for example, the amplification product fragment may be subjected to ultrasonication, transposase, hydraulic shearing, enzymatic cleavage, or the like. Chemical.
  • the technique of fragmenting the amplified product by the above method is known to those skilled in the art, and can be carried out by selecting appropriate conditions as needed.
  • a DNA library for sequencing is constructed using the product of the step E or the step F as a DNA fragment to be sequenced.
  • the construction of a DNA library for sequencing generally requires 0.001 to 1000 ng of DNA.
  • the DNA library for sequencing can be constructed using, for example, a standard Illumina DNA small fragment construction method, a PCR free method, a one-step method, and the like.
  • Various methods of constructing DNA libraries for sequencing are known to those skilled in the art and can be carried out by those skilled in the art in accordance with conventional procedures.
  • the standard Illumina DNA mini-fragment method usually includes steps such as end-repair, end-addition A, Adapter ligation, amplification, and amplification product purification, which can be performed according to the method recommended by Illumina.
  • the Hi-C library construction method of the present invention does not use biotin to fish for recombination site DNA fragments, but may use informatics analysis to filter out fragments in which non-rejoining sites are located. Since the standard Hi-C sequencing data analysis method has the filtering of such a fragment, the library constructed by the Hi-C library construction method of the present invention can perform bioinformatics analysis without additional filtering conditions, and obtain a chromosome at Interaction information in three dimensions.
  • the DNA was fragmented and the amplified product was ultrasonically interrupted using a Diagenod Bioruptor UCD-600 (NGS).
  • the interrupting procedure was: 30 second ultrasound, 30 second rest, 22 cycles.
  • the previously amplified DNA fragment was fragmented into a DNA fragment of the target fragment between 100 and 700 bp.
  • the sample was placed in a Thermomixer at 20 ° C for 30 min. After completion of the reaction, the DNA in the purified reaction system was recovered using a Beckman Agencourt AMPure XP nucleic acid purification kit, and dissolved in 32 ⁇ L of water.
  • the sample was placed in a Thermomixer at 37 ° C for 30 min.
  • the DNA in the purified reaction system was recovered using a Beckman Agencourt AMPure XP nucleic acid purification kit and dissolved in 18 ⁇ L of water.
  • the sample was placed in a Thermomixer bath at 20 ° C for 15 min.
  • the DNA in the purified reaction system was recovered using a Beckman Agencourt AMPure XP nucleic acid purification kit and dissolved in 30 ⁇ L of water.
  • Purification was carried out using an Agencourt AMPure XP nucleic acid purification kit; dissolved in 15 ⁇ L of water; and purified after concentration.
  • Non-Patent Document 1 Unlike Non-Patent Document 1, the present invention does not use biotin to fish for recombination site DNA fragments, but uses informatics analysis to filter out fragments in which non-rejoining sites are located. Since the standard Hi-C sequencing data analysis method has the filtering of such a fragment (this filtering condition is in Non-Patent Document 1 in order to remove residual biotin-labeled unreconnected fragments in the library), the present invention The constructed library can perform bioinformatics analysis without additional filtering conditions and obtain information on the interaction of chromosomes in three dimensions.
  • the advantages and disadvantages of the method of the present invention and the method of Non-Patent Document 1 are as follows.
  • any technical feature or combination of technical features described in the specification as a component of a certain technical solution may also be applied to the embodiments that can be implemented without obscuring the gist of the present invention.
  • Other technical solutions; and, while being able to implement and not clearly deviating from the gist of the present invention, the technical features described as the constituent parts of the different technical solutions may be combined in any manner to constitute other technical solutions.
  • the present invention also encompasses the technical solutions obtained by the combination in the above case, and these technical solutions are equivalent to those described in the present specification.

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Abstract

L'invention concerne un procédé pour construire une banque Hi-C. Le procédé ne fait pas appel à une étiquette biotine ni à des billes magnétiques streptavidine.
PCT/CN2015/092182 2015-10-19 2015-10-19 Procédé simple, rapide et à faible coût pour construire une banque hi-c Ceased WO2017066909A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802205A (zh) * 2024-01-09 2024-04-02 广西壮族自治区人民医院 一种单细胞Hi-C文库构建方法
CN117845339A (zh) * 2024-01-09 2024-04-09 广西壮族自治区人民医院 一种用于检测与目标基因座相互作用的dna片段的文库构建方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010036323A1 (fr) * 2008-09-25 2010-04-01 University Of Massachusetts Medical School Procédé d'identification des interactions entre des loci génomiques
WO2015123588A1 (fr) * 2014-02-13 2015-08-20 Bio-Rad Laboratories, Inc. Capture de conformation chromosomique dans des fragments

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010036323A1 (fr) * 2008-09-25 2010-04-01 University Of Massachusetts Medical School Procédé d'identification des interactions entre des loci génomiques
WO2015123588A1 (fr) * 2014-02-13 2015-08-20 Bio-Rad Laboratories, Inc. Capture de conformation chromosomique dans des fragments

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, YIFENG ET AL.: "Chromosome Conformation Capture and Its Development", LETTERS IN BIOTECHNOLOGY, vol. 21, no. 6, 31 December 2010 (2010-12-31), pages 864 - 867 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117802205A (zh) * 2024-01-09 2024-04-02 广西壮族自治区人民医院 一种单细胞Hi-C文库构建方法
CN117845339A (zh) * 2024-01-09 2024-04-09 广西壮族自治区人民医院 一种用于检测与目标基因座相互作用的dna片段的文库构建方法

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