WO2017066908A1 - 构建高分辨率、大信息量单细胞Hi-C文库的方法 - Google Patents

构建高分辨率、大信息量单细胞Hi-C文库的方法 Download PDF

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WO2017066908A1
WO2017066908A1 PCT/CN2015/092181 CN2015092181W WO2017066908A1 WO 2017066908 A1 WO2017066908 A1 WO 2017066908A1 CN 2015092181 W CN2015092181 W CN 2015092181W WO 2017066908 A1 WO2017066908 A1 WO 2017066908A1
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chromatin
immobilized
cells
dna
small amount
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PCT/CN2015/092181
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French (fr)
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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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安诺优达基因科技(北京)有限公司
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Priority to US15/769,073 priority Critical patent/US10900031B2/en
Priority to EP15906427.8A priority patent/EP3366818B1/en
Priority to PCT/CN2015/092181 priority patent/WO2017066908A1/zh
Publication of WO2017066908A1 publication Critical patent/WO2017066908A1/zh

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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
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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

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 sequencing, which can capture the gap between different gene loci in the genome-wide range. Spatial interaction to study DNA elements that regulate genes in three-dimensional space.
  • 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 enzymes, 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. Finally, a library of high-throughput sequencing was constructed and detected by double-end sequencing to obtain spatial interaction information of total chromatin.
  • the method mainly comprises the following steps: 1) Firstly, more than 10 6 cell samples are fixed by formaldehyde cross-linking, and DNA having a relatively close internal space is cross-linked by proteins and collects cells; 2) using a lysing system And the cells are lysed by grinding to obtain the separated nuclei; 3) the chromatin of the cross-linked cells is digested with a restriction endonuclease (such as EcoR I); 4) the biotin is labeled with the end of the digested end, and formed Flat end; 5) using DNA ligase to join the blunt ends, DNA fragments on the same cross-linking molecule will have a greater probability of joining to form new molecules; 6) high temperature (65 ° C) treatment reverse cross-linking, Release of double-stranded DNA molecules; 7) removal of unligated end biotin labeling; 8) fragmentation of DNA and biotin-specific uptake, enrichment of hybrid junction sites; and 9) construction of Illumina sequencing The library was subjected to
  • the method starts with a large number of samples above 10 6 cell levels, and large-scale grinding and extraction methods are used throughout the process, which is not suitable for small samples (under 10 5 cells), especially single cell levels. Hi-C detection.
  • this method is directed to a large number of cell samples, only the total chromatin conformation of the population cells can be detected, and the differences between the cell chromatin conformation individuals cannot be detected and compared.
  • Non-Patent Document 2 reports a single nuclear Hi-C method, but the method is still a large number of initial sample cell sample 106 orders of magnitude.
  • the pre-treatment uses formaldehyde to immobilize the chromatin structure, then breaks the original genome sequence by restriction endonuclease, and biotinylation, and then rejoins to form 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 pretreated nuclear nuclei are selected under a microscope to obtain a single cell nucleus, and then the cross-linking removal, biotin fishing, and restriction endonuclease digestion of the DNA molecule are performed on a single nuclear sample, and enrichment is obtained. Space-hybrid molecules that interact on the desired space. Finally, a library of high-throughput sequencing was constructed and detected by double-end sequencing to obtain spatial interaction information of total chromatin.
  • the method mainly comprises the following steps: 1) Firstly, more than 10 6 cell samples are fixed by formaldehyde cross-linking, and DNA having a relatively close internal space is cross-linked by proteins and collects cells; 2) using a lysing system And the cells are lysed by grinding to obtain the separated nuclei; 3) the chromatin of the cross-linked cells is digested with a restriction endonuclease (such as EcoR I); 4) biotin labeling is performed on the end of the enzyme digestion, and formation Flat end; 5) using DNA ligase to ligate the blunt ends, DNA fragments on the same cross-linking molecule will have a greater probability of joining to form new molecules; 6) single-core nucleus pick-up under the microscope Hi-C-treated single-cell nuclear sample; 7) High-temperature removal of formaldehyde cross-linking of single-cell nuclear sample to release DNA; 8) Biotin-specific acquisition of DNA molecules to enrich heterozygous molecular attachment sites; 9) Frag
  • the method undergoes multiple steps such as digestion, end repair, biotin labeling, blunt-end ligation, biotin uptake, and ligation before the library is amplified, these steps are all in a set of genome copies.
  • the efficiency of each step will affect the amount of information captured last time, resulting in serious information loss during the whole experiment, and the number of DNA interaction fragments finally captured is also low.
  • the method employs two restriction nucleases for chromatin digestion and recombination product fragmentation, respectively. In order to ensure the efficiency of fragmentation of the re-ligated product, the endonuclease used in this step appears much more frequently in the genome than the frequency of endonuclease used for chromatin digestion.
  • Non-patent document 2 has also attempted to perform chromatin digestion using a four-base enzyme which occurs at a high frequency in the genome, but since the subsequent recombination product fragmentation is not available with a higher frequency endonuclease, the resolution and the amount of information cannot be improved.
  • the method for studying single cells in this method is to pick a single cell nucleus, and the operation difficulty and the requirements for instruments and techniques are much higher than picking single cells.
  • Patent Document 1
  • the inventors have intensively studied to solve the above technical problems, and have made ingenious improvements on the basis of the traditional method of constructing Hi-C library, and created a Hi-C library construction method suitable for a small amount of chromatin, thereby completing the present invention. invention.
  • the present invention includes:
  • a method of constructing a Hi-C library comprising the steps of:
  • Step B obtaining a small amount of 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 F amplifying the DNA fragment released in the step E to obtain an amplification product
  • Step H Using the amplification product as a DNA fragment to be sequenced, 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 G Fragmenting the amplification product obtained in the step F to obtain a smaller DNA fragment
  • step H a smaller DNA fragment obtained in the step G is used as a DNA fragment to be sequenced, and a DNA library for sequencing is constructed.
  • Step A obtaining a small amount of cells in which chromatin is immobilized
  • the cells obtained in the step A are cleaved to obtain a small amount of immobilized chromatin.
  • step A comprises:
  • Step A-1 Immobilizing a certain amount of chromatin of a cell to obtain a certain amount of cells in which chromatin is immobilized;
  • Step A-2 A small amount of chromatin-immobilized cells are picked from a certain amount of chromatin-immobilized cells obtained in the step A-1.
  • step A comprises:
  • Step A-3 Immobilization of chromatin of a small amount of cells is performed to obtain a small amount of cells in which chromatin is immobilized.
  • a method of determining a chromatin region that may interact spatially comprising:
  • a method of constructing a Hi-C library with a small starting sample amount, high resolution, large amount of information, and simple operation is provided.
  • the present invention can operate on a single cell or a small sample (1-1000 cells) from the initial step, and solves the small sample amount of cells for Hi- The problem of C detection.
  • the present invention can perform Hi-C analysis on individual cells, detecting and studying the chromatin conformation of individual cells and the difference in chromatin conformation between different cells.
  • the present invention abandons the steps of end-repairing, biotin labeling and retrieval after enzymatic cleavage, and can replace the connection efficiency by using a sticky end connection with high connection efficiency.
  • the lower blunt end connection reduces the loss of information and captures more chromatin conformational information.
  • the present invention can adopt a random interruption method in fragment fragmentation, and the fragmentation effect is much higher than that of restriction endonuclease digestion.
  • This operation allows for the use of higher resolution restriction enzymes such as restriction endonucleases recognizing the 4 base sequence in chromatin digestion, thereby enhancing chromatin conformational resolution.
  • the present invention can pick a single cell for library construction, which is less difficult than picking a single cell nucleus.
  • the invention provides a method of constructing a Hi-C library (method of the invention), the method comprising the steps of:
  • Step B obtaining a small amount of 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 F amplifying the DNA fragment released in the step E to obtain an amplification product
  • Step H Using the amplification product as a DNA fragment to be sequenced, a DNA library for sequencing is constructed.
  • the method of the present invention further comprises:
  • Step G Fragmenting the amplification product obtained in the step F to obtain a smaller DNA fragment; and in the step H, The smaller DNA fragment obtained in the step G is used as a DNA fragment to be sequenced, and a DNA library for sequencing is constructed.
  • the method of the present invention further includes:
  • Step A A small amount of cells in which chromatin is immobilized is obtained; and in the step B, the cells obtained in the step A are cleaved to obtain a small amount of immobilized chromatin.
  • the chromatin of a certain amount of cells can be immobilized first, and a certain amount of cells in which chromatin is immobilized are obtained, and then A small amount of chromatin-immobilized cells are picked up from a certain amount of chromatin obtained by immobilization, or a small amount of chromatin can be directly immobilized to obtain a small amount of chromatin-immobilized cells.
  • the cells herein may also be nuclei.
  • 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.
  • a small amount of chromatin means an amount of chromatin which is as small as the prior art Hi-C method cannot operate, and generally refers to a chromatin of 1 to 10,000 cells, or 1 to 1000 cells. Chromatin, or chromatin of 1 to 100 cells, even chromatin of 1 cell (single cell), and even part of the chromatin of 1 cell (single cell).
  • the "small amount of chromatin” may be 10 -6 to 10 2 ng, preferably 10 -5 to 10 ng, by mass, based on the naked DNA.
  • a small amount of cells means a number of cells which are as small as the prior art Hi-C method cannot operate, and generally may refer to 1 to 10,000 cells, or 1 to 1000 cells, or 1 to 100. One cell, even one cell (single cell).
  • 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 chromatin of a certain amount (for example, 10 5 or more, preferably 10 6 to 10 8 ) of cells may be immobilized to obtain a certain amount of cells in which chromatin is immobilized, and then A small amount of cells in which chromatin is immobilized are picked up; or the chromatin of a small amount of cells can be directly immobilized, thereby obtaining a small amount of cells in which chromatin is immobilized.
  • the picking of the small amount of cells can be carried out by capillary method, dilution method, gradient dilution method or flow cytometry sorting method.
  • 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.
  • the deoxyribonuclease a type I restriction endonuclease, a type II restriction endonuclease or a type III restriction endonuclease is preferred.
  • a restriction enzyme that recognizes a four-base sequence in a subsequent step, only a restriction within a 6-base sequence can be used. Dicers digest chromatin. This type of digestion has a low resolution and a small amount of information retained.
  • step C since DNA fragmentation can be performed by a method such as ultrasonication, transposase, hydraulic shearing or the like in a subsequent step, a higher resolution can be used in this step C.
  • the restriction endonuclease or other nuclease that recognizes the 4 base sequence digests chromatin, which theoretically increases the resolution by a factor of ten, and the amount of chromatin interaction information available is greatly increased.
  • chromatin digestion can also be carried out using a restriction endonuclease that recognizes a 6-base sequence.
  • 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, and preferably, the method of the present invention is not as in the prior art. These sticky ends are repaired to make them blunt-ended, but the chromosomal ends are used to rejoin these chromatin fragments, which is more efficient than blunt-end ligation.
  • the method can also be used for 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 blunt-end restriction endonuclease digestion followed by blunt-end ligation .
  • the terminal ligation can be carried out, for example, by using a DNA ligase having terminal ligation activity such as T4 DNA ligase, T3 DNA ligase, Escherichia coli DNA ligase, thermostable DNA ligase, etc. .
  • 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, Withered It is carried out with a protease, proteinase K, DTT, NaCl, KCl or a combination thereof.
  • step E may be performed together with the step F.
  • step F the DNA fragment released in the step E is amplified to obtain an amplification product.
  • the amplification method is not particularly limited as long as a sufficient amount (for example, 0.001 to 1000 ng) of amplification product for constructing a DNA library for sequencing can be obtained.
  • an amplification method suitable for a small amount of cells, a single cell or a trace amount of DNA such as MDA, MALBAC, NEB-WGA, GenomePlex (preferably MALBAC) or the like can be employed, and specific conditions of these amplification methods can be appropriately selected by those skilled in the art as needed.
  • the above amplification method can generally be carried out based on a PCR reaction (polymerase chain reaction), which is generally carried out by a certain PCR reaction procedure (temperature cycle).
  • the PCR reaction procedure generally includes steps of denaturation, annealing, extension, and the like.
  • the design of the primers used in the PCR reaction is well known to those skilled in the art, for example, in accordance with the Guide to Molecular Cloning (J. Sambrook, J., et al., Huang Peitang et al., 3rd edition, 2005).
  • the instruction is to design, or use computer software (such as Primer Premier 6.0 developed by Premier) to design.
  • the amplification product obtained in the step F is 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 amplification product was fragmented by a method, a transposase method, or a hydraulic shear method. This improves the resolution and gives you more information.
  • 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 fragment for sequencing is constructed using the smaller DNA fragment obtained in the step G as a DNA fragment to be sequenced.
  • 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.
  • Single cells or a small number of cells are picked by capillary method and placed in the lysate.
  • the amplification product of the re-ligated DNA was obtained by performing DNA amplification on the previously obtained sample using the Yikang single-cell whole genome amplification kit based on the MALBAC method according to the kit instructions.
  • the amplified product was ultrasonically interrupted using a Diagenode Bioruptor UCD-600 (NGS) with a 30 second ultrasound, 30 second rest, and 22 cycles.
  • NGS Diagenode Bioruptor UCD-600
  • the previously amplified DNA fragment was fragmented into a DNA fragment having a fragment size 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.
  • Non-Patent Documents 1 and 2 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 a filter for such a fragment (this filter condition is in Non-Patent Documents 1 and 2 to remove residual biotin-labeled unreconnected fragments in the library),
  • the library produced by the present invention can perform bioinformatics analysis and obtain interaction information of chromosomes in three dimensions without additional filtering conditions.
  • the present invention uses Mbo I (the recognition site is GATC but sensitive to Dam, Dcm and CpG methylation) for chromatin digestion, and captures the interaction between chromatin regions under the sequencing amount of the 14M-19M double-end sequencing reads pair.
  • the logarithm of action is 270-331K.
  • Non-Patent Document 2 uses Dpn II in the single-cell nuclear Hi-C (the recognition site is also GATC but sensitive to Dam, Dcm methylation, that is, its recognition site number should be higher than Mbo I, so the theory More information should be obtained on the fragment and chromatin region interaction information)
  • the recognition site is also GATC but sensitive to Dam, Dcm methylation, that is, its recognition site number should be higher than Mbo I, so the theory More information should be obtained on the fragment and chromatin region interaction information
  • the remaining single nuclei are digested with BglII endonuclease for chromatin digestion.
  • the number of interactions between the chromatin regions captured by the M-15.3M double-end sequencing reads was 11.7k-30.6k.
  • the chromatin conformation information obtained by the present invention is remarkably higher than the single cell nuclear Hi-C method of Non-Patent Document 2.
  • 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.
  • the detection of chromatin conformation can be performed on samples of a small number of cells.
  • Cell types include, but are not limited to, animal cells, plant cells, microbial cells, viruses, cancer cells; such cell sources include, but are not limited to, primary culture, cell line culture, tissue, organism, environmental source, fossil.
  • the difference in chromosome conformation between different cells can be compared.
  • the differences between these cells include, but are not limited to, different species sources, different organ sources, different cell types, different cell cycles, different developmental stages, different culture conditions, different treatment conditions, and different cell individuals.
  • chromatin conformational changes in cells and changes in other DNA, RNA, and proteins can be analyzed.
  • changes include, but are not limited to, DNA mutations, DNA methylation changes, gene knockouts, gene knock-ins, transgenes, RNA expression changes, RNA silencing, microRNA expression changes, long non-coding RNA expression changes, 16s rDNA changes, mRNA expression change, ribose Changes in RNA expression, RNA conformational changes, DNA conformational changes, changes in DNA regulatory elements, chromosomal abnormalities, chromosomal deletions, chromosomal duplication, chromosomal ectopic, chromosome conformational changes, CNV, protein expression changes, antigen-antibody changes, secreted protein changes, membranes Protein changes.
  • the relationship between the chromatin conformation of a cell and a specific gene, DNA, RNA, and protein can be studied, and the interaction between a specific protein in the cell and DNA and RNA in the nucleus can be analyzed.
  • RNA sequencing it is possible to study cell characteristics or functions, as well as chromatin conformation, DNA, RNA and protein functions in combination with other methods for studying DNA, RNA, and proteins.
  • Other methods for studying DNA, RNA, and protein include, but are not limited to, gene chip, QPCR, first generation sequencing, second generation sequencing, third generation sequencing, fourth generation sequencing, gene sequencing, genome sequencing, metagenomic sequencing, exon sequencing, and Sequencing, target gene capture sequencing, RNA sequencing, expression profiling, transcriptome sequencing, small RNA transcriptome, microRNA sequencing, macro transcriptome sequencing, LncRNA sequencing, tumor gene sequencing, tumor genome sequencing, Bisulfite methylation sequencing , ChIP-DNA sequencing, MeDIP sequencing, RRBS sequencing, Target-BS sequencing, hmC sequencing.

Abstract

本发明提供一种构建高分辨率、大信息量单细胞Hi-C文库的方法，包括下述步骤：步骤B：获得少量的被固定化的染色质；步骤C：对所述步骤B中获得的被固定化的染色质进行消化，得到被固定化的染色质片段；步骤D：将所述步骤C中得到的被固定化的染色质片段直接进行重新连接，得到重新连接的被固定化的染色质片段；步骤E：使所述步骤D中得到的重新连接的被固定化的染色质片段解除固定化，释放DNA片段；步骤F：对所述步骤E中释放的DNA片段进行扩增，得到扩增产物；以及步骤H：以所述扩增产物作为待测序DNA片段，构建测序用DNA文库。

Description

构建高分辨率、大信息量单细胞Hi-C文库的方法 技术领域
本发明涉及一种可在全基因组范围内对染色质三维构象进行捕获的文库构建方法，属于基因测序技术领域。
背景技术
DNA是细胞遗传信息的载体，在生物体内以染色质的形式存在于每个细胞中，并控制着整个生命活动的进程。目前绝大多数对于DNA信息的研究是以研究DNA分子内碱基的序列(DNA的一维信息)来进行，通过分析碱基排列信息来探究生命活动的规律。
真实状态中的细胞核是一个狭小的三维立体空间，直链分子结构的DNA会以复杂的卷曲方式位于细胞核内，原一维DNA序列被赋予三维空间构象，并导致了大量复杂的基因调控作用方式。对此，简单的一维DNA序列信息由于不能提供真实DNA空间分布相关的信息，因此也无法解释由于空间构象导致的一系列基因调控现象。
为解决这一问题，目前已有一系列的检测方法。如3c(染色体结构捕获)方法及衍生的4c、5c方法。这些方法均以测序为基本检测手段，利用细胞核内蛋白形成DNA结构固定因子，之后通过对DNA的片段重联等构建带有空间结构信息的DNA序列，最后利用测序技术来检测染色质DNA信息，并计算其在空间中分布和相互作用。尽管此类方法在一定程度上能够提供部分染色质相互作用信息，但由于其方法与技术限制，这一类的方法仅能检测定点或部分的DNA相互作用位点，无法探究全细胞核水平上的立体互作信息。因此不可避免地大量信息将会遗漏。在对于未知相互作用信息的发现上，这一点尤为重要。
近年来随着高通量测序技术的出现，大规模基因组信息的获得变得更加容易。Hi-C技术便是结合高通量测序的方法，对整个细胞核内染色质的信息进行检测的技术。Hi-C技术是染色体构象捕获(Chromosome conformation capture，简称为3C)的一种衍生技术，是指基于高通量测序进行染色体构象的捕获，它能够在全基因组范围内捕捉不同基因座位之间的空间交互，研究三维空间中调控基因的DNA元件。
例如，专利文献1和非专利文献1报导了一种Hi-C方法，该方法利用甲醛固定染色质结构，然后通过限制性内切酶打断原基因组序列，并进行生物素标记后，再重新连接形成带有结构信息的新DNA分子。在这一过程中，如果两个不同基因组位置的DNA分子片段连接形成一个杂合分子，这将被认为是这两个DNA分子在空间上相互临近的证据。然后对DNA进行纯化并打碎，再针对标记的生物素分子进行钓取，富集获得所需的空间上相互作用的DNA杂合分子。最后构建高通量测序的文库及通过双端测序检测，获得全染色质在空间上的相互作用信息。该方法主要包括下述步骤：1)首先对106个以上的细胞样本进行甲 醛交联固定，使其内部空间距离较近的DNA通过蛋白交联在一起，并收集细胞；2)使用裂解体系并配合研磨进行细胞裂解，获得分离的细胞核；3)使用限制型内切酶(如EcoR I)对交联后细胞的染色质进行酶切；4)对酶切末端进行标记生物素，并形成平末端；5)使用DNA连接酶对平末端进行连接，处于同一个交联分子上的DNA片段间将有较大的概率连接形成新的分子；6)高温(65℃)处理逆转交联，释放双链DNA分子；7)去除未连接的末端生物素标记；8)对DNA进行片段化，并进行生物素特异性调取，富集杂合分子连接位点区域；以及9)构建Illumina测序文库，进行双端测序，获得数据。
但是，该方法以106个细胞水平以上的大量样本起始，整个流程中使用了大规模的研磨及提取方法，并不适用于小量样本(105个细胞以下)、特别是单细胞水平的Hi-C检测。此外，由于该方法针对大量细胞样本，只能检测群体细胞总的染色质构象，无法对细胞染色质构象个体间的区别进行检测和比较。
另一方面，非专利文献2报导了一种单细胞核Hi-C方法，但该方法起始样本依然为106数量级的大量细胞样本。前期处理利用甲醛固定染色质结构，然后通过限制性内切酶打断原基因组序列，并进行生物素标记后，再重新连接形成带有结构信息的新DNA分子。在这一过程中，如果两个不同基因组位置的DNA分子片段连接形成一个杂合分子，这将被认为是这两个DNA分子在空间上相互临近的证据。接下来，对经过前处理的细胞核在显微镜下进行挑选，获得单个的细胞核，再针对单个细胞核样本进行交联去除、生物素钓取及限制性内切酶对DNA分子进行片段化，富集获得所需的空间上相互作用的DNA杂合分子。最后构建高通量测序的文库及通过双端测序检测，获得全染色质在空间上的相互作用信息。该方法主要包括下述步骤：1)首先对106个以上的细胞样本进行甲醛交联固定，使其内部空间距离较近的DNA通过蛋白交联在一起，并收集细胞；2)使用裂解体系并配合研磨进行细胞裂解，获得分离的细胞核；3)使用限制型内切酶(如EcoR I)对交联后细胞的染色质进行酶切；4)对酶切末端进行生物素标记，并形成平末端；5)使用DNA连接酶对平末端进行连接，处于同一个交联分子上的DNA片段间将有较大的概率连接形成新的分子；6)在显微镜下进行单细胞核的挑取获得经过Hi-C化处理的单细胞核样本；7)对单细胞核样本进行高温去除甲醛交联，释放DNA；8)对DNA分子进行生物素特异性调取，富集杂合分子连接位点区域；9)使用第二种限制性内切酶对DNA进行片段化，使其适应于Illumina测序文库插入片段大小；10)以结合生物素标记片段(空间上相互作用片段的杂合分子)的磁珠为载体，构建Illumina测序文库，进行双端测序，获得数据。
尽管此方法在后期的去交联及文库构建阶段开始针对单细胞核进行操作，并且最终获得的结果为单细胞水平的Hi-C结果，但是在处理初期，由于需要依靠大体积的甲醛交联处理及研磨破碎细胞等手段获取细胞核，因此该方法仍然需要106个细胞以上的大量样本作为起始进行Hi-C化处理，因此并未真正实现针对单细胞样本起始水平的Hi-C检测，仍然无法适用于小量样本(105个细胞以下)、特别是单细胞样本。再者，由于该方法在文库扩增之前要经历酶切、末端修复、生物素标记、平末端连接、生物素调取、加接头等多个步骤，而这些步骤都是在一套基因组拷贝的情况下操作的，每一步骤的效率都会影响到最后捕获 的信息量，导致整个实验过程中信息损失严重，最终捕获到的DNA互作片段数目也很低。此外，由于该方法采用两种限制性核酸酶分别进行染色质消化和重连产物片段化。为了保证重连产物片段化的效率，该步骤所用内切酶在基因组中出现的频率要远高于染色质消化所用内切酶出现的频率。这个原因导致该方法无法使用基因组中高频率出现的四碱基酶进行染色质消化，最终导致所得Hi-C文库数据分辨率较低。非专利文献2中也尝试过使用基因组中高频出现的四碱基酶进行染色质消化，但由于后面的重连产物片段化无更高频的内切酶可用，无法提高分辨率和信息量。而且，该方法对单细胞进行研究的方案是挑取单细胞核，其操作难度和对仪器、技术的要求远高于挑取单细胞。
因此，上述现有技术中的Hi-C方法均不是能够适用于单细胞或少量细胞的、实用化的Hi-C方法。
专利文献1
国际公开号WO2010036323A1
非专利文献1
Lieberman-Aiden E et al.Comprehensive mapping of long-range interactions reveals folding principles of the human genome.Science 326，289-293(2009)
非专利文献2
Takashi Nagano et al.Single-cell Hi-C reveals cell-to-cell variability in chromosome structure.Nature 502，59-64(2013)
发明内容
鉴于上述现有技术中存在的不足，本发明的目的在于：提供一种能够适用于少量染色质(少量细胞甚至是单细胞的染色质)的构建Hi-C文库的方法。
本发明人为解决上述技术问题进行了深入研究，在传统的构建Hi-C文库的方法的基础上进行了巧妙的改进，首创了适用于少量染色质的Hi-C文库构建方法，从而完成了本发明。
即，本发明包括：
1.一种构建Hi-C文库的方法，该方法包括下述步骤：
步骤B：获得少量的被固定化的染色质；
步骤C：对所述步骤B中获得的被固定化的染色质进行消化，得到被固定化的染色质片段；
步骤D：将所述步骤C中得到的被固定化的染色质片段直接进行重新连接，得到重新连接的被固定化的染色质片段；
步骤E：使所述步骤D中得到的重新连接的被固定化的染色质片段解除固定化，释放DNA片段；
步骤F：对所述步骤E中释放的DNA片段进行扩增，得到扩增产物；以及
步骤H：以所述扩增产物作为待测序DNA片段，构建测序用DNA文库。
2.根据项1所述的方法，其中，所述少量的被固定化的染色质是10-6～102ng的染色质，以裸DNA计。
3.根据项1或2所述的方法，其中，所述少量的被固定化的染色质是10-5～10ng的染色质，以裸DNA计。
4.根据项1～3中任一项所述的方法，其中，所述步骤C中使用脱氧核糖核酸酶对所述被固定化的染色质进行消化。
5.根据项4所述的方法，其中，所述脱氧核糖核酸酶是I型限制性内切酶、II型限制性内切酶或III型限制性内切酶。
6.根据项1～5中任一项所述的方法，其中，所述步骤D中采用粘末端或平末端连接方法将所述步骤C中得到的被固定化的染色质片段进行重新连接。
7.根据项1～6中任一项所述的方法，其还包括：
步骤G：将所述步骤F中得到的扩增产物片段化，得到更小的DNA片段；且
所述步骤H中，以所述步骤G中得到的更小的DNA片段作为待测序DNA片段，构建测序用DNA文库。
8.根据项7所述的方法，其中，所述步骤G中采用超声打断法、转座酶法、内切酶酶切法或液压剪切法将所述扩增产物片段化。
9.根据项8所述的方法，其中，所述步骤G中得到的更小的DNA片段的大小为50～1000bp。
10.根据项1～9中任一项所述的方法，其还包括：
步骤A：获取少量的染色质被固定化的细胞；且
在所述步骤B中，裂解所述步骤A中获取的细胞，获得少量的被固定化的染色质。
11.根据项10所述的方法，其中，所述少量的染色质被固定化的细胞是1～10000个细胞。
12.根据项10所述的方法，其中，所述少量的染色质被固定化的细胞是1～1000个细胞。
13.根据项10所述的方法，其中，所述少量的染色质被固定化的细胞是单个细胞。
14.根据项10所述的方法，其中，所述步骤A包括：
步骤A-1：对一定量的细胞的染色质进行固定化，得到一定量的染色质被固定化的细胞；以及
步骤A-2：从所述步骤A-1得到的一定量的染色质被固定化的细胞中挑取少量的染色质被固定化的细胞。
15.根据项10所述的方法，其中，所述步骤A包括：
步骤A-3：对少量的细胞的染色质进行固定化，得到少量的染色质被固定化的细胞。
16.一种测定可能在空间上相互作用的染色质区域的方法，该方法包括：
采用项1～15中任一项所述的方法构建Hi-C文库；以及
对所述Hi-C文库的全部或部分进行测序，并将所获得信息与染色质DNA一级序列信息进行比对。
发明效果
根据本发明，提供了一种起始样本量少、分辨率高、信息量大、操作简便的构建Hi-C文库的方法。
针对现有技术无法处理微量样本和单细胞样本的问题，本发明从最起始的步骤便可针对单一细胞或微量样本(1-1000个细胞)进行操作，解决了小样本量细胞进行Hi-C检测的难题。
针对现有技术无法检测单细胞染色质构象的问题，本发明可针对单个细胞进行Hi-C分析，检测和研究单个细胞的染色质构象以及不同细胞间染色质构象的区别。
针对现有技术中DNA相互作用信息损失较大的问题，本发明摒弃了酶切后的末端修复、生物素标记和调取等步骤，而且可以采用连接效率较高的粘末端连接来代替连接效率较低的平末端连接，因而降低了信息量的损失，可以捕获到更多的染色质构象信息。
针对现有技术获得染色质构象分辨率较低的问题，本发明可在文库片段化时采用随机打断的方法，其片段化效果远高于限制性内切酶酶切。这一操作使得染色质消化时可选用更高分辨率的限制性内切酶如识别4碱基序列的限制性内切酶，从而能够提高染色质构象分辨率。
针对现有技术中挑取单细胞核操作困难的问题，本发明可以挑取单个细胞进行文库构建，其难度小于挑取单个细胞核。
发明的具体实施方式
本说明书中提及的科技术语具有与本领域技术人员通常理解的含义相同的含义，如有冲突以本说明书中的定义为准。
在一个方面中，本发明提供一种构建Hi-C文库的方法(本发明的方法)，该方法包括下述步骤：
步骤B：获得少量的被固定化的染色质；
步骤C：对所述步骤B中获得的被固定化的染色质进行消化，得到被固定化的染色质片段；
步骤D：将所述步骤C中得到的被固定化的染色质片段直接进行重新连接，得到重新连接的被固定化的染色质片段；
步骤E：使所述步骤D中得到的重新连接的被固定化的染色质片段解除固定化，释放DNA片段；
步骤F：对所述步骤E中释放的DNA片段进行扩增，得到扩增产物；以及
步骤H：以所述扩增产物作为待测序DNA片段，构建测序用DNA文库。
优选地，本发明的方法还包括：
步骤G：将所述步骤F中得到的扩增产物片段化，得到更小的DNA片段；且所述步骤H中，以所 述步骤G中得到的更小的DNA片段作为待测序DNA片段，构建测序用DNA文库。
本发明的方法还包括：
步骤A：获取少量的染色质被固定化的细胞；且在所述步骤B中，裂解所述步骤A中获取的细胞，获得少量的被固定化的染色质。
对于步骤A中获取少量的染色质被固定化的细胞的方式没有特殊限制，例如，可以首先对一定量的细胞的染色质进行固定化，得到一定量的染色质被固定化的细胞，然后从得到的一定量的染色质被固定化的细胞中挑取少量的染色质被固定化的细胞；或者可以直接对少量的细胞的染色质进行固定化，得到少量的染色质被固定化的细胞。需要说明的是，这里的细胞也可是细胞核。
在本说明书中，Hi-C是指染色质三维空间互作组，它是一种可以在全基因组范围内进行染色质空间构象捕获，研究染色质的三维结构和不同DNA区域在空间上相互关系的方法。Hi-C文库是指：Hi-C方法中通过高通量测序来获取可能的染色质相互作用信息，用于这种高通量测序的DNA文库即为Hi-C文库。
在本说明书中，“少量的染色质”是指少至现有技术的Hi-C方法无法进行操作的量的染色质，通常可以指1～10000个细胞的染色质，或者1～1000个细胞的染色质，或者1～100个细胞的染色质，甚至是1个细胞(单细胞)的染色质，再甚至是1个细胞(单细胞)的染色质的一部分。如果以质量计，所述“少量的染色质”可以是10-6～102ng，优选是10-5～10ng，以裸DNA计。
在本说明书中，“少量的细胞”是指少至现有技术的Hi-C方法无法进行操作的数量的细胞，通常可以指1～10000个细胞，或者1～1000个细胞，或者1～100个细胞，甚至是1个细胞(单细胞)。
在本说明书中，“固定化”是指：细胞中，染色质在三维空间上相互接近的部分被以接近天然构象的状态被固定。在本说明书中，染色质也包括染色体形态。所述固定化通常可以通过将染色质上的蛋白质交联来进行。染色质上的蛋白质的交联方法是本领域技术人员已知的，例如，可以单独使用紫外线，或者可以单独使用四硝基甲烷、碳二酰亚胺类、甲醛、甲醇、乙醇、戊醛、氮芥、二甲基硫酸、甲醛释放剂、酰亚胺酯类、有丝裂酶素C、芥子气和扫若仑等化学试剂，还可以采用以上化学试剂结合紫外线照射的方法进行交联。例如，在通过甲醛交联来进行所述固定化的情况下，可以将细胞置于适当量(例如1～10000000μL)的水、TE缓冲液、生理盐水、PBS或细胞培养基中制成细胞悬浮液滴，再加入适当量(例如1-10000000μL)的甲醛溶液(对其浓度没有限制，例如可以是1～20重量％)，室温静置一定时间(例如1-100min)，进行交联。然后，向上述反应液滴中加入一定量的氨基酸(一种氨基酸或多种氨基酸的混合物)或蛋白质((例如BSA等)来终止交联反应。
在所述步骤A中，可以先对一定量(例如105个以上，优选106～108个)的细胞的染色质进行固定化，得到一定量的染色质被固定化的细胞，然后再从中挑取少量的染色质被固定化的细胞；也可以直接对少量的细胞的染色质进行固定化，从而得到少量的染色质被固定化的细胞。所述少量细胞的挑取可以通过毛细管法、稀释法、梯度稀释法或流式细胞仪分选法来进行。
在所述步骤B中，对所述步骤A中获取的细胞进行裂解，从而得到被固定化的染色质。细胞裂解通常可以通过将所述细胞置于适当的细胞裂解液中来进行。所述细胞裂解液的配方及用量可由本领域技术人员根据所述细胞的种类及量适宜确定。
在所述步骤C中，对所述步骤B中获得的被固定化的染色质进行消化，得到被固定化的染色质片段。所述消化可以使用脱氧核糖核酸酶来进行。作为所述脱氧核糖核酸酶优选I型限制性内切酶、II型限制性内切酶或III型限制性内切酶。在非专利文献2所记载的Hi-C方法中，因为后续步骤中还要使用识别4碱基序列的限制性内切酶进行DNA片段化，所以只能使用识别6碱基序列的限制性内切酶对染色质进行消化。这种消化方式的分辨率低，保留的信息量小。另一方面，在本发明的方法中，因为后续步骤中可以采用超声打断法、转座酶法、液压剪切法等方法进行DNA片段化，所以在该步骤C中可以使用分辨率更高的识别4碱基序列的限制性内切酶或其他核酸酶对染色质进行消化，这使得理论上分辨率提高了十数倍，可获得的染色质互作信息量大幅增加。当然，在步骤C中同样可以使用识别6碱基序列的限制性内切酶进行染色质消化。
在所述步骤D中，将所述步骤C中得到的被固定化的染色质片段直接进行重新连接，得到重新连接的被固定化的染色质片段。在这里，“直接进行重新连接”是指：不对所述被固定化的染色质片段进行生物素标记，而将这些片段重新连接起来。此外，在所述步骤C中使用粘性末端限制性内切酶进行染色质消化时，得到的被固定化的染色质片段具有粘性末端，优选地，本发明的方法中不像现有技术那样对这些粘性末端进行修复使之成为平末端，而是采用粘性末端连接方式将这些染色质片段重新连接起来，这样效率要高于平末端连接。当然，本方法也可以采用修复后进行平末端连接(但不进行连接点标记如加入带生物素标记的核苷酸修复粘末端)或者采用平末端限制性内切酶酶切然后进行平末端连接。末端连接可以例如通过使用具有末端连接活性的DNA连接酶来进行，所述具有末端连接活性的DNA连接酶例如T4 DNA连接酶、T3 DNA连接酶、大肠杆菌DNA连接酶、热稳定DNA连接酶等。连接反应中使用的酶和底物的量、以及反应条件可由本领域技术人员视需要适宜选择。例如，通常可以在0.1～10×连接酶缓冲液中于0～80℃(优选10～40℃)进行约1分钟～200小时(优选1～30小时)。
在所述步骤E中，使所述步骤D中得到的重新连接的被固定化的染色质片段解除固定化，释放DNA片段。在本说明书中，“解除固定化”是指：解除所述被固定化的染色质片段中、在三维空间上相互接近的部分的被固定状态。例如，在所述固定化是通过将染色质上的蛋白质交联而实现的情况下，所述“解除固定化”是指蛋白质去交联。蛋白质去交联的方法是本领域技术人员已知的，通常可以采用生物、化学处理进行解交联的方法和/或高温处理解交联的方法，从而释放DNA片段。例如，作为高温处理解交联的方法，可以通过将上述连接反应后的体系置于50～100℃(优选60～80℃)、1分钟～200小时(优选1～30小时)来进行蛋白质解交联。作为生物、化学处理解交联的方法，可以通过向所述体系中加入内肽酶、丝氨酸蛋白酶、巯基蛋白酶、金属蛋白酶、天冬氨酸蛋白酶、胃蛋白酶、胰蛋白酶、组织蛋白酶、木瓜蛋白酶、枯 草杆菌蛋白酶、蛋白酶K、DTT、NaCl、KCl或它们的组合来进行。当然，也可以通过组合使用生物、化学处理解交联的方法和高温处理解交联的方法来进行蛋白质解交联。此外，在下述步骤F的DNA片段扩增步骤中存在高温处理步骤的情况下，所述步骤E也可以与步骤F一并进行。
在步骤F中，对所述步骤E中释放的DNA片段进行扩增，得到扩增产物。对于扩增方法没有特殊限制，只要能够获得对构建测序用DNA文库而言为充足量(例如0.001～1000ng)的扩增产物即可。例如，可以采用MDA、MALBAC、NEB-WGA、GenomePlex(优选MALBAC)等适合少量细胞、单细胞或微量DNA的扩增方法，这些扩增方法的具体条件可由本领域技术人员视需要适宜选择。上述扩增方法通常可基于PCR反应(聚合酶链反应)来进行，所述PCR反应一般通过一定的PCR反应程序(温度循环)来实现。所述PCR反应程序一般包括变性、退火、延伸等步骤。PCR反应中使用的引物的设计是本领域技术人员熟知的，例如可以按照《分子克隆实验指南》(J.萨姆布鲁克(Sambrook.J.)等著，黄培堂等译，第3版，2005)的教导来进行设计，或者利用计算机软件(例如Premier公司开发的Primer Premier 6.0)来进行设计。
在所述步骤G中，将所述步骤F中得到的扩增产物片段化，得到更小的DNA片段。在本说明书中，“更小的DNA片段”是指其大小适于构建测序(例如二代测序、三代测序或四代测序)用DNA文库，例如Illumina DNA测序文库。“更小的DNA片段”的具体大小可以是例如10～50000bp、优选50～1000bp。在非专利文献2所记载的Hi-C方法中，只适合使用识别4碱基序列的限制性内切酶进行扩增产物片段化，但是，在本发明的方法中，还可以采用超声打断法、转座酶法、液压剪切法将所述扩增产物片段化。这提高了分辨率，能够获得更多的信息。采用上述方法将扩增产物片段化的技术是本领域技术人员已知的，可视需要选择适宜条件来进行。
在所述步骤H中，以所述步骤G中得到的更小的DNA片段作为待测序DNA片段，构建测序用DNA文库。可以采用例如标准Illumina DNA小片段建库方法、PCR free方法、一步法等DNA小片段建库方法来构建所述测序用DNA文库。各种构建测序用DNA文库的方法是本领域技术人员已知的，可以由本领域技术人员按照常规操作来进行。例如，标准Illumina DNA小片段建库方法通常包括末端修复、末端加A、Adapter连接、扩增、扩增产物纯化等步骤，可以按照Illumina公司推荐的方法来进行。
需要说明的是，在上述步骤C～G中均可以视需要取一部分产物进行下一步骤。
通过对采用本发明的方法构建的测序用DNA文库的全部或部分进行测序，再与染色质DNA一级序列信息进行比对，即可获得可能在空间上相互作用的染色质区域的信息。
实施例
以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例是用于解释本发明，并非对本发明的限定。
实施例1
1.样本处理
1.1交联
1.1.1将1-10个人白细胞样本置于10μL的PBS中制成细胞悬浮液滴；
1.1.2加入10μL 4％的甲醛溶液进行交联，室温静置5min；
1.1.3向反应液滴中加入5μL浓度为0.25M的甘氨酸溶液终止交联。在室温下静置5min，然后冰浴15min。
1.2单细胞(少量细胞)挑取
采用毛细管法挑出单个细胞或少量细胞置于裂解液中。
裂解液配方：
10mM Tris-HCl pH 7.4
30mM NaCl
0.2％NP-40
10％protease inhibitor cocktail
无菌水
2.细胞裂解
2.1将含有单细胞样本的细胞裂解缓冲液微离心将液体汇聚于管底，之后冰浴45min；
2.2加入4％的SDS溶液使SDS终浓度为0.35％，37℃温浴60min；
2.3加入20％的Triton X-100溶液使Triton X-100终浓度为3％，37℃温浴60min。
3.染色质消化
3.1对每个样品，加入8U(国际单位)的Mbo I，以及终浓度为1×的NEBuffer 3.1缓冲液。37℃温浴16小时。
3.2失活内切酶。65℃，温浴20min。
4.DNA重连和DNA、蛋白解交联
4.1对每个样品，加入8U(国际单位)T4 DNA连接酶以及终浓度为1x的连接酶缓冲液。16℃温浴16小时。
4.2 65℃温浴16小时。
5.DNA扩增
使用基于MALBAC方法的亿康单细胞全基因组扩增试剂盒，按照试剂盒说明书、对前面得到的样品进行DNA扩增，获得重连DNA的扩增产物。
6.DNA片段化
使用Diagenode Bioruptor UCD-600(NGS)对扩增产物进行超声打断，打断程序为：30秒超声，30秒休息，22个循环。将前面扩增的DNA片段化成片段大小在100-700bp之间的DNA片段。
7.小片段文库构建
7.1末端修复
根据下表向上步产物中加入加末端修复反应体系：
Figure PCTCN2015092181-appb-000001
将样本置于Thermomixer中20℃温浴30min。反应结束后使用Beckman Agencourt AMPure XP核酸纯化试剂盒回收纯化反应体系中的DNA，溶于32μL的水中。
7.2末端加“A”
根据下表向上步产物中加入加“A”反应体系：
Figure PCTCN2015092181-appb-000002
将样本置于Thermomixer中37℃温浴30min。使用Beckman Agencourt AMPure XP核酸纯化试剂盒回收纯化反应体系中的DNA，溶于18μL的水中。
7.3“Adapter”连接：
根据下表向上步产物中加入“Adapter”反应体系：
Figure PCTCN2015092181-appb-000003
Figure PCTCN2015092181-appb-000004
将样本置于Thermomixer中20℃温浴15min。使用Beckman Agencourt AMPure XP核酸纯化试剂盒回收纯化反应体系中的DNA，溶于30μL的水中。
7.4文库扩增
根据下表向上步产物中加入以下反应体系：
Figure PCTCN2015092181-appb-000005
PCR反应的程序设定如下：
Figure PCTCN2015092181-appb-000006
使用Beckman Agencourt AMPure XP核酸纯化试剂盒进行纯化；15μL水溶解；纯化后测定DNA浓度。
7.5文库质检，上机进行双端测序，获得数据
与非专利文献1和2不同，本发明不使用生物素钓取重连位点DNA片段，而是利用信息学分析过滤掉非重连位点所处的片段。由于标准的Hi-C测序数据分析方法中即有对这种片段的过滤(该过滤条件在非专利文献1和2中是为了去除文库中残余的被生物素标记的未重连片段)，所以本发明产生的文库无需额外的过滤条件即可进行生物信息学分析并获得染色体在三维空间上的互作信息。
7.6获得的信息量及与现有文献中的单细胞核Hi-C对比
本发明使用Mbo I(识别位点是GATC但对Dam、Dcm和CpG甲基化敏感)进行染色质消化，在14M-19M双端测序reads对的测序量下，捕获到染色质区域间的相互作用对数为270-331K。与之相对比，非专利文献2的单细胞核Hi-C中使用Dpn II(识别位点也是GATC但对Dam、Dcm甲基化敏感，也就是它的识别位点数理应高于Mbo I，因此理论上应该获得更多的酶切片段和染色质区域互作信息)进行染色 质消化，在13M双端测序reads对的测序量下只能捕获到12K的染色质区域间的相互作用个数；非专利文献2中其余单细胞核采用BglII内切酶进行染色质消化，在5.5M-15.3M双端测序reads对的测序量下捕获到染色质区域间的相互作用个数为11.7k-30.6k。采用本发明的获得的染色质构象信息显著高于非专利文献2的单细胞核Hi-C的方法。
Figure PCTCN2015092181-appb-000007
还需要说明的是，在可实施且不明显违背本发明的主旨的前提下，在本说明书中作为某一技术方案的构成部分所描述的任一技术特征或技术特征的组合同样也可以适用于其它技术方案；并且，在可实施且不明显违背本发明的主旨的前提下，作为不同技术方案的构成部分所描述的技术特征之间也可以以任意方式进行组合，来构成其它技术方案。本发明也包含在上述情况下通过组合而得到的技术方案，并且这些技术方案相当于记载在本说明书中。
上述说明示出并描述了本发明的优选实施例，如前所述，应当理解本发明并非局限于本文所披露的形式，不应看作是对其他实施例的排除，而可用于各种其他组合、修改和环境，并能够在本文所述发明构想范围内，通过上述教导或相关领域的技术或知识进行改动。而本领域技术人员所进行的改动和变化不脱离本发明的精神和范围，则都应在本发明所附权利要求的保护范围内。
工业实用性
根据本发明，可以对少量细胞的样本进行染色质构象的检测。细胞类型包括但不限于：动物细胞、植物细胞、微生物细胞、病毒、癌细胞；这些细胞来源包括但不限于：原代培养、细胞系培养、组织、生物体、环境来源、化石。
根据本发明，可以比较不同细胞之间染色体构象的区别。这些细胞之间的区别包括但不限于：不同物种来源、不同器官来源、不同细胞种类、不同细胞周期、不同发育阶段、不同培养条件、不同处理条件、不同细胞个体。
根据本发明，可以分析细胞内染色质构象变化与其他DNA、RNA、蛋白质的变化之间的联系。这些变化包括但不限于以下方面：DNA突变、DNA甲基化变化、基因敲除、基因敲入、转基因、RNA表达变化、RNA沉默、microRNA表达变化、长非编码RNA表达变化，16s rDNA变化、mRNA表达变化、核糖 体RNA表达变化、RNA构象变化、DNA构象变化、DNA调控元件变化、染色体异常、染色体缺失、染色体重复、染色体异位、染色体构象变化、CNV、蛋白表达变化、抗原抗体变化、分泌蛋白变化、膜蛋白变化。
根据本发明，可以研究细胞染色质构象与特定基因、DNA、RNA、蛋白之间的关系，分析细胞内特定蛋白与细胞核内DNA、RNA之间的相互作用。
根据本发明，可以与其他研究DNA、RNA、蛋白的方法联合使用研究细胞特性或功能，以及染色质构象、DNA、RNA和蛋白质的功能。这些其他研究DNA、RNA、蛋白的方法包括但不限于：基因芯片、QPCR、一代测序、二代测序、三代测序、四代测序、基因测序、基因组测序、宏基因组测序、外显子测序、内含子测序、目标基因捕获测序、RNA测序、表达谱测序、转录组测序、小RNA转录组、微RNA测序、宏转录组测序、LncRNA测序、肿瘤基因测序、肿瘤基因组测序、Bisulfite甲基化测序、ChIP-DNA测序、MeDIP测序、RRBS测序、Target-BS测序、hmC测序。

Claims (16)

  1. 一种构建Hi-C文库的方法，该方法包括下述步骤：
    步骤B：获得少量的被固定化的染色质；
    步骤C：对所述步骤B中获得的被固定化的染色质进行消化，得到被固定化的染色质片段；
    步骤D：将所述步骤C中得到的被固定化的染色质片段直接进行重新连接，得到重新连接的被固定化的染色质片段；
    步骤E：使所述步骤D中得到的重新连接的被固定化的染色质片段解除固定化，释放DNA片段；
    步骤F：对所述步骤E中释放的DNA片段进行扩增，得到扩增产物；以及
    步骤H：以所述扩增产物作为待测序DNA片段，构建测序用DNA文库。
  2. 根据权利要求1所述的方法，其中，所述少量的被固定化的染色质是10-6～102ng的染色质，以裸DNA计。
  3. 根据权利要求1所述的方法，其中，所述少量的被固定化的染色质是10-5～10ng的染色质，以裸DNA计。
  4. 根据权利要求1所述的方法，其中，所述步骤C中使用脱氧核糖核酸酶对所述被固定化的染色质进行消化。
  5. 根据权利要求4所述的方法，其中，所述脱氧核糖核酸酶是I型限制性内切酶、II型限制性内切酶或III型限制性内切酶。
  6. 根据权利要求1所述的方法，其中，所述步骤D中采用粘末端或平末端连接方法将所述步骤C中得到的被固定化的染色质片段进行重新连接。
  7. 根据权利要求1所述的方法，其还包括：
    步骤G：将所述步骤F中得到的扩增产物片段化，得到更小的DNA片段；且
    所述步骤H中，以所述步骤G中得到的更小的DNA片段作为待测序DNA片段，构建测序用DNA文库。
  8. 根据权利要求7所述的方法，其中，所述步骤G中采用超声打断法、转座酶法、内切酶酶切法或液压剪切法将所述扩增产物片段化。
  9. 根据权利要求8所述的方法，其中，所述步骤G中得到的更小的DNA片段的大小为50～1000bp。
  10. 根据权利要求1所述的方法，其还包括：
    步骤A：获取少量的染色质被固定化的细胞；且
    在所述步骤B中，裂解所述步骤A中获取的细胞，获得少量的被固定化的染色质。
  11. 根据权利要求10所述的方法，其中，所述少量的染色质被固定化的细胞是1～10000个细胞。
  12. 根据权利要求10所述的方法，其中，所述少量的染色质被固定化的细胞是1～1000个细胞。
  13. 根据权利要求10所述的方法，其中，所述少量的染色质被固定化的细胞是单个细胞。
  14. 根据权利要求10所述的方法，其中，所述步骤A包括：
    步骤A-1：对一定量的细胞的染色质进行固定化，得到一定量的染色质被固定化的细胞；以及
    步骤A-2：从所述步骤A-1得到的一定量的染色质被固定化的细胞中挑取少量的染色质被固定化的细胞。
  15. 根据权利要求10所述的方法，其中，所述步骤A包括：
    步骤A-3：对少量的细胞的染色质进行固定化，得到少量的染色质被固定化的细胞。
  16. 一种测定可能在空间上相互作用的染色质区域的方法，该方法包括：
    采用权利要求1～15中任一项所述的方法构建Hi-C文库；以及
    对所述Hi-C文库的全部或部分进行测序，并将所获得信息与染色质DNA一级序列信息进行比对。
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