WO2018103025A1 - 单细胞测序文库的构建方法及其应用 - Google Patents

单细胞测序文库的构建方法及其应用 Download PDF

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WO2018103025A1
WO2018103025A1 PCT/CN2016/108940 CN2016108940W WO2018103025A1 WO 2018103025 A1 WO2018103025 A1 WO 2018103025A1 CN 2016108940 W CN2016108940 W CN 2016108940W WO 2018103025 A1 WO2018103025 A1 WO 2018103025A1
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amplification
chromatin
library
fragmentation
dna
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PCT/CN2016/108940
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French (fr)
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刘龙奇
刘传宇
吴亮
商周春
成梦南
袁月
徐礼钦
刘心
徐讯
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深圳华大智造科技有限公司
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Priority to CN201680091050.2A priority Critical patent/CN109996892B/zh
Priority to PCT/CN2016/108940 priority patent/WO2018103025A1/zh
Priority to US16/466,741 priority patent/US11434483B2/en
Priority to DK16923153.7T priority patent/DK3553180T3/da
Priority to ES16923153T priority patent/ES2922281T3/es
Priority to EP16923153.7A priority patent/EP3553180B1/en
Publication of WO2018103025A1 publication Critical patent/WO2018103025A1/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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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    • 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
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention relates to the field of high-throughput sequencing. Specifically, the present invention relates to a method for constructing a single-cell sequencing library, and a sequencing method and application based on Single Cell Multi-Omics Sequencing (SCMOS).
  • SCMOS Single Cell Multi-Omics Sequencing
  • Single-cell sequencing technology is one of the most interesting sequencing technologies in recent years.
  • the single-cell sequencing technologies that have been published include single-cell DNA and DNA methylation sequencing, single-cell RNA sequencing, and single-cell chromatin accessibility. Chromatin accessibility) sequencing.
  • Fuchou Tang et al. report a single-cell RNA sequencing technique that involves: full lysis of a single cell by a strong lysate followed by polyadenosine in the tail. The RNA is subjected to reverse transcription and PCR amplification, and finally constructed and sequenced based on a specific sequencing platform.
  • Buenrostro et al. report a single-cell chromatin accessibility sequencing technique that includes a micro-scaled reaction system for 50,000 cells using microfluidic chip technology. Is proportionally reduced to a single-cell nanoscale reaction system; by lysis of single cells and Tn5 transposase treatment, a sequencing linker is inserted into the open region of chromatin, and then all open chromatin regions are performed by PCR amplification. Amplify and build and sequence.
  • the DNA in the remaining liquid is subjected to bisulfite treatment, and then the treated DNA is constructed.
  • Hou et al. (Cell Research 26: 304-319 (2016)) report another method for simultaneous sequencing of single-cell DNA methylation and RNA, which involves first splitting the cell membrane with a weak lysate to release the cytoplasm. But reserved Cell nucleus. Then, the cytoplasm is separated for RNA construction, and the nucleus is subjected to sub-sulfuric acid treatment, and further DNA construction is performed.
  • the reaction system is in the nano-upgrading reaction system of the microfluidic chip. This is not conducive to the efficient separation of chromatin and RNA, and therefore it is not possible to perform chromatin accessibility and transcriptome formation in a single cell.
  • the present invention provides a method for constructing a single cell sequencing library, a sequencing method based on Single Cell Multi-Omics Seqencing (SCMOS) and an application thereof.
  • SCMOS Single Cell Multi-Omics Seqencing
  • the invention is fully modified and optimized on the basis of the traditional single cell chromatin accessibility technology, and the single cell chromatin accessibility library can be effectively carried out in the micro-upgrading reaction system, and can be effectively separated.
  • Chromatin and RNA in turn, achieve chromatin accessibility and RNA simultaneous library construction, fully capture the chromatographic state and gene expression of the omics information.
  • the "single cell multi-omics" method provided by the present invention refers to separating the nucleus and cytoplasm of a single cell, and then performing chromatin accessibility construction on the nucleus, and at the same time, constructing a transcriptome for the total cytoplasm. For high-throughput sequencing and bioinformatics analysis.
  • the present invention provides a method of constructing a single-cell sequencing library capable of simultaneously compiling a chromatin accessibility and a transcriptome of a single cell, comprising the steps of:
  • step b) separating the nucleus and cytoplasm of the single cell lysate obtained in step a) to obtain a nuclear solution and a total RNA solution;
  • step c) performing chromatin DNA library construction on the nuclear solution obtained in step b) to obtain a chromatin-accessible sequencing library of the single cell; and performing transcriptome library construction on the total RNA solution obtained in step b) to obtain the single A transcriptome sequencing library of cells.
  • a single lysis system for lysing single cells comprises:
  • the rest is water.
  • the final concentration of NP-40 is from 0.1 to 0.3%, and the value includes any discrete value between 0.1 and 0.3%, such as 0.12%, 0.14%, 0.16. %, 0.18%, 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, and the like.
  • the final concentration of Tris-HCl, pH 7.5 is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the final concentration of NaCl is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the final concentration of the RNase inhibitor is 1-2 U/ ⁇ l, which includes any discrete value between 1-2 U/ ⁇ l, such as 1.1 U/ ⁇ l, 1.2 U/ ⁇ l, 1.3 U/ ⁇ l, 1.4 U/ ⁇ l, 1.5 U/ ⁇ l, 1.6 U/ ⁇ l, 1.7 U/ ⁇ l, 1.8 U/ ⁇ l, 1.9 U/ ⁇ l, and the like.
  • step c) chromatin DNA library construction is performed using Tn5 transposase or by DNase digestion.
  • the step of constructing the chromatin DNA library using the Tn5 transposase comprises:
  • C1 cleavage of an open region of chromatin using a Tn5 transposase comprising the steps of fragmenting chromatin DNA using a Tn5 transposase and terminating fragmentation after fragmentation;
  • a single fragmentation system for fragmenting chromatin DNA comprises:
  • the final concentration of Tris-HCl, pH 7.5 is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5. mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the final concentration of NaCl is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the following components are added to the single fragmentation system for fragmentation termination: EDTA, pH 8.0, 50-70 mM, preferably 60 mM; Tris-HCl, pH 8.0, 10-14 mM, preferably 12 mM The rest is water.
  • the final concentration of EDTA, pH 8.0 is 50-70 mM, which includes any discrete value between 50-70 mM, such as 52 mM, 55 mM, 58 mM, 60 mM. 63 mM, 65 mM, 68 mM, and the like.
  • Tris-HCl, pH 8.0 has a final concentration of 10-14 mM, and this value includes any discrete value between 10-14 mM, such as 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, and the like.
  • the step of constructing the chromatin DNA library using the Tn5 transposase further comprises: after step c1), subjecting the resulting product to a second cleavage; further preferably, using the RLT Plus buffer for a second time Crack solution;
  • the first lysis uses a weak lysate that lyses the cell membrane without affecting the nucleus (ie, the nucleus is intact), which facilitates the integrity of chromatin in the cell;
  • second lysis Using a strong lysate, the nucleus can be completely lysed and performed after the Tn5 completes the attack, which is beneficial to fully release the chromatin in the nucleus and achieve the most maximal chromatin effect in a single cell.
  • the carrier DNA is added to the system while the second cleavage is carried out; preferably, the carrier DNA is added in an amount of 4-6 ng/ ⁇ l system, preferably 5 ng/ ⁇ l system; one of ordinary skill in the art can It is understood that the final concentration of the above vector DNA is 4-6 ng/ ⁇ l system, and the value includes any discrete value between 4-6 ng/ ⁇ l system, for example, 4.2 ng/ ⁇ l system, 4.4 ng/ ⁇ l system, 4.6 ng/ ⁇ l system. 4.8 ng/ ⁇ l system, 5.0 ng/ ⁇ l system, 5.2 ng/ ⁇ l system, 5.4 ng/ ⁇ l system, 5.6 ng/ ⁇ l system, 5.8 ng/ ⁇ l system, and the like.
  • Advantages of adding vector DNA include: due to the very small amount of chromatin DNA in a single cell (pg level), the rate of loss of purification during subsequent purification is greater; however, when a large amount of exogenous DNA is added before DNA purification The total amount of DNA is significantly increased, thereby significantly increasing the yield of purification; at the same time, since the vector DNA is added after Tn5 is attacked and the reaction is terminated, there is no sequencing linker on the DNA, so there is no subsequent step of building the database. interference.
  • the step of constructing the chromatin DNA library using the Tn5 transposase further comprises: purifying the second cleavage product prior to amplification in step c2);
  • the construction of the chromatin DNA library using the Tn5 transposase further comprises: performing a second amplification of the amplification product of step c2); since the total amount of DNA of the single cell is small, the inventors have thought of Two amplification strategies; for example, in a specific embodiment, the first use of shorter specific primers for 8 cycles of initial amplification, which can significantly increase the total amount of DNA; on this basis, The second amplification is performed using the primer sequence containing the sequencing linker, thereby ensuring that the PCR proceeds smoothly with a high template amount, thereby ensuring the quality of the library.
  • the amplification of step c2) is carried out by the nucleotide sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1) and the nucleotide sequence 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2) a primer pair consisting of;
  • the second amplification is carried out by the nucleotide sequence 5'-phos- GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC -3' (underlined partial sequence SEQ ID NO: 3) and the nucleotide sequence 5'- TGTGAGCCAAGGAGTTGTTGTCTTC (underlined The partial sequence is the primer pair consisting of SEQ ID NO: 4) - barcode sequence - GTCTCGTGGGCTCGG (italic part sequence, SEQ ID NO: 5) - 3'.
  • step c2) further comprising the step of determining the number of amplification cycles required for the second amplification using real-time fluorescent quantitative PCR; in particular, Preferably comprising: amplification by step c2)
  • the product is a template and is subjected to real-time fluorescence using a primer pair consisting of the nucleotide sequence 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 1) and the nucleotide sequence 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 2).
  • Quantitative PCR in the obtained linear amplification curve, find the number of cycles corresponding to 1/3 of the plateau fluorescence intensity, which is the number of amplification cycles required for the second amplification.
  • the construction of the chromatin DNA library using the Tn5 transposase further comprises amplifying the constructed chromatin DNA library after completion of the construction of the chromatin DNA library.
  • transcriptome library construction comprises:
  • the cDNA amplification product obtained in the step c1') is fragmented using a Tn5 transposase, and the resulting fragmented product is amplified to obtain a transcriptome sequencing library of the single cell.
  • the method of constructing a single-cell sequencing library preferably, the chromatin DNA library construction and transcriptome library construction of step c) are carried out in a micro-upgraded reaction system.
  • the invention provides a method for high throughput sequencing of a single cell sequencing library comprising the steps of:
  • the single-cell sequencing library constructed by the method described in the first aspect is subjected to high-throughput sequencing to obtain chromatin accessibility information and transcriptome sequence information of the single cells, respectively.
  • the present invention provides a single cell multi-omics analysis method comprising:
  • Bioinformatics analysis was performed on the chromatin accessibility information and transcriptome sequence information of the obtained single cells.
  • the present invention provides the method of constructing a single-cell sequencing library according to the first aspect or the single-cell multi-omics analysis method according to the third aspect, in at least one of the following: a tumor target sieve Investigation, disease monitoring, and preimplantation embryo diagnosis.
  • the present invention provides a kit comprising one or more reagents as used in the method of the first aspect;
  • the kit comprises a single cell lysis reagent, a chromatin DNA fragmentation reagent, a fragmentation termination reagent, a second cleavage reagent after chromatin DNA fragmentation, and a vector DNA;
  • the single cell lysis reagent comprises:
  • the rest is water
  • the final concentration of NP-40 in the above single cell lysis reagent is 0.1-0.3%, and the value includes any discrete value between 0.1-0.3%, such as 0.12%, 0.14%, 0.16%. , 0.18%, 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, and the like.
  • the final concentration of Tris-HCl, pH 7.5 is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the final concentration of NaCl is 8-12 mM, which includes any discrete value between 8-12 mM, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the final concentration of the RNase inhibitor is 1-2 U/ ⁇ l, which includes any discrete value between 1-2 U/ ⁇ l, such as 1.1 U/ ⁇ l, 1.2 U/ ⁇ l, 1.3 U/ ⁇ l, 1.4 U/ ⁇ l, 1.5 U/ ⁇ l, 1.6 U/ ⁇ l, 1.7 U/ ⁇ l, 1.8 U/ ⁇ l, 1.9 U/ ⁇ l, and the like.
  • the chromatin DNA fragmentation reagent comprises:
  • the rest is water
  • the final concentration of the fragmented Tn5 enzyme is 0.05-0.09 U/ ⁇ l, and the value includes any discrete value between 0.05-0.09 U/ ⁇ l, for example, 0.055. U/ ⁇ l, 0.06 U/ ⁇ l, 0.065 U/ ⁇ l, 0.07 U/ ⁇ l, 0.075 U/ ⁇ l, 0.08 U/ ⁇ l, 0.085 U/ ⁇ l, and the like.
  • Tris-HCl a final concentration of pH 7.5 of 14-20 mM, includes any discrete value between 14-20 mM, such as 14.5 mM, 15 mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM. 18.5 mM, 19 mM, 19.5 mM, and the like.
  • the final concentration of NaCl is 14-20 mM, which includes any discrete value between 14-20 mM, such as 14.5 mM, 15 mM, 15.5 mM, 16 mM, 16.5 mM, 17 mM, 17.5 mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM, etc.
  • the fragmentation termination reagent comprises:
  • EDTA pH 8.0 50-70 mM, preferably 60 mM;
  • Tris-HCl pH 8.0 8-12 mM, preferably 10 mM
  • the rest is water
  • the final concentration of EDTA, pH 8.0 is 50-70 mM, which includes any discrete value between 50-70 mM, such as 52 mM, 55 mM, 58 mM, 60 mM, 63 mM, 65 mM, 68 mM, and the like.
  • Tris-HCl, pH 8.0 final concentration is 8-12 mM, which includes between 8-12 mM Intentional discrete values, such as 8.5 mM, 9 mM, 9.5 mM, 10 mM, 10.5 mM, 11 mM, 11.5 mM, and the like.
  • the second lysis reagent comprises RLT Plus buffer.
  • the method for constructing the single-cell sequencing library can efficiently build the library of chromatin DNA and construct the RNA of the same cell, not only obtaining the chromatin open information and gene expression in a single cell for the first time.
  • the information also reduces the omics information lost during the sequencing process to a very low level, which is beneficial to fully integrate and analyze the epigenetic and transcriptome data in individual cells, mine multiple omics information of single cells, and establish single cell genes.
  • Expression regulation network The simultaneous database construction and sequencing method of the present invention brings a very useful research tool for the study of single cell gene expression regulation, and has a very good application prospect in basic research such as tumor, development and new disease monitoring technology development.
  • the existing chromatin accessibility technology is carried out in the microfluidic chip nano-reaction reaction system, which cannot effectively separate chromatin and RNA; and the microfluidic chip technology is not suitable.
  • the microfluidic chip technology is not suitable.
  • the micro The upgrade reaction system can be operated in a single tube (i.e., a single reaction tube), in a single well (i.e., a single reaction well), or in another container that can accommodate a micro-upgraded liquid.
  • the method of the invention greatly optimizes the chromatin accessibility technology by introducing two steps of cleavage, adding carrier DNA and secondary amplification, and achieves the purpose of effectively separating chromatin and RNA, and has achieved remarkable results compared with the prior art. improvement.
  • Figure 1 shows an example of the number of amplification cycles required for real-time PCR to determine the second amplification of a chromatin DNA sample
  • Figure 2 shows an example of the result of amplification detection of a fragmented product of a chromatin DNA sample
  • Figure 3 shows an example of the result of amplification of a cDNA sample.
  • Figure 4 shows an example of the result of amplification detection of a fragmentation product of a cDNA sample.
  • a single cell chromatin accessibility and transcriptome sequencing library simultaneous construction method of the present invention was carried out by taking the H9ES cell line as an example to prepare a single cell library. Specific steps are as follows:
  • the adherent H9ES cells were digested with Accutase; 1 ml of the digested cell suspension was taken at After centrifugation at 1000 rpm for 5 min, the cell pellet was resuspended in 1 x PBS, repeated once; the resuspended cells were diluted to a suitable density, and single cells (volume less than 0.5 ⁇ l) were aspirated using a glass needle having a diameter of less than 200 ⁇ m to complete cell sample preparation. 100 cells were used as a positive control and PBS was used as a negative control.
  • the vortex is shaken for 1 min, 4 ° C, and centrifuged at 1000 g for 5 min;
  • the vector DNA is an exogenous DNA having a fragment size between lkb and 10 kb which is different from the genome of the cell of interest, which may be linear or circular.
  • a DNA fragment of a bacterial origin that satisfies the requirements, or a conventional plasmid cloning vector can be used as the vector DNA.
  • DNA purification was carried out in this step. Specifically, Agencourt AMPure XP magnetic beads were purified (1.8x), and finally 9 ul of sterile ultrapure water was used to dissolve the DNA.
  • primer B sequence (shown as SEQ ID NO: 1) is:
  • the primer C sequence (shown as SEQ ID NO: 2) is:
  • the N5 primer sequence is:
  • the N7 primer sequence is:
  • the barcode sequence may be 5'-ATTTATGACA-3' (ie SEQ ID NO: 6).
  • the constructed chromatin DNA library can be amplified, and the specific amplification system is shown in Table 8:
  • primer 1 the sequence of primer 1 is:
  • the primer 2 sequence is:
  • pre-processing and sequencing on the machine are performed based on a specific sequencing platform.
  • RNA solution solution obtained in 4.1.1) 6 SuperScript II reverse transcriptase 200U/ ⁇ l (Invitrogen, Cat. No. 18064) 0.75 RNAse inhibitor 40U/ ⁇ l (Takara) 0.375 5xSuperscript II First-Strand Buffer (Invitrogen, Catalog No. 18064) 3 100mM DTT (Invitrogen) 0.75 5M betaine (SIGMA, article number B0300-1VL) 3 0.2M MgCl2 (MILLIPORE) 0.45 100uM Template-Switching Oligo (LNA) (EXIQON, article number 500100) 0.15 Nuclease-free water (Ambion) 0.525 Total 15
  • Primer 1 Primer 2, N5 primer and N7 primer were used for amplification in this step, and their sequences were as described above.
  • Primer 1 (10 ⁇ M) 1 Primer 2 (10 ⁇ M) 1 Primer N5 (0.5 ⁇ M) 1 Primer N7 (0.5 ⁇ M) 1 2x KAPA HiFi HotStart ReadyMix 25 Nuclease-free water (Ambion) 8.5 Total 50
  • the PCR product was selectively purified using Agencourt AMPure XP magnetic beads.
  • the libraries constructed above can be sequenced using current mainstream second generation sequencing platforms (eg, BGISEQ-500, Hiseq2000, Hiseq4000, etc.).
  • the sequencing platform used in this case was BGISEQ-500.
  • the analysis after sequencing was performed by filtering single cell accessibility and transcriptome data, data comparison, and downstream personalized data mining analysis.

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Abstract

本发明提供了一种构建单细胞测序文库的方法,包括如下步骤：a)裂解单细胞，获得单细胞裂解物；b)对步骤a)所得单细胞裂解物进行细胞核与细胞质的分离，获得细胞核溶液与总RNA溶液；c)对步骤b)所得细胞核溶液进行染色质DNA文库构建，获得所述单细胞的染色质可接近性测序文库；并且，对步骤b)所得总RNA溶液进行转录组文库构建，获得所述单细胞的转录组测序文库。

Description

单细胞测序文库的构建方法及其应用 技术领域
本发明涉及高通量测序领域，具体地，本发明涉及一种构建单细胞测序文库的方法，基于单细胞多组学(Single Cell Multi-Omics Sequencing，SCMOS)的测序方法及应用。
背景技术
单细胞测序技术是近几年最受关注的测序技术之一，目前已经发表的单细胞测序技术主要包括单细胞DNA和DNA甲基化测序，单细胞RNA测序，单细胞染色质可接近性(Chromatin accessibility)测序。
例如，Fuchou Tang等人(Nature Methods 6，377-382(2009))报道了单细胞RNA测序技术，其包括：通过强裂解液对单细胞进行充分裂解，然后对其中尾部带有多聚腺嘌呤的RNA进行反转录和PCR扩增，最后基于特定的测序平台进行建库和测序。
再例如，Buenrostro等人(Nature 523，486-490(2015))报道了单细胞染色质可接近性测序技术，其包括：利用微流控芯片技术，将针对5万细胞的微升级的反应体系等比例缩减至单个细胞的纳升级的反应体系；通过对单细胞进行裂解和Tn5转座酶处理，在染色质中开放区域插入测序接头，然后通过PCR扩增的方法进行全部开放染色质区域的扩增，并进行建库和测序。
除了单个组学水平上的测序之外，如何在单个细胞中充分捕获单细胞多个组学的信息是当前的难题。单细胞多组学测序过程面临的主要问题是单个细胞中的目标物质(如DNA，RNA，染色质)总量非常少，在不同维度的操作过程中非常容易带来损失，直接导致多组学建库的失败或者出现检测灵敏度不高、组学信息丢失严重、技术噪音高、操作失误率高、重复性差等问题。
目前，已经实现了在单细胞DNA或DNA甲基化与单细胞RNA之间的整合性测序，例如，Macaulay等人(Nature Methods 12，519-522(2015))报道了一种单细胞DNA与RNA同时测序的方法，其包括：用强裂解液进行裂解，然后利用磁珠捕获带有polyA的mRNA，分离完之后，再对剩余液体中的DNA进行全基因组扩增，进一步进行全基因组DNA建库，RNA进行cDNA建库。与此类似的是，Angermueller等人(Nature Methods 13，229-232(2016))基于此开发了单细胞DNA甲基化和RNA同时测序的方法，与前一种方法不同的地方在于DNA和RNA分离后，对剩余液体中的DNA进行重亚硫酸盐处理，然后对处理后的DNA进行建库。再例如，Hou等人(Cell Research 26：304-319(2016))报道了另一种单细胞DNA甲基化与RNA同时测序的方法，其包括：首先利用弱裂解液裂开细胞膜，释放细胞质，但保留 细胞核。然后分离细胞质进行RNA建库，对细胞核进行重亚硫酸亚处理，进一步进行DNA建库。
然而，对于单细胞的其他不同组学之间的整合，例如单细胞RNA和染色质可接近性同时测序，目前还无任何相关报道。
现有技术中，由于单细胞的基于转座酶的染色质可接近性测序分析(assay for transposase-accessible chromatin，ATAC-seq)，其反应体系都是在微流控芯片的纳升级反应体系中进行，这不利于有效分离染色质和RNA，因此也无法在单细胞中进行染色质可接近性和转录组的同时建库。
发明内容
针对现有技术中所存在的缺陷，本发明提供了一种构建单细胞测序文库的方法，基于单细胞多组学(Single Cell Multi-Omics Seqencing，SCMOS)的测序方法及应用。本发明在传统的单细胞染色质可接近性技术的基础上进行了充分的改造和优化，不仅使单细胞染色质可接近性建库可以在微升级反应体系中有效进行，同时还能有效分离染色质和RNA，进而实现染色质可接近性和RNA同时建库，充分捕获染色质状态和基因表达的组学信息。
本发明提供的“单细胞多组学”方法是指，对单个细胞的细胞核和细胞质进行分离，然后对细胞核进行染色质可接近性建库，与此同时，对总细胞质进行转录组建库，用于高通量测序和生物信息学分析。
第一方面，本发明提供了一种构建单细胞测序文库的方法，其能够同时对单细胞的染色质可接近性和转录组进行建库，包括如下步骤：
a)裂解单细胞，获得单细胞裂解物；
b)对步骤a)所得单细胞裂解物进行细胞核与细胞质的分离，获得细胞核溶液与总RNA溶液；
c)对步骤b)所得细胞核溶液进行染色质DNA文库构建，获得所述单细胞的染色质可接近性测序文库；并且，对步骤b)所得总RNA溶液进行转录组文库构建，获得所述单细胞的转录组测序文库。
在优选的具体实施方案中，步骤a)中，用于裂解单细胞的单个裂解体系包括：
Figure PCTCN2016108940-appb-000001
其余为水。
本领域普通技术人员可以理解的是，上述单个裂解体系中，NP-40的终浓度为0.1-0.3％，该值包括0.1-0.3％之间的任意离散值，例如0.12％、0.14％、0.16％、0.18％、0.2％、0.22％、0.24％、0.26％、0.28％等。同样，Tris-HCl，pH7.5的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。NaCl的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。RNA酶抑制剂的终浓度为1-2U/μl，该值包括1-2U/μl之间的任意离散值，例如1.1U/μl、1.2U/μl、1.3U/μl、1.4U/μl、1.5U/μl、1.6U/μl、1.7U/μl、1.8U/μl、1.9U/μl等。
在优选的具体实施方案中，步骤c)中，利用Tn5转座酶或者利用DNase酶切进行染色质DNA文库构建。
优选地，利用Tn5转座酶进行染色质DNA文库构建的步骤包括：
c1)利用Tn5转座酶切割染色质开放区域，其包括利用Tn5转座酶对染色质DNA进行片段化以及在片段化后进行片段化终止的步骤；以及
c2)对经片段化的染色质DNA进行扩增。
在优选的实施方案中，步骤c1)中，用于使染色质DNA片段化的单个片段化体系包括：
细胞核溶液；5×片段化缓冲液，0.2μl/μl；反应体系；TTE Mix V5S，0.03-0.05μl/μl、优选0.04μl/μl反应体系；Tris-HCl，pH7.5，8-12mM、优选10mM；NaCl，8-12mM、优选10mM；其余为水；
本领域普通技术人员可以理解，上述单个片段化体系中，Tris-HCl，pH7.5的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。同样，NaCl的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。
优选地，在片段化后，向单个片段化体系中加入以下组分进行片段化终止：EDTA，pH8.0，50-70mM、优选60mM；Tris-HCl，pH8.0，10-14mM、优选12mM；其余为水。
本领域普通技术人员可以理解，上述片段化终止组分中，EDTA，pH8.0的终浓度为50-70mM，该值包括50-70mM之间的任意离散值，例如52mM、55mM、58mM、60mM、63mM、65mM、68mM等。同样，Tris-HCl，pH8.0终浓度为10-14mM，该值包括10-14mM之间的任意离散值，例如10.5mM、11mM、11.5mM、12mM、12.5mM、13mM、13.5mM等。
在优选的实施方案中，利用Tn5转座酶进行染色质DNA文库构建的步骤还包括：在步骤c1)后，对所得产物进行第二次裂解；进一步优选地，采用RLT Plus buffer进行第二次裂 解；
在具体的优选实施方案中，第一次裂解采用弱裂解液，其裂解细胞膜，但不影响细胞核(即，细胞核还是完整的)，这有利于保证细胞内染色质的完整性；第二次裂解采用强裂解液，可对细胞核进行彻底裂解，并且在Tn5完成攻击以后进行，有利于充分释放核内的染色质，实现在单个细胞中最大程度获得最多的染色质的效果。
进一步优选地，在进行第二次裂解的同时，向体系中加入载体DNA；优选地，所述载体DNA的加入量为4-6ng/μl体系、优选5ng/μl体系；本领域普通技术人员可以理解，上述载体DNA的终浓度为4-6ng/μl体系，该值包括4-6ng/μl体系之间的任意离散值，例如4.2ng/μl体系、4.4ng/μl体系、4.6ng/μl体系、4.8ng/μl体系、5.0ng/μl体系、5.2ng/μl体系、5.4ng/μl体系、5.6ng/μl体系、5.8ng/μl体系等。加入载体DNA的优点包括：由于单个细胞中的染色质DNA非常少(pg级别)，在后续纯化过程中，其纯化损失率会较大；然而，当在DNA纯化之前加入大量的外源DNA时，DNA的总量会显著增加，从而显著提高了纯化的产率；同时，因为载体DNA是在Tn5攻击完并终止反应后加入的，DNA上并没有测序接头，所以对于后续的建库步骤没有干扰。
在优选的实施方案中，利用Tn5转座酶进行染色质DNA文库构建的步骤还包括：在步骤c2)扩增之前，对第二次裂解产物进行纯化；
在优选的实施方案中，利用Tn5转座酶进行染色质DNA文库构建还包括：对步骤c2)的扩增产物进行第二次扩增；由于单细胞的DNA总量很少，发明人想到了两次扩增策略；例如，在一个具体的实施方案中，第一次使用较短的特异性引物进行8个循环的初步扩增，这一步能显著提高DNA总量；在此基础上，再利用含测序接头的引物序列进行第二次扩增，从而保证PCR在高模板量的情况下顺利进行，从而保证了文库的质量。
在一个具体的优选实施方案中，步骤c2)的扩增采用由核苷酸序列5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：1)和核苷酸序列5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：2)组成的引物对；
在一个具体的优选实施方案中，第二次扩增采用由核苷酸序列5′-phos-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC-3′(下划线部分序列SEQ ID NO：3)和核苷酸序列5′-TGTGAGCCAAGGAGTTGTTGTCTTC(下划线部分序列即SEQ ID NO：4)-条形码序列-GTCTCGTGGGCTCGG(斜体部分序列即SEQ ID NO：5)-3′组成的引物对。
在优选的实施方案中，在步骤c2)的扩增与第二次扩增之间，还包括使用实时荧光定量PCR确定第二次扩增所需要的扩增循环数的步骤；具体的，其优选包括：以步骤c2)的扩增 产物为模板，采用由核苷酸序列5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′(SEQ ID NO：1)和核苷酸序列5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′(SEQ ID NO：2)组成的引物对进行实时荧光定量PCR，在所得线性扩增曲线中，找到平台期荧光强度1/3所对应的循环数，即为第二次扩增所所需要的扩增循环数。
在优选的实施方案中，利用Tn5转座酶进行染色质DNA文库构建还包括：在染色质DNA文库构建完成后，对所构建的染色质DNA文库进行扩增。
上述构建单细胞测序文库的方法，作为优选，步骤c)中，所述转录组文库构建包括：
c1’)将总RNA反转录为cDNA，并对cDNA进行扩增，获得cDNA扩增产物；
c2’)利用Tn5转座酶，将步骤c1’)所得cDNA扩增产物进行片段化，并对所得片段化产物进行扩增，获得所述单细胞的转录组测序文库。
上述构建单细胞测序文库的方法，作为优选，步骤c)的染色质DNA文库构建和转录组文库构建均在微升级反应体系中进行。
第二方面，本发明提供了一种对单细胞测序文库进行高通量测序的方法，其包括如下步骤：
对由第一方面所述的方法所构建的单细胞测序文库进行高通量测序，分别获得所述单细胞的染色质可接近性信息和转录组序列信息。
第三方面，本发明提供了一种单细胞多组学分析方法，其包括：
实施如第二方面所述的方法；以及，
对所获得的单细胞的染色质可接近性信息和转录组序列信息进行生物信息学分析。
第四方面，本发明提供了如第一方面所述的构建单细胞测序文库的方法或如第三方面所述的单细胞多组学分析方法在以下至少一项中的应用：肿瘤靶点筛查、疾病监测以及植入前胚胎诊断中。
第五方面，本发明提供了一种试剂盒，其包括如第一方面所述的方法中所使用的一种或多种试剂；
优选地，所述试剂盒包括单细胞裂解试剂、染色质DNA片段化试剂、片段化终止试剂、染色质DNA片段化后的第二次裂解试剂以及载体DNA；
进一步优选地，所述单细胞裂解试剂包括：
Figure PCTCN2016108940-appb-000002
其余为水；
本领域普通技术人员可以理解，上述单细胞裂解试剂中，NP-40的终浓度为0.1-0.3％，该值包括0.1-0.3％之间的任意离散值，例如0.12％、0.14％、0.16％、0.18％、0.2％、0.22％、0.24％、0.26％、0.28％等。同样，Tris-HCl，pH7.5的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。同样，NaCl的终浓度为8-12mM，该值包括8-12mM之间的任意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。RNA酶抑制剂的终浓度为1-2U/μl，该值包括1-2U/μl之间的任意离散值，例如1.1U/μl、1.2U/μl、1.3U/μl、1.4U/μl、1.5U/μl、1.6U/μl、1.7U/μl、1.8U/μl、1.9U/μl等。
进一步优选地，所述染色质DNA片段化试剂包括：
Figure PCTCN2016108940-appb-000003
其余为水；
本领域普通技术人员可以理解，上述染色质DNA片段化试剂中，片段化Tn5酶的终浓度为0.05-0.09U/μl，该值包括0.05-0.09U/μl之间的任意离散值，例如0.055U/μl、0.06U/μl、0.065U/μl、0.07U/μl、0.075U/μl、0.08U/μl、0.085U/μl等。同样，Tris-HCl，pH7.5的终浓度为14-20mM，该值包括14-20mM之间的任意离散值，例如14.5mM、15mM、15.5mM、16mM、16.5mM、17mM、17.5mM、18mM、18.5mM、19mM、19.5mM等。同样，NaCl的终浓度为14-20mM，该值包括14-20mM之间的任意离散值，例如14.5mM、15mM、15.5mM、16mM、16.5mM、17mM、17.5mM、18mM、18.5mM、19mM、19.5mM等。
进一步优选地，所述片段化终止试剂包括：
EDTA，pH8.0               50-70mM，优选60mM；
Tris-HCl，pH8.0           8-12mM、优选10mM；
其余为水；
本领域普通技术人员可以理解，上述片段化终止试剂中，EDTA，pH8.0的终浓度为50-70mM，该值包括50-70mM之间的任意离散值，例如52mM、55mM、58mM、60mM、63mM、65mM、68mM等。同样，Tris-HCl，pH8.0终浓度为8-12mM，该值包括8-12mM之间的任 意离散值，例如8.5mM、9mM、9.5mM、10mM、10.5mM、11mM、11.5mM等。
进一步优选地，所述第二次裂解试剂包括RLT Plus buffer。
有益效果
本发明提供的单细胞测序文库的构建方法在高效地对染色质DNA进行建库的同时又对同一细胞的RNA进行建库，不仅首次实现在单个细胞中同时获得染色质开放性信息和基因表达的信息，也将测序过程中所丢失的组学信息降到很低水平，有利于充分整合分析单个细胞中表观基因组和转录组的数据，挖掘单细胞多个组学信息，建立单细胞基因表达调控网络。本发明的同时建库及测序方法为单细胞基因表达调控的研究带来了非常有用的研究工具，在肿瘤，发育等基础研究和新的疾病监测技术开发中具有非常好的应用前景。
此外，值得注意的是，现有的染色质可接近性技术都是在微流控芯片纳升级反应体系中进行的，无法有效地分离染色质和RNA；并且，微流控芯片技术也不适合用于进行自动化核酸纯化操作；本发明的发明人直接绕过微流控芯片中无法进行液体物理分离的缺陷，开发了一种在微升级反应体系中进行的染色质可接近性技术，该微升级反应体系可以是在单管(即单个反应管)中进行操作，也可以是在单孔(即单个反应孔)中进行操作，也可以是在其他可容纳微升级液体的容器中进行操作。本发明的方法通过引入两次裂解、加入载体DNA以及二次扩增等步骤，大大优化了染色质可接近性技术，实现了有效分离染色质和RNA的目的，相对于现有技术取得了显著的进步。
附图说明
图1显示荧光定量PCR确定染色质DNA样品第二次扩增所需要的扩增循环数的实例；
图2显示染色质DNA样品片段化产物的扩增检测结果的实例；
图3显示cDNA样品扩增检测结果的实例。
图4显示cDNA样品片段化产物的扩增检测结果的实例。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例H9ES细胞系的单细胞文库制备
本实施例以H9ES细胞系为例，实施本发明的单细胞染色质可接近性和转录组测序文库同时构建方法，以进行单细胞文库的制备。具体步骤如下：
一、单细胞制备及裂解
1.1、单细胞制备
使用Accutase酶对贴壁的H9ES细胞进行消化；将消化好的细胞悬浮液取1ml，在 1000rpm条件下离心5min，用1×PBS重悬细胞沉淀，重复一次；将重悬的细胞稀释至合适密度，使用直径小于200μm的玻璃针吸取单细胞(体积小于0.5μl)，完成细胞样品制备。使用100个细胞作为阳性对照，PBS作为阴性对照。
1.2、单细胞裂解
按表1配制单细胞裂解体系，每个单细胞悬浮液样本(0.5μl)加入6.5μl裂解液，轻弹管壁混匀，快速离心3s，转移至热循环仪中，4℃反应30min进行细胞裂解。
表1
试剂 体积(μl)
单细胞悬浮液 0.5
10％NP-40(Sigma，货号I8896) 0.07
1M Tris-HCl(pH7.5)(Invitrogen) 0.07
1M NaCl(Sigma) 0.07
RNAse抑制剂(40U/μl)(Takara，2313A) 0.25
无核酸酶水(Ambion AM9932) 6.04
总共 7
二、细胞核/RNA分离
2.1裂解结束后，漩涡震荡1min，4℃，1000g离心5min；
2.2小心吸取4μl上清到新PCR管中，剩余3μl为细胞核溶液，RNA在上清中。
三、利用Tn5进行细胞核染色质DNA建库
3.1Tn5切割染色质开放区域
3.1.1于室温解冻5×Tagament Buffer L，上下颠倒混匀后备用。
3.1.2按表2配制Tn5片段化反应体系，轻弹管壁混匀，快速离心3s。
表2
试剂 体积(μl)
DNA溶液(2.2所得细胞核溶液) 3
5xTagament buffer L(5x片段化缓冲液，BGE005B01) 1.4
TTE Mix V5S(片段化Tn5酶V5S，BGE005S) 0.3
1M Tris，pH7.5(Invitrogen) 0.07
1M NaCl(Sigma) 0.07
无核酸酶水(Ambion) 2.16
总共 7
3.1.3转移至热循环仪，37℃反应30min。
3.1.4按表3配制Tn5酶片段化终止试剂，混合均匀，向上述片段化反应产物中加入片段化终止试剂3.5μl，轻弹管壁混匀，快速离心3s。
表3
试剂 体积(μl)
0.1M EDTA，pH8.0(Ambion) 2.1
0.1M Tris，pH8.0(Ambion) 0.42
无核酸酶水(Ambion) 0.98
总共 3.5
3.1.5转移至热循环仪，50℃反应30min。
3.2第二次裂解与载体DNA的加入
3.2.1按表4配制第二次裂解试剂，混合均匀。向3.1.5所得反应产物中加入该试剂6μl(总体积16.5μl)，快速离心3s，室温静置15min。
表4
试剂 体积(μl)
RLT Plus buffer(Qiagen，货号1053393) 3
10ng/μl载体DNA 3
总共 6
表4中，所述载体DNA为与目的细胞的基因组不同源的、片段大小在lkb～10kb之间的外源DNA，其可以是线性的或环状的。例如，满足要求的细菌来源的DNA片段，或常规的质粒克隆载体都可以作为载体DNA。
3.2.2补加灭菌超纯水至40μl，轻弹管壁混匀，快速离心3s。
3.3DNA纯化
为了使后续PCR反应体系中没有其他物质干扰，本步骤进行了DNA的纯化。具体地，使用Agencourt AMPure XP磁珠纯化(1.8x)，最后使用9ul灭菌超纯水溶解DNA。
3.4第一次DNA扩增
3.4.1按表5配制第一次扩增试剂，混合均匀。向3.3纯化产物中加入第一次扩增试剂11μl；轻弹管壁混匀，快速离心3s。
表5
试剂 体积(μl)
High-Fidelity 2X PCR Master Mix(NEB，货号M0541L) 10
引物B(20μM) 0.5
引物C(20μM) 0.5
总共 11
其中，引物B序列(如SEQ ID NO：1所示)为：
5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′；
引物C序列(如SEQ ID NO：2所示)为：
5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′
3.4.2转移至热循环仪中，72℃5min；98℃1min；98℃15s，63℃30s，72℃1min，重复8次。
3.4.3使用Agencourt AMPure XP磁珠纯化(1.8x)，最后使用20μl灭菌超纯水溶解DNA。
3.5使用实时荧光定量PCR确定第二次扩增需要的PCR循环数
3.5.1从3.4.3所得纯化产物中取出4μl到新的PCR管中。
3.5.2按表6配制扩增试剂，混合均匀。，向3.5.1中含有纯化产物的PCR管加入16μl扩增试剂；轻弹管壁混匀，快速离心3s。
表6
Figure PCTCN2016108940-appb-000004
3.5.3转移至荧光定量PCR仪中，72℃5min；98℃30s；98℃10s，63℃30s，72℃1min，重复40次。
3.5.4在线性扩增Rn/Cycle曲线中，平台期荧光强度1/3对应的循环数即为第二次扩增需要的循环数N，如图1。
3.6第二次DNA扩增
3.6.1按表7配制第二次扩增试剂，混合均匀。向3.4.3所得纯化产物加入第二次扩增试剂17.1μl，再加入条形码引物N70.7μl，轻弹管壁混匀，快速离心3s。
表7
试剂 体积(μl)
High-Fidelity 2X PCR Master Mix(NEB，货号M0541L) 16.4
N5(25μM) 0.7
总共 17.1
其中，N5引物序列为：
5′-phos-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC-3′(即SEQ ID NO：3)；
N7引物序列为：
5′-TGTGAGCCAAGGAGTTGTTGTCTTC(即SEQ ID NO：4)-条形码序列-GTCTCGTGGGCTCGG(即SEQ ID NO：5)-3′。
具体地，条形码序列可以是5′-ATTTATGACA-3′(即SEQ ID NO：6)。
3.6.2转移至热循环仪中，72℃5min；98℃1min；98℃15s，63℃30s，72℃1min，重复N次(N为3.5.4中确定的循环数)。
3.6.3使用Agencourt AMPure XP磁珠纯化(1x)，最后使用20μl灭菌超纯水溶解DNA。
3.7Qubit检测产物浓度，Agilent 2100检测文库构建结果，符合预期的文库应该分布在100bp以上，如图2。
任选地，可对所构建的染色质DNA文库进行扩增，具体扩增体系如表8所示：
表8
Figure PCTCN2016108940-appb-000005
其中，引物1序列为：
5′-phos-GAACGACATGGCTACGATCCGACTT-3′(下划线部分序列即SEQ ID NO：7)；
引物2序列为：
5′-TGTGAGCCAAGGAGTTGTTGTCTTC-3′(即SEQ ID NO：4)。
3.8进一步，基于特定测序平台进行上机前处理及上机测序。
四、由总RNA制备cDNA样品，并对所述cDNA样品进行片段化文库构建
4.1总RNA反转录为cDNA
参考引物名称及序列：
Oligo-dT：
5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′(下划线部分序列即SEQ ID NO：8)；
Template-Switching Oligo(LNA)：
5’-AAGCAGTGGTATCAACGCAGAGTACrGrG+G-3’(其中，下划线部分序列即SEQ ID NO：9，rG为RNA碱基，+G为锁核酸LNA修饰)。
4.1.1向2.2转移出的RNA上清中加入1μl oligo-dT(10μM)，1μl dNTP(10mM)(ENZYMATICS)，轻弹管壁混匀，快速离心3s，转移至热循环仪中，72℃反应3min，结束后离心至管底，放置冰上备用；
4.1.2按表9配制反转录体系，轻弹管壁混匀，快速离心3s。
表9
试剂 体积(μl)
RNA溶液(4.1.1所得溶液) 6
SuperScript II反转录酶200U/μl(Invitrogen，货号18064) 0.75
RNAse抑制剂40U/μl(Takara) 0.375
5xSuperscript II First-Strand Buffer(Invitrogen，货号18064) 3
100mM DTT(Invitrogen) 0.75
5M甜菜碱(SIGMA，货号B0300-1VL) 3
0.2M MgCl2(MILLIPORE) 0.45
100uM Template-Switching Oligo(LNA)(EXIQON，货号500100) 0.15
无核酸酶水(Ambion) 0.525
总共 15
4.1.3转移至热循环仪中，42℃90min；50℃2min，42℃2min，重复10次；72℃5min。
4.2cDNA扩增
参考引物名称及序列
IS引物：5′-AAGCAGTGGTATCAACGCAGAGT-3′(SEQ ID NO：10)。
4.2.1按表10配置cDNA扩增体系，轻弹管壁混匀，快速离心3s。
表10
试剂 体积(μl)
cDNA溶液(4.1.3所得产物) 15
2x KAPA HiFi HotStart ReadyMix(KAPA BIOSYSTEMS，货号KK2602) 14.7
10uM IS引物 0.3
总共 30
4.2.2转移至热循环仪中，98℃3min；98℃20s，67℃20s，72℃6min，重复20次；72℃5min。
4.2.3cDNA纯化
使用Agencourt AMPure XP磁珠纯化。
4.2.4Agilent 2100检测cDNA扩增结果，如图3。
4.3利用Tn5进行cDNA建库
4.3.1cDNA片段化
4.3.1.1于室温解冻5x Tagment Buffer L，上下颠倒混匀后备用。
4.3.1.2按表11配制Tn5片段化体系，使用移液器轻轻吹打20次以将各组分充分混匀，快速离心3s。
表11
Figure PCTCN2016108940-appb-000006
4.3.1.355℃热循环仪中温浴10min，待样品温度降至4℃后，取出PCR管。
4.3.1.4向4.3.1.3反应产物中加入2.5ul 5x NT Solution，使用移液器轻轻吹打20次充分混匀，室温放置5min。
4.3.2cDNA片段化产物扩增
参考引物名称及序列：本步骤的扩增使用了引物1、引物2、N5引物及N7引物，它们的序列如前所述。
4.3.2.1按表12配制cDNA片段化产物扩增反应体系，轻弹管壁以混匀各组分，快速离心3s。
表12
试剂 体积(μl)
cDNA溶液(4.3.1.4反应产物) 12.5
引物1(10μM) 1
引物2(10μM) 1
引物N5(0.5μM) 1
引物N7(0.5μM) 1
2x KAPA HiFi HotStart ReadyMix 25
无核酸酶水(Ambion) 8.5
总共 50
4.3.2.2转移至热循环仪中，72℃5min；95℃3min；98℃20s，60℃15s，72℃30s，重复16次；72℃5min。
4.3.2.3将PCR产物使用Agencourt AMPure XP磁珠，进行选择性纯化。
4.3.2.4Qubit检测产物浓度，Agilent 2100检测文库构建结果，符合预期的文库主峰大小在150-350bp之间。如图4。
五、高通量测序与数据分析
以上构建的文库可以使用目前主流的二代测序平台(如BGISEQ-500，Hiseq2000，Hiseq4000等)进行测序。本案例中使用的测序平台为BGISEQ-500。测序下机后的分析包括对单细胞可接近性和转录组的数据分别进行过滤，数据比对，以及下游的个性化数据挖掘分析。
申请人声明，本发明通过上述实施例来说明本发明的方法及其应用，但本发明并不局限于此，所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明方法中所涉参数或步骤的等效替换、添加及具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (15)

  1. 一种构建单细胞测序文库的方法，其包括如下步骤：
    a)裂解单细胞，获得单细胞裂解物；
    b)对步骤a)所得单细胞裂解物进行细胞核与细胞质的分离，获得细胞核溶液与总RNA溶液；
    c)对步骤b)所得细胞核溶液进行染色质DNA文库构建，获得所述单细胞的染色质可接近性测序文库；并且，对步骤b)所得总RNA溶液进行转录组文库构建，获得所述单细胞的转录组测序文库。
  2. 根据权利要求1述的方法，其特征在于，步骤c)中，利用Tn5转座酶或者利用DNase酶切进行染色质DNA文库构建。
  3. 根据权利要求2所述的方法，其特征在于，利用Tn5转座酶进行染色质DNA文库构建的步骤包括：
    c1)利用Tn5转座酶切割染色质开放区域，其包括利用Tn5转座酶对染色质DNA进行片段化以及在片段化后进行片段化终止的步骤；以及
    c2)对经片段化的染色质DNA进行扩增。
  4. 根据权利要求3所述的方法，其特征在于，利用Tn5转座酶进行染色质DNA文库构建还包括：
    在步骤c1)后，对所得产物进行第二次裂解的步骤；优选地，采用RLT Plus buffer进行第二次裂解；
    优选地，在进行第二次裂解的同时，向体系中加入载体DNA；优选地，所述载体DNA的加入量为4-6ng/μl体系、优选5ng/μl体系；
    优选地，在步骤c2)之前，对第二次裂解产物进行纯化。
  5. 根据权利要求4所述的方法，其特征在于，利用Tn5转座酶进行染色质DNA文库构建还包括：
    对步骤c2)的扩增产物进行第二次扩增；
    优选地，在染色质DNA文库构建完成后，对所构建的染色质DNA文库进行扩增。
  6. 根据权利要求3-5任一项所述的方法，其特征在于，步骤c1)中，用于使染色质DNA片段化的单个片段化体系包括：
    细胞核溶液；5×片段化缓冲液，0.2μl/μl反应体系；TTE Mix V5S，0.03-0.05μl/μl、优选0.04μl/μl反应体系；Tris-HCl，pH7.5，8-12mM、优选10mM；NaCl，8-12mM、优选10mM；其余为水；
    优选地，在片段化后，向单个片段化体系中加入以下组分进行片段化终止：EDTA，pH8.0，50-70mM、优选60mM；Tris-HCl，pH8.0，10-14mM、优选12mM；其余为水。
  7. 根据权利要求5-6任一项所述的方法，其特征在于，步骤c2)的扩增采用由核苷酸序列5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′和核苷酸序列5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′组成的引物对；
    优选地，第二次扩增采用由核苷酸序列5′-phos-GAACGACATGGCTACGATCCGACTTTCGTCGGCAGCGTC-3′和核苷酸序列5′-TGTGAGCCAAGGAGTTGTTGTCTTC-条形码序列-GTCTCGTGGGCTCGG-3′组成的引物对。
  8. 根据权利要求5-7任一项所述的方法，其特征在于，在步骤c2)的扩增与第二次扩增之间，还包括使用实时荧光定量PCR确定第二次扩增所需要的扩增循环数的步骤；
    优选地，所述确定第二次扩增所需要的扩增循环数的步骤包括：以步骤c2)的扩增产物为模板，采用由核苷酸序列5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′和核苷酸序列5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′组成的引物对进行实时荧光定量PCR，在所得线性扩增曲线中，找到平台期荧光强度1/3所对应的循环数，即为第二次扩增所需要的扩增循环数。
  9. 根据权利要求1-8任一项所述的方法，其特征在于，所述转录组文库构建包括：
    c1’)将总RNA反转录为cDNA，并对cDNA进行扩增，获得cDNA扩增产物；
    c2’)利用Tn5转座酶，将步骤c1’)所得cDNA扩增产物进行片段化，并对所得片段化产物进行扩增，获得所述单细胞的转录组测序文库。
  10. 根据权利要求1-9任一项所述的方法，其特征在于，步骤c)的染色质DNA文库构建和转录组文库构建均在微升级反应体系中进行。
  11. 根据权利要求1-10任一项所述的方法，其特征在于，步骤a)中，用于裂解单细胞的单个裂解体系包括：
    Figure PCTCN2016108940-appb-100001
  12. 一种对单细胞测序文库进行高通量测序的方法，其包括如下步骤：
    对由权利要求1-11任一项所述的方法所构建的单细胞测序文库进行高通量测序，分别获得所述单细胞的染色质可接近性信息和转录组序列信息。
  13. 一种单细胞多组学分析方法，其包括：
    实施如权利要求12所述的方法；以及，
    对所获得的单细胞的染色质可接近性信息和转录组序列信息进行生物信息学分析。
  14. 如权利要求1-11任一项所述的构建单细胞测序文库的方法或如权利要求13所述的单细胞多组学分析方法在以下至少一项中的应用：肿瘤靶点筛查、疾病监测以及植入前胚胎诊断。
  15. 一种试剂盒，其包括如权利要求1-11任一项所述的方法中所使用的一种或多种试剂；
    优选地，所述试剂盒包括单细胞裂解试剂、染色质DNA片段化试剂、片段化终止试剂、染色质DNA片段化后的第二次裂解试剂以及载体DNA；
    进一步优选地，所述单细胞裂解试剂包括：
    Figure PCTCN2016108940-appb-100002
    进一步优选地，所述染色质DNA片段化试剂包括：
    Figure PCTCN2016108940-appb-100003
    进一步优选地，所述片段化终止试剂包括：
    EDTA，pH8.0                  50-70mM，优选60mM；
    Tris-HCl，pH8.0              8-12mM、优选10mM；
    其余为水；
    进一步优选地，所述第二次裂解试剂包括RLT Plus buffer。
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