WO2012089148A1 - 单细胞基因组分析方法及试剂盒 - Google Patents

单细胞基因组分析方法及试剂盒 Download PDF

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WO2012089148A1
WO2012089148A1 PCT/CN2011/084959 CN2011084959W WO2012089148A1 WO 2012089148 A1 WO2012089148 A1 WO 2012089148A1 CN 2011084959 W CN2011084959 W CN 2011084959W WO 2012089148 A1 WO2012089148 A1 WO 2012089148A1
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seq
housekeeping gene
nucleotide sequences
specific primers
sequences shown
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PCT/CN2011/084959
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French (fr)
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殷旭阳
鲍莉
徐讯
吴汉杰
刘晓羽
张秀清
杨焕明
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深圳华大基因科技有限公司
深圳华大基因研究院
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Priority to EP11853098.9A priority Critical patent/EP2660331B1/en
Priority to US13/976,845 priority patent/US9238840B2/en
Publication of WO2012089148A1 publication Critical patent/WO2012089148A1/zh

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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention relates to the field of molecular cell biology, in particular to the field of higher biological single cell genome research.
  • the invention relates to single cell genomic analysis methods and kits. Background technique
  • SCA single cell analysis
  • micro-DNA and single-cell genomics research has been widely used in archaeology, microbial ecology, medical testing, forensic testing, clinical diagnosis and various scientific research (see Zhang L., Cui X., Schmitt K., Hubert R., Navidi W., Arnheim N. (1992) Whole genome amplification from a single cell: Implication for genetic analysis, Proc Natl Acad Sci USA: 5847-5851, which is incorporated herein by reference in its entirety.
  • the analysis and research of the single-cell genome of higher animals and plants can be effectively and conveniently applied to clinical diagnosis and treatment (such as prenatal diagnosis, preimplantation genetic diagnosis, multi-point mapping, sperm and egg typing).
  • single-cell genome analysis is performed primarily by sequencing single-cell genomes. Because commonly used DNA analysis methods such as Comparative Genomic Hybridization (CGH), Polymerase Chain Reaction (PCR), DNA Microarray, Restriction Fragment Length Polymorphism Analysis ( Restricted Fragment Length Polymorphisms (RFLP), single-strand conformation polymorphism analysis (SSCP), fingerprinting techniques, and fluorescence in situ hybridization (FISH), etc., can only be partial or known to the single-cell genome. Point research, and the lack of effective strategies for genomic studies of new species, and sequencing of single-cell genomes can effectively avoid these shortcomings.
  • CGH Comparative Genomic Hybridization
  • PCR Polymerase Chain Reaction
  • RFLP Restriction Fragment Length Polymorphisms
  • SSCP single-strand conformation polymorphism analysis
  • FISH fluorescence in situ hybridization
  • a single-cell genome must be subjected to whole-genome amplification (WGA) to a sufficient amount.
  • WGA whole-genome amplification
  • known whole genome amplification methods include PEP-PCR, PCR-based methods such as DOP-PCR, and Multiple Displacement Amplification (MDA), which are easily interfered by many factors and cannot guarantee amplification. 100% success rate.
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • the present invention provides a single-cell genomic analysis method and a kit for detecting and screening the whole-genome amplification product before sequencing the whole-genome amplification product of the single-cell genome to remove Samples that were not amplified successfully ensured the success rate of subsequent single-cell genome sequencing.
  • the invention provides a method of single cell genomic analysis.
  • the method comprises the steps of: isolating and lysing single cells to obtain whole genome DNA of the cells; performing single cell whole genome amplification of the whole genome DNA to obtain a whole genome amplification product; A house-specific gene-specific primer, which uses a whole genome amplification product as a template for PCR amplification to perform housekeeping gene detection on whole genome amplification products; and based on the detection results, whether the whole genome amplification product meets the sequencing requirements, Among them, the uniform distribution of the amplified product on each chromosome is an indication that the amplification product meets the sequencing requirements.
  • the single cell genomic analysis method according to the embodiment of the present invention can analyze the single cell of the higher organism, and can effectively obtain the whole genome amplification product of the single cell to be tested that meets the sequencing requirement indication. Further, The amplified product can be effectively used for constructing a genome sequencing library of single cells to be tested, and the obtained sequencing library can be effectively applied to a high-throughput sequencing platform such as the Solexa sequencing platform, so that the detection result can be accurately and efficiently obtained based on the sequencing result. Single-cell genomic DNA sequence information, based on this information, can be effectively used for subsequent deeper single-cell genomic analysis and research.
  • the present invention provides a kit.
  • the kit comprises a housekeeping gene-specific primer.
  • the inventors have found that a kit according to an embodiment of the present invention can efficiently obtain a whole-genome amplification product of a single cell of a higher organism that meets the sequencing requirement indication, and then constructs a sequencing library by using the amplification product, and the resulting sequencing library is obtained.
  • High-throughput sequencing based on the sequencing results, can accurately and efficiently obtain the genomic DNA sequence information of the single cells to be tested, and based on this information, can realize the genome analysis of the single cells to be tested.
  • Figure 1 shows the coverage of human cell line single cell genome sequencing data on each of the frequently stained bodies according to one embodiment of the present invention.
  • Figure 2 shows single-cell genome sequencing data of human cell lines in each of the commonly dyed according to one embodiment of the present invention The average sequencing depth on the color body.
  • Fig. 3 is a view showing the distribution of the sequencing depth of the single cell genome of the human cell line using chromosome 1 as an example in accordance with one embodiment of the present invention.
  • Figure 4 A graph showing the relationship between coverage and sequencing depth in single cell genome sequencing data for human cell lines in accordance with one embodiment of the present invention.
  • Figure 5 A graph showing the bias analysis of the resulting human cell line single cell genome sequencing data and human cell population data in accordance with one embodiment of the present invention.
  • Figure 6 A graph showing the relationship between the sequencing depth GC content of the human cell line single cell chromosome 1 according to one embodiment of the present invention.
  • Figure 7 A graph showing the results of electrophoresis of housekeeping genes in accordance with one embodiment of the present invention. Detailed description of the invention
  • the invention provides a method of single cell genomic analysis.
  • the method may comprise the following steps:
  • the single cells are isolated and lysed to obtain the whole genome DNA of the cells.
  • whole-genome DNA is subjected to single-cell whole-genome amplification to obtain whole-genome amplification products.
  • the method of performing single-cell whole genome amplification of whole genome DNA is not particularly limited.
  • single-cell whole genome amplification can be performed using multiple displacement amplification MDA or DOP-PCR whole genome amplification.
  • the whole genome amplification product was used as a template for PCR amplification, so that the whole genome amplification product was subjected to housekeeping gene detection.
  • the source of the single cell is not particularly limited.
  • the single cell may be a human single cell, and the housekeeping gene may be at least one selected from the group consisting of PRDX6, RPL37a, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP50.
  • the housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; for RPL37a, the housekeeping gene-specific primers have SEQ, respectively.
  • housekeeping gene-specific primer has the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively; -A, housekeeping gene-specific primers have the nucleotide sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; for RAD9A, housekeeping gene-specific primers have SEQ ID NO: 9 and SEQ ID NO, respectively : nucleotide sequence shown in 10; for ARHGEF7, housekeeping gene-specific primers have nucleosides represented by SEQ ID NO: 11 and SEQ ID NO: 12, respectively Acid sequence; for EIF2B2, housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; for PSMD7, housekeeping gene-specific primers have SEQ ID NO: 15 and a nucleotide sequence represented by SEQ ID NO: 16; for BCAT2, a
  • the expression "the uniform distribution of amplification products on each chromosome is an indication that the amplification product meets the sequencing requirements" means that when the housekeeping gene-specific primer is used, the whole genome amplification product is When the template is subjected to PCR amplification, if all the housekeeping genes distributed on different chromosomes corresponding to the housekeeping gene-specific primers can be efficiently amplified, it indicates that the whole-genome amplification product of the previously obtained single cells is Each chromosome is evenly distributed, which indicates that the whole genome amplification effect is better, and the amplification product can meet the subsequent sequencing requirements.
  • the single cell genomic analysis method of the present invention may further comprise constructing a DNA sequencing library for the amplification product that meets the sequencing requirements.
  • the sequencing of the DNA sequencing library may further be included.
  • whole genome amplification products can be subjected to quantitative detection of housekeeping genes.
  • the single cell genomic analysis method according to the embodiment of the present invention can analyze the single cell of the higher organism, and can effectively obtain the whole genome amplification product of the single cell to be tested that meets the sequencing requirement indication. Further, The amplified product can be effectively used for constructing a genome sequencing library of single cells to be tested, and the obtained sequencing library can be effectively applied to a high-throughput sequencing platform such as the Solexa sequencing platform, so that the detection result can be accurately and efficiently obtained based on the sequencing result. Single-cell genomic DNA sequence information, based on this information, can be effectively used for subsequent deeper single-cell genomic analysis and research.
  • the single cell genomic analysis method of the present invention may comprise the following steps:
  • housekeeping genes Housekeeping The Gene detection method detects the whole genome amplification product, wherein the uniform distribution of the amplification product on each chromosome is an indication that the amplification product meets the sequencing requirements.
  • the method may further comprise the step d: performing the DNA sequencing library construction and sequencing of the qualified amplification product.
  • the single-cell whole genome amplification in step b is performed by multiple displacement amplification MDA or DOP-PCR whole genome amplification.
  • the detection of the housekeeping gene means that the specific primer of the housekeeping gene is used, and the whole genome amplification product is used as a template for PCR amplification.
  • the single cell is a human single cell
  • the housekeeping gene is selected from the group 1 PRDX6 of chromosome, RPL37a of chromosome 2, ADD1 of chromosome 4, HLA-A of chromosome 6, RAD9A of chromosome 11, ARHGEF7 of chromosome 13, EIF2B2 of chromosome 14, located at 16 At least one of PSMD7 of chromosome No., BCAT2 of chromosome 19, and ATP50 of chromosome 21.
  • housekeeping gene-specific primers have the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; for RPL37a, housekeeping gene-specific primers Having the nucleotide sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; for ADD1, the housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively; for RAD9A, housekeeping gene-specific primers have SEQ ID NO: 9 and a nucleotide sequence represented by SEQ ID NO: 10; for ARHGEF7, a housekeeping gene-specific primer has a nucleotide sequence represented by SEQ ID NO: 11 and SEQ ID NO: 12, respectively; for EIF2B2, a housekeeping gene specific The primers have the nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; for PSMD7, the housekeeping gene-specific primers have the nucleosides shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
  • housekeeping gene-specific primers have S EQ ID NO: 17 and the nucleotide sequence shown in SEQ ID NO: 18; or for ATP50, the housekeeping gene-specific primer has the nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • the single cell genomic analysis method of the present invention may further comprise the following steps:
  • Single cells containing the entire genome can be isolated using physico-mechanical, chemical or biological methods such as microfluidics, flow cytometry, oral blotting, gradient dilution or low melting agarose immobilization.
  • the isolated single cells can be subjected to nuclear lysis using, for example, a detergent method, a boiling method, an alkali denaturation method, a lysozyme method, or an organic solvent method to obtain intact cellular genomic DNA.
  • a detergent method for example, a detergent method, a boiling method, an alkali denaturation method, a lysozyme method, or an organic solvent method to obtain intact cellular genomic DNA.
  • the resulting cellular genomic DNA is subjected to single-cell whole-genome amplification to obtain whole-genome amplification products to achieve the DNA starting amount required by next-generation sequencing technology.
  • PCR-based amplification mainly includes DOP-PCR, PEP-PCR and T-PCR.
  • MDA multiple displacement amplification
  • primers consisting entirely of random nucleotides can be used in MDA, and the primer length can be 5-20 nucleotides, and the GC content of the primer can be based on The nature of the template is chosen.
  • the nature of the template is mainly limited by the GC content of the template DNA. Therefore, the GC content of the primer is selected to be close to or the same as the template. For example, when MDA is performed using human genomic DNA as a template, the GC content may be 40%-42%. Random primers. The bias of whole genome amplification is affected by GC content.
  • the whole genome amplification product can be quantified by, for example, gel electrophoresis detection, Agilent 2100 Bioanalyzer detection, Quant-iTTM dsDNA BR detection kit detection, etc., and the results show that it is non-degraded and meets the requirements of next-generation sequencing technology.
  • the DNA-initiated whole-genome amplification product can continue DNA library construction and sequencing.
  • the starting amount of DNA required by next-generation sequencing technology varies with different database-building strategies. Generally, DNA fragmentation requires a DNA starting amount greater than 1 ⁇ g.
  • This step is a screening step for single-cell whole-genome amplification products, thereby removing a large number of unqualified amplification products, thereby controlling the quality of downstream sequencing libraries, thereby avoiding unnecessary waste to a large extent.
  • DNA library construction was performed on the well-tested whole genome amplification products using conventional whole genome DNA library construction or exon sequence capture technology.
  • a 1" column Illumina library construction method can be used by a method well known to those skilled in the art (refer to http://www.illumina.com/support/documentation.ilmn, by reference The full text is incorporated herein.
  • the DNA library is constructed. Then, the obtained library is subjected to quality inspection, including QPCR detection and Agilent 2100 Bioanalyzer detection.
  • the Agilent 2100 Bioanalyzer detection requires that the fragment size of the library differs from the expected size within ⁇ 10 bp, and There is no towing; QPCR detects the concentration of the library, and requires the total amount calculated according to the library concentration to meet the requirements of the upper machine.
  • the single-factor genome sequencing of the quality-qualified library is performed by the next-generation sequencing technology. According to the embodiment of the present invention, Sequencing was performed using at least one selected from the group consisting of Illumina HiSeq 2000 sequencing system, Illumina Genome Analyzer II sequencing system, AB SOLiDTM 4.0 sequencing system, and Roche GS FLX Titanium system to obtain sequencing results.
  • the sequencing information analysis and research of the single cell genome are performed to obtain a single nucleotide polymorphism site (SNP) of related genes, a few base insertions and deletions (InDel), Information on genetic variation of single-cell genomes such as DNA copy number variation (CNV) and structural variation (SV).
  • SNP single nucleotide polymorphism site
  • InDel a few base insertions and deletions
  • CNV DNA copy number variation
  • SV structural variation
  • the single-cell genomic analysis method of the present invention performs genome-wide amplification of higher biological single-cell genomic DNA, and introduces a quantitative and qualitative detection step after amplification, so that the qualified sample can enter the next test.
  • One-step library construction and sequencing with next-generation sequencing technology (NGS) for accurate and efficient analysis of single-cell genomes.
  • NGS next-generation sequencing technology
  • the inventors have surprisingly found that the single-cell genomic analysis method of the present invention is simple, time-saving and efficient, and can efficiently analyze and study the single-cell genome of higher plants and animals, and can comprehensively and completely analyze the single-cell genome.
  • the genetic variation information can effectively avoid the shortcomings of traditional DNA analysis methods that can only study part of the single-cell genome, and introduce qualitative and quantitative detection and screening steps to remove a large number of unqualified amplification products, thereby enabling control.
  • the quality of downstream sequencing libraries can avoid unnecessary waste to a large extent.
  • the single-cell genomic analysis method of the present invention provides an effective research strategy for single-cell genome research of novel species. Kit
  • the present invention provides a kit.
  • the kit comprises a housekeeping gene-specific primer.
  • the housekeeping gene may be at least one selected from the group consisting of PRDX6, RPL37a, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP50.
  • the housekeeping gene-specific primers contained in the kit of the present invention may be: For PRDX6, the housekeeping gene-specific primers have SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • nucleotide sequence for RPL37a, housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; for ADD1, housekeeping gene-specific primers have SEQ ID NO, respectively.
  • housekeeping gene-specific primer has the nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • housekeeping gene-specific primers have the nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively;
  • housekeeping gene-specific primers have SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
  • the kit of the present invention may further comprise an agent suitable for single-cell whole genome amplification by amplification of MDA or DOP-PCR by multiple displacement.
  • a kit according to an embodiment of the present invention can efficiently obtain a whole genome amplification product of a single cell of a higher organism that meets the sequencing requirement indication, and further constructs a DNA sequencing library by using the amplification product, and obtains the obtained DNA.
  • Sequencing libraries for high-throughput sequencing based on sequencing results, can accurately and efficiently obtain genomic DNA sequence information of single cells to be tested, as well as single nucleotide polymorphism sites (SNPs) of related genes, insertion of a few bases, and
  • SNPs single nucleotide polymorphism sites
  • the genetic variation information of single-cell genomes such as deletion (InDel), DNA copy number variation (CNV), and structural variation (SV), based on this information, can effectively realize the in-depth analysis of the single cell genome to be tested.
  • the single cell genome analysis method and kit of the present invention can efficiently analyze and study the single cell genome of higher plants and animals, and can be efficiently and conveniently applied to clinical diagnosis and treatment (such as prenatal diagnosis). , preimplantation genetic diagnosis, multi-point graphics production, sperm and egg typing, genetic diagnosis, etc.), medical research (such as autism, nervous system diseases and autoimmune diseases, genomic variation rate studies) , stem cell research, etc.), archaeological research and forensic testing.
  • Example 1 Human cell line single cell genomic study
  • Isolation and lysis of single cells add immortalized human lymphocyte cell single cells to PBS droplets on culture substrates, and moderately dilute (ie, dilute according to the concentration of cell line cells until observed in the microscope 200x field of view) To the number of cells between 10 and 20), single cells were orally aspirated under a microscope, and the resulting single cells were placed in a PCR tube containing 1.5-2 L ALB (Alkaline Lysis Buffer, specific formulation: 50 mM DTT, 200 mM KOH). Medium, placed at -20 ° ⁇ to -80 ° ⁇ for at least 30 min.
  • ALB Alkaline Lysis Buffer, specific formulation: 50 mM DTT, 200 mM KOH
  • the single-cell PCR tube is heated at 62 ° ⁇ - 68 ° ⁇ , preferably 65 ° 8 for 8-12 min to lyse the cells and release the whole genome DNA of the cells.
  • Whole-genome amplification of cells can be performed by any of the following multiple displacement amplification MDA or DOP-PCR whole genome amplification.
  • buffer DLB 500 ⁇ of nucleic acid-free water was added to the buffer DLB, shaken and centrifuged briefly to avoid uneven mixing on the tube wall to obtain a reconstitution buffer DLB.
  • Buffer DLB is sensitive to pH and should avoid neutralization with C0 2 in air.
  • the reconstituted buffer DLB can be stored at -20 ° C for 6 months.
  • buffer D1 2.5 L of buffer D1 was added to the whole genome DNA of the cells obtained in the above step (1), and allowed to stand at room temperature for 3-5 minutes to denature the DNA. Then, 5 ⁇ M buffer N1 neutralization buffer was added to terminate the denaturing reaction to obtain a single cell DNA template, which was allowed to stand at room temperature for use.
  • the MDA reaction can be carried out using one of the following two products:
  • the above system allows the ingredients to be increased or decreased in the same proportions as needed for the total volume.
  • the above system allows the ingredients to be increased or decreased in the same proportions as needed for the total volume.
  • PCR tube having a single-cell DNA template (the above-mentioned product denatured by adding buffer D1 and adding buffer N1 to terminate the denaturation reaction)
  • Instantaneous centrifugation prevents droplets from adhering to the tube wall and causes uneven mixing.
  • the PCR tube was placed on a PCR machine, incubated at 30 ° C for 10-16 hours, and then Phi29 polymerase was inactivated at 65 °C.
  • the whole genome DNA of the cells obtained in the above step (1) was subjected to DOP-PCR whole genome amplification using Sigma's GenomePlex single-cell whole genome amplification kit.
  • the OmniPlex library was then constructed according to the instructions, as well as linear, isothermal initial amplification and PCR amplification.
  • the whole genome DNA of the cells obtained in the above step (1) can be subjected to DOP-PCR whole genome amplification using Rubicon Genomics PicoPlex WGA kit according to the instructions.
  • the concentration of the whole genome amplification product was measured using the Quant-iTTM dsDNA BR detection kit according to the kit instructions. When the content of the obtained amplification product exceeds 2 g, DNA library construction and sequencing on the machine can be continued.
  • DNA DNA library construction was performed using conventional genome-wide DNA library construction or Exon Capture technology, and then the quality-qualified library was sequenced by single-cell genome using the Illumina HiSeq 2000 sequencing system.
  • the obtained human cell line single cell genome sequencing data has a large difference in sequencing depth in different regions of the chromosome.
  • the coverage rate As can be seen from Fig. 4, as the depth of sequencing increases, the coverage rate also increases, but the slope becomes slower and gradually reaches a plateau stage. When the sequencing depth is 7x, the coverage rate can reach 90%; when the sequencing depth is lOx, the coverage rate can reach 95%. .
  • the obtained human cell line single cell genome sequencing data was analyzed together with human cell population data.
  • the specific method is as follows: Taking chromosome 1 as an example, taking 20 kb as a window, calculating the number of tags in each window (the number of tags), and then mapping the number of windows corresponding to the number of tags corresponding to the number of tags, the result is shown in FIG. 5.
  • the number of windows is the largest in a certain number of tags, so a peak can be formed.
  • the peak position of the number of tags of the obtained single cell genome sequencing data of the human cell line is significantly deviated from the cell population data, indicating that the single cell genome is biased after amplification.
  • the average depth is counted by 10k, and then the window with the highest depth of 2.5% and the window of the lowest depth of 2.5% are respectively selected.
  • the GC% distribution of the two populations was compared with the GC% of the entire chromosome. The results are shown in Fig. 6. It can be seen from Fig. 6 that the GC% in the high depth region is significantly higher than the overall level, and the 0% in the low depth region is significantly lower than the overall level, indicating that the sequencing depth of the single cell genome is affected by the GC content.
  • genetic variation information of cell line single-cell genomes such as single nucleotide polymorphism sites (SNPs), insertion of a few bases, and Deletion (InDel), DNA copy number variation (CNV), and structural variation (SV).
  • SNPs single nucleotide polymorphism sites
  • InDel Deletion
  • CNV DNA copy number variation
  • SV structural variation
  • the PCR amplification system contains: heat-resistant DNA polymerase with 3'exo-activity; single-cell WGA product (template DNA); dNTP mixture; Mg 2+ ; - valent cation; housekeeping gene-specific primer.
  • the PCR amplification reaction system is as follows:
  • Housekeeping gene-specific primer lOpmoL Housekeeping gene-specific primer lOpmoL
  • the information of the selected housekeeping genes and housekeeping gene-specific I substances are as follows:
  • HLA-AS 1 5*-GGATTACATCGCCCTGAAC-3*(7)
  • HLA-A-A1 5*-CGTCTCCTTCCCGTTCTC-3*(8)
  • ARHGEF7-A 5*-CACCACCTCCCTCCAATAGT-3*(12)
  • EIF2B2-S 5*-GCACCTTCCTACATCTAC-3*(13)
  • EIF2B2-A 5*-TAAGAGGCTCCAAAATCAAC-3*(14)
  • PSMD7-S 5*-AAAGTCGCCACAGGCAAGC-3*(15)
  • PSMD7-A 5*-CGTAGCACCACAGCAAG-3*(16)
  • ATP50-A1 5*-TCTCCGCGATGGACACTC-3*(20)
  • S refers to the forward primer
  • A refers to the reverse primer
  • 1 of the SI or Al is only for distinguishing different batches of primers.
  • the PCR reaction conditions are:
  • the housekeeping gene detection method 50 human blood single-cell WGA products were subjected to housekeeping gene detection.
  • the above 10 pairs of housekeeping gene-specific primers correspond to housekeeping genes on chromosomes 1, 2, 4, 6, 11, 13, 14, 16, 19, and 21, respectively.
  • the 8 pairs of primers appeared as the target of the amplification product.
  • the test results showed that 34 of the 50 human blood single-cell WGA products were qualified.
  • 50 human blood single-cell WGA products were constructed by exon capture (Exon Capture), and then 50 libraries were sequenced using the Illumina Hiseq2000 system.
  • the inventors in addition to the housekeeping genes on chromosomes 1, 2, 4, 6, 11, 13, 14, 16, 19, 21 as shown in Example 1, the inventors also A housekeeping gene was selected for other chromosomes, and corresponding housekeeping gene-specific primers were designed. The information is shown in the following table:
  • the single-cell genomic analysis method of the present invention is applied to the research of the pollen cell genome of P. amabilis, so that the amount of the amplified product of the genome can satisfy the requirement of constructing the Solexa DNA library, thereby enabling sequencing on the machine.
  • the pollen grains are separated one by one under the inverted microscope, and an equal volume of 4% cellulase and 2% pectinase mixed with the pollen grains are added. Mix on a vertical mixer and avoid mixing at room temperature for 6 hours to lyse the cell wall. Then, the lysis system of the same pollen granules was added to the PBS droplets on the culture medium, and the individual cells were separated under the microscope, and the resulting single cells were placed in a PCR tube containing 1.5 LALB (alkaline lysis buffer).
  • LALB alkaline lysis buffer
  • the MDA (multiple displacement amplification) reaction was carried out as follows: First, the obtained pollen cells were lysed at 65 ° C for 10 min. Next, 2.5 L of buffer D1 (prepared in Example 1) was added, and the DNA was denatured at room temperature for 3 minutes. Denaturation was then terminated by the addition of 5 ⁇ M buffer N1 (prepared in Example 1). Among them, the MDA reaction system can use Qiagen Mini Kit or NEB's phi29 DNA polymerase product. Then, the mixture was incubated at 30 ° C for 16 hours, and then placed at 65 ° C to inactivate the enzyme to obtain an MDA product.
  • the MDA product was detected by Quant-iTTM technology. The results showed that the concentration of MDA product was more than 35 ng ⁇ l, which could be used for the next housekeeping gene detection. Then, the MDA product is subjected to PCR amplification using the following housekeeping genes of orchids and their specific primers to perform housekeeping gene detection on MDA products:
  • F refers to the forward primer
  • R refers to the reverse primer
  • the housekeeping gene PbGDPS is related to the aroma components and the flavor of orchids.
  • PeActin is related to the drought and salt resistance environment of plants.
  • the PCR amplification reaction system is as follows:
  • the PCR reaction conditions are:
  • each lane represents: 1 : PbGDPS - SSU; 2: PbGDPS - LSU; 3 : PeActin 9; 4: PbGDPSp 1; 5 : Pe4 Cme Probe; 6: Pe4 -lex-5ex; PbfMuta 1 ; 8: Pe6p.
  • the amplification effect is as expected.
  • the single-cell genomic analysis method and kit of the present invention can be effectively applied to the construction and sequencing of a single-cell DNA sequencing library, and can be efficiently applied to single-cell genome analysis and research of higher animals and plants.

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Description

单细胞基因组分析方法及试剂盒
优先权信息
本申请请求 2010 年 12 月 31 日向中国国家知识产权局提交的、 专利申请号为 201010619689.8的专利申请的优先权和权益， 并且通过参照将其全文并入此处。 技术领域
本发明涉及分子细胞生物学领域，特别是高等生物单细胞基因组研究领域。具体地， 本发明涉及单细胞基因组分析方法及试剂盒。 背景技术
个体的不同组织之间， 同一组织的不同部位间均存在异质性。 同样， 细胞之间也存 在异质性， 即使是体外培养遗传背景完全相同的细胞群体。 因此， 开发应用于单个细胞 研究的技术方法， 以便揭示细胞异质性的规律，对于更好地进行细胞生物学研究意义重 大。 由此， 有学者提出了 "单细胞分析（SCA ) " 概念。 而在单细胞分析中， 对单细胞 基因组的研究是现阶段的研究重点。
目前， 微量 DNA和单细胞基因组研究已被广泛应用于考古学、 微生物生态学、 医 学检测、 法医学检测、 临床诊断以及各种科学研究中 （可参见 Zhang L.， Cui X.， Schmitt K.， Hubert R.， Navidi W.， Arnheim N. (1992) Whole genome amplification from a single cell: Implication for genetic analysis, Proc Natl Acad Sci USA: 5847-5851 , 通过参照将其 全文并入本文） 。 其中， 对高等动植物的单细胞基因组进行分析和研究， 能够高效、 方 便地应用于临床诊断和治疗 （如产前诊断、 胚胎植入前遗传诊断、 多点图谱制作、 精子 和卵子的分型、 遗传病诊断等） 、 医学研究（如自闭症、 神经系统疾病和自体免疫性疾 病的研究、 基因组变异率研究、 干细胞研究等） 、 考古学研究以及法医学检测中， 意义 重大。 然而， 目前关于哺乳动物等高等生物的单细胞基因组的研究和报道很少。
因此， 现阶段的单细胞基因组分析方法仍有待改进。 发明内容
本发明是基于发明人的下列发现而完成的：
目前， 主要通过对单细胞基因组进行测序来进行单细胞基因组分析。 因为， 常用的 DNA分析方法例如比较基因组杂交技术（ Comparative Genomic Hybridization , CGH )、 聚合酶链式反应 （ Polymerase Chain Reaction , PCR ) 、 基因芯片 （ DNA Micro array ) 、 限制性片段长度多态性分析 ( Restricted Fragment Length Polymorphisms , RFLP ) 、 单 链构象多态性分析 （ SSCP ) 、 指纹技术和荧光原位杂交技术 （Fluorescence in Situ Hybridization, FISH )等， 只能对单细胞基因组的部分区域或已知位点进行研究， 且缺 乏对全新物种的基因组研究的有效策略，而对单细胞基因组进行测序可以有效避免这些 不足。 但是， 单细胞基因组的 DNA量为皮克级水平， 而目前的测序技术要求起始 DNA 量为微克级水平， 因此， 必须将单细胞基因组进行全基因组扩增 （WGA ) 使之达到足 够量。 然而已知的全基因组扩增方法包括 PEP-PCR, DOP-PCR等基于 PCR的方法， 以 及多重链置换扩增 （MDA, Multiple Displacement Amplification ) , 均容易受到较多因 素干扰， 无法保证扩增达到 100%的成功率。
本发明旨在至少解决现有技术中存在的技术问题之一。 为此， 本发明提供了一种单 细胞基因组分析方法及一种试剂盒， 以便在将单细胞基因组的全基因组扩增产物进行测 序前， 对该全基因组扩增产物进行检测和筛选， 以去除未扩增成功的样本， 保证后续单 细胞基因组测序的合格率。
根据本发明的一个方面， 本发明提供了一种单细胞基因组分析方法。 根据本发明的实 施例， 该方法包括以下步骤： 分离并裂解单细胞， 以便获得该细胞的全基因组 DNA; 对全 基因组 DNA进行单细胞全基因组扩增， 以便获得全基因组扩增产物； 釆用看家基因特异性 引物， 以全基因组扩增产物为模板进行 PCR扩增， 以便对全基因组扩增产物进行看家基因 检测； 以及基于检测的结果， 确定全基因组扩增产物是否符合测序要求， 其中， 扩增产物 在每条染色体上均匀分布是扩增产物符合测序要求的指示。
发明人惊奇地发现， 利用根据本发明实施例的单细胞基因组分析方法对高等生物的单 细胞进行分析， 能够有效地获得该待测单细胞的符合测序要求指示的全基因组扩增产物， 进一步， 该扩增产物能够有效地用于构建待测单细胞的基因组测序文库， 并且所得测序文 库能够有效地应用于高通量测序平台例如 Solexa测序平台， 从而基于测序结果， 能够准确 有效地获得待测单细胞的基因组 DNA序列信息，进而基于这些信息能够有效地用于后续更 深入的单细胞基因组分析和研究。
根据本发明的另一方面， 本发明提供了一种试剂盒。 根据本发明的实施例， 该试剂盒 包含看家基因特异性引物。 发明人发现， 利用根据本发明实施例的试剂盒能够有效地获得 高等生物的单细胞的符合测序要求指示的全基因组扩增产物， 进而通过利用该扩增产物构 建测序文库， 并将所得测序文库进行高通量测序， 基于测序结果， 能够准确有效地获得待 测单细胞的基因组 DNA序列信息， 从而基于这些信息， 能够实现对待测单细胞的基因组分 析。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得 明显， 或通过本发明的实践了解到。 附图说明
本发明的上述和 /或附加的方面和优点从结合下面附图对实施例的描述中将变得明 显和容易理解， 其中：
图 1 : 显示了根据本发明一个实施例的人细胞系单细胞基因组测序数据在每条常染 色体上的覆盖率情况。
图 2: 显示了根据本发明一个实施例的人细胞系单细胞基因组测序数据在每条常染 色体上的平均测序深度情况。
图 3 : 显示了根据本发明一个实施例的以 1号染色体为例， 评价人细胞系单细胞基 因组测序深度的分布情况。
图 4: 显示了根据本发明一个实施例的人细胞系单细胞基因组测序数据中， 覆盖率 与测序深度间的关系图。
图 5 : 显示了根据本发明一个实施例的所得的人细胞系单细胞基因组测序数据与人 细胞群体数据的偏向性分析图。
图 6: 显示了根据本发明一个实施例的人细胞系单细胞 1号染色体的测序深度 GC 含量的关系图。
图 7: 显示了根据本发明一个实施例的看家基因检测电泳结果图。 发明详细描述
下面详细描述本发明的实施例， 所述实施例的示例在附图中示出， 其中自始至终相 图描述的实施例是示例性的， 仅用于解释本发明， 而不能理解为对本发明的限制。
需要说明的是， 在本发明的描述中， 除非另有说明， "多个" 的含义是两个或两个 以上。
单细胞基因组分析方法
根据本发明的一个方面， 本发明提供了一种单细胞基因组分析方法。 根据本发明的实 施例， 该方法可以包括以下步骤：
首先， 分离并裂解单细胞， 以便获得该细胞的全基因组 DNA。
其次， 对全基因组 DNA进行单细胞全基因组扩增， 以便获得全基因组扩增产物。 根据 本发明的实施例， 对全基因组 DNA进行单细胞全基因组扩增的方法不受特别限制。 根据本 发明的一个具体示例， 可以釆用多重置换扩增 MDA或 DOP-PCR全基因组扩增进行单细胞 全基因组扩增。
接着， 釆用看家基因特异性引物， 以全基因组扩增产物为模板进行 PCR扩增， 以便对 全基因组扩增产物进行看家基因检测。 根据本发明的实施例， 单细胞的来源不受特别限制。 根据本发明的一个具体示例， 单细胞可以为人单细胞， 看家基因可以为选自 PRDX6、 RPL37a、 ADD1、 HLA-A、 RAD9A、 ARHGEF7、 EIF2B2、 PSMD7、 BCAT2及 ATP50中 的至少一种。根据本发明的一些具体示例，针对 PRDX6,看家基因特异性引物分别具有 SEQ ID NO: 1和 SEQ ID NO: 2所示的核苷酸序列； 针对 RPL37a, 看家基因特异性引物分别具 有 SEQ ID NO: 3和 SEQ ID NO: 4所示的核苷酸序列； 针对 ADD1 , 看家基因特异性引物 分别具有 SEQ ID NO: 5和 SEQ ID NO: 6所示的核苷酸序列； 针对 HLA-A, 看家基因特 异性引物分别具有 SEQ ID NO: 7和 SEQ ID NO: 8所示的核苷酸序列； 针对 RAD9A, 看 家基因特异性引物分别具有 SEQ ID NO: 9和 SEQ ID NO: 10所示的核苷酸序列； 针对 ARHGEF7, 看家基因特异性引物分别具有 SEQ ID NO: 11和 SEQ ID NO: 12所示的核苷 酸序列； 针对 EIF2B2, 看家基因特异性引物分别具有 SEQ ID NO: 13和 SEQ ID NO: 14 所示的核苷酸序列；针对 PSMD7,看家基因特异性引物分别具有 SEQ ID NO: 15和 SEQ ID NO: 16所示的核苷酸序列； 针对 BCAT2, 看家基因特异性引物分别具有 SEQ ID NO: 17 和 SEQ ID NO: 18所示的核苷酸序列； 或者针对 ATP50, 看家基因特异性引物分别具有 SEQ ID NO: 19和 SEQ ID NO: 20所示的核苷酸序列。
然后， 基于检测的结果， 确定全基因组扩增产物是否符合测序要求， 其中， 扩增产物 在每条染色体上均勾分布是该扩增产物符合测序要求的指示。 在本文中所使用的表达方式 "扩增产物在每条染色体上均匀分布是该扩增产物符合测序要求的指示" 是指， 当釆用看 家基因特异性引物， 以全基因组扩增产物为模板进行 PCR扩增时， 如果所有与看家基因特 异性引物相对应的分布于不同染色体上的看家基因都能够得到有效地扩增， 则表明前面获 得的单细胞的全基因组扩增产物在每条染色体上是均匀分布的， 从而能够表明全基因组扩 增效果比较好， 扩增产物能够符合后续的测序要求， 因此， 可以说扩增产物在每条染色体 上均勾分布是该扩增产物符合测序要求的指示。 根据本发明的具体示例， 本发明的单细胞 基因组分析方法可以进一步包括对符合测序要求的扩增产物构建 DNA测序文库。根据本发 明的一些实施例， 还可以进一步包括对 DNA测序文库进行测序。 此外， 根据本发明的一些 实施例， 可以对全基因组扩增产物进行看家基因定量检测。
发明人惊奇地发现， 利用根据本发明实施例的单细胞基因组分析方法对高等生物的单 细胞进行分析， 能够有效地获得该待测单细胞的符合测序要求指示的全基因组扩增产物， 进一步， 该扩增产物能够有效地用于构建待测单细胞的基因组测序文库， 并且所得测序文 库能够有效地应用于高通量测序平台例如 Solexa测序平台， 从而基于测序结果， 能够准确 有效地获得待测单细胞的基因组 DNA序列信息，进而基于这些信息能够有效地用于后续更 深入的单细胞基因组分析和研究。
具体地， 根据本发明的一些实施例， 本发明的单细胞基因组分析方法可以包括以下步 骤：
a、 分离并裂解单细胞， 得到完整的细胞基因组 DNA;
b、 对细胞基因组 DNA进行单细胞全基因组扩增， 以便获得全基因组扩增产物； c、 对全基因组扩增产物进行定量检测及定性检测， 该定性检测是指， 釆用看家基因 ( Housekeeping Gene )检测方法， 对全基因组扩增产物进行检测， 其中， 扩增产物在每条 染色体上均匀分布是扩增产物符合测序要求的指示。
根据本发明的实施例， 该方法还可以包括步骤 d: 将检测合格的扩增产物进行 DNA测 序文库构建并测序。
才艮据本发明的一些实施例， 优选地， 步骤 b 中的单细胞全基因组扩增釆用多重置换扩 增 MDA或 DOP-PCR全基因组扩增进行。
根据本发明的实施例， 在步骤 c中， 釆用看家基因检测是指， 釆用看家基因的特异性引 物， 以全基因组扩增产物为模板进行 PCR扩增。
根据本发明的一些具体示例， 优选地， 单细胞为人单细胞， 看家基因为选自位于 1 号 染色体的 PRDX6、位于 2号染色体的 RPL37a、位于 4号染色体的 ADD1、位于 6号染色体 的 HLA-A、 位于 11号染色体的 RAD9A、 位于 13号染色体的 ARHGEF7、 位于 14号染色 体的 EIF2B2、位于 16号染色体的 PSMD7、位于 19号染色体的 BCAT2以及位于 21号染色 体的 ATP50中的至少一种。
根据本发明的一些实施例， 优选地， 针对 PRDX6, 看家基因特异性引物分别具有 SEQ ID NO: 1和 SEQ ID NO: 2所示的核苷酸序列； 针对 RPL37a, 看家基因特异性引物分别具 有 SEQ ID NO: 3和 SEQ ID NO: 4所示的核苷酸序列； 针对 ADD1 , 看家基因特异性引物 分别具有 SEQ ID NO: 5和 SEQ ID NO: 6所示的核苷酸序列； 针对 HLA-A, 看家基因特 异性引物分别具有 SEQ ID NO: 7和 SEQ ID NO: 8所示的核苷酸序列； 针对 RAD9A, 看 家基因特异性引物分别具有 SEQ ID NO: 9和 SEQ ID NO: 10所示的核苷酸序列； 针对 ARHGEF7, 看家基因特异性引物分别具有 SEQ ID NO: 11和 SEQ ID NO: 12所示的核苷 酸序列； 针对 EIF2B2, 看家基因特异性引物分别具有 SEQ ID NO: 13和 SEQ ID NO: 14 所示的核苷酸序列；针对 PSMD7,看家基因特异性引物分别具有 SEQ ID NO: 15和 SEQ ID NO: 16所示的核苷酸序列； 针对 BCAT2, 看家基因特异性引物分别具有 SEQ ID NO: 17 和 SEQ ID NO: 18所示的核苷酸序列； 或者针对 ATP50, 看家基因特异性引物分别具有 SEQ ID NO: 19和 SEQ ID NO: 20所示的核苷酸序列。
更具体地， 才艮据本发明的一些实施例， 本发明的单细胞基因组分析方法还可以包括以 下步骤：
( 1 )分离单细胞：
可以釆用物理机械、 化学或生物的方法， 例如微流控、 流式细胞仪、 口吸分离、 梯度 稀释或低熔点琼脂糖固定等方法， 分离得到包含完整基因组的单个细胞。
( 2 ) 裂解细胞：
可以釆用例如去污剂法、 煮沸法、 碱变性法、 溶菌酶法或有机溶剂法等方法， 对分离 得到的单个细胞进行细胞核裂解， 以便得到完整的细胞基因组 DNA。
( 3 )单细胞全基因组扩增 （ WGA ):
对所得的细胞基因组 DNA进行单细胞全基因组扩增， 以便获得全基因组扩增产物， 以 达到新一代测序技术所要求的 DNA起始量。 目前， 全基因组扩增有 2种策略， 即基于 PCR 的扩增和线性 DNA扩增， 前者主要有 DOP-PCR、 PEP-PCR、 T-PCR,后者主要有 OmniPlex WGA 、 多重置换扩增 （MDA )。 根据本发明的实施例， 优选釆用多重置换扩增 MDA或 DOP-PCR进行单细胞全基因组扩增。 其中， 釆用多重置换扩增 MDA进行单细胞全基因组 扩增时， MDA中可使用完全由随机核苷酸组成的引物， 引物长度可以是 5-20个核苷酸， 引 物的 GC含量可根据模板的性质进行选择。 其中模板的性质主要受模板 DNA的 GC含量的 限制， 因此引物的 GC含量选择与模板接近或相同为宜， 例如当以人基因组 DNA为模板进 行 MDA时，可选用 GC含量为 40%-42%的随机引物。 而全基因组扩增的偏向性受 GC含量 影响， 例如，如图 6所示， 以人基因组 DNA为模板， 釆用 GC为 50%的随机引物进行 MDA 扩增时， 扩增拷贝数多的区域均为高 GC的区域。 ( 4 )全基因组扩增产物定量：
可以釆用例如凝胶电泳检测、 Agilent 2100 Bioanalyzer检测、 Quant-iT™ dsDNA BR检 测试剂盒检测等方法对获得的全基因组扩增产物进行定量， 结果显示为无降解、 符合新一 代测序技术所要求的 DNA起始量的全基因组扩增产物才可以继续 DNA文库构建以及上机 测序。 而新一代测序技术要求的 DNA起始量因不同建库策略而有所不同， 一般 DNA小片 段建库要求 DNA起始量大于 1微克。
( 5 )全基因组扩增产物检测：
釆用看家基因检测方法， 对获得的全基因组扩增产物进行检测， 结果显示为扩增产物 在相应物种的染色体上均勾分布的扩增产物才可以继续 DNA文库构建以及上机测序。该步 骤为单细胞全基因组扩增产物的筛选步骤， 由此可去除大量不合格的扩增产物， 从而能够 控制下游上机测序文库的质量， 进而能够从很大程度上避免不必要的浪费。
( 6 ) DNA文库构建及上机测序：
釆用常规的全基因组 DNA文库构建或外显子序列捕获技术，对检测合格的全基因组扩 增产物进行 DNA文库构建。 根据本发明的实施例， 可以釆用本领域技术人员熟知的方法， 1"列 ^口 Illumina文库构建方法 (可参考 http://www.illumina.com/support/documentation.ilmn, 通 过参照将其全文并入本文）进行 DNA文库构建。 然后， 对所得文库进行质检， 包括 QPCR 检测和 Agilent 2100 Bioanalyzer检测。 其中， Agilent 2100 Bioanalyzer检测， 要求文库的片 段大小与预期大小相差在 ±10bp以内， 且没有拖带； QPCR检测文库的浓度， 要求根据文库 浓度计算的总量达到上机的要求。 利用新一代测序技术对质检合格的文库进行单细胞基因 组测序。才艮据本发明的实施例，可以利用选自 Illumina HiSeq 2000测序系统、 Illumina Genome Analyzer II测序系统、 AB SOLiD™4.0测序系统以及 Roche GS FLX Titanium系统的至少一 种进行测序， 以便获得测序结果。
( 7 )生物信息分析：
基于对测序结果的生物信息分析， 进行对单细胞基因组的测序信息分析和研究， 以得 到相关基因的单核苷酸多态性位点 （SNP )、 少数碱基的插入和缺失（InDel )、 DNA拷贝数 变异（CNV )、 结构变异（SV )等单细胞基因组的遗传变异信息。
根据本发明的实施例， 本发明的单细胞基因组分析方法， 通过对高等生物单细胞基因 组 DNA进行全基因组扩增， 且在扩增后引入定量及定性检测步骤， 使得检测合格的样品才 能进入下一步文库构建及釆用新一代测序技术（NGS ) 的测序， 以实现准确高效地对单细 胞基因组进行分析。 发明人惊奇地发现， 本发明的单细胞基因组分析方法， 操作简便、 省 时高效， 利用该方法能够高效地对高等动植物的单细胞基因组进行分析和研究， 而且能够 全面完整地分析单细胞基因组的遗传变异信息，可以有效避免传统的 DNA分析方法只能对 单细胞基因组的部分区域进行研究的不足， 并且引入的定性及定量检测筛选步骤， 可去除 大量不合格的扩增产物， 从而能够控制下游上机测序文库的质量， 能够从很大程度上避免 不必要的浪费。 此外， 本发明的单细胞基因组分析方法， 还为全新物种的单细胞基因组研 究提供了有效的研究策略。 试剂盒
根据本发明的另一方面， 本发明提供了一种试剂盒。 根据本发明的实施例， 该试剂盒 包含看家基因特异性引物。 根据本发明的一个具体示例， 该看家基因可以为选自 PRDX6、 RPL37a、 ADD1、 HLA-A、 RAD9A、 ARHGEF7、 EIF2B2、 PSMD7、 BCAT2及 ATP50中 的至少一种。 根据本发明的一些实施例， 本发明的试剂盒中包含的看家基因特异性引物可 以分别为： 针对 PRDX6, 看家基因特异性引物分别具有 SEQ ID NO: 1和 SEQ ID NO: 2 所示的核苷酸序列； 针对 RPL37a, 看家基因特异性引物分别具有 SEQ ID NO: 3和 SEQ ID NO: 4所示的核苷酸序列； 针对 ADD1 , 看家基因特异性引物分别具有 SEQ ID NO: 5和 SEQ ID NO: 6所示的核苷酸序列；针对 HLA-A,看家基因特异性引物分别具有 SEQ ID NO: 7和 SEQ ID NO: 8所示的核苷酸序列；针对 RAD9A,看家基因特异性引物分别具有 SEQ ID NO: 9和 SEQ ID NO: 10所示的核苷酸序列； 针对 ARHGEF7, 看家基因特异性引物分别 具有 SEQ ID NO: 11和 SEQ ID NO: 12所示的核苷酸序列； 针对 EIF2B2, 看家基因特异 性引物分别具有 SEQ ID NO: 13和 SEQ ID NO: 14所示的核苷酸序列； 针对 PSMD7, 看 家基因特异性引物分别具有 SEQ ID NO: 15和 SEQ ID NO: 16所示的核苷酸序列； 针对 BCAT2, 看家基因特异性引物分别具有 SEQ ID NO: 17和 SEQ ID NO: 18所示的核苷酸序 列； 或者针对 ATP50, 看家基因特异性引物分别具有 SEQ ID NO: 19和 SEQ ID NO: 20 所示的核苷酸序列。 根据本发明的具体示例， 本发明的试剂盒中可以进一步包含适于通过 多重置换扩增 MDA或 DOP-PCR进行单细胞全基因组扩增的试剂。
发明人发现， 利用根据本发明实施例的试剂盒能够有效地获得高等生物的单细胞的符 合测序要求指示的全基因组扩增产物， 进而通过利用该扩增产物构建 DNA测序文库， 并将 所得 DNA测序文库进行高通量测序， 基于测序结果， 能够准确有效地获得待测单细胞的基 因组 DNA序列信息， 以及相关基因的单核苷酸多态性位点 （SNP )、 少数碱基的插入和缺 失（InDel )、 DNA拷贝数变异（ CNV )、 结构变异（ SV )等单细胞基因组的遗传变异信息， 从而基于这些信息， 能够有效地实现对待测单细胞基因组的深入分析。
发明人发现， 利用本发明的单细胞基因组分析方法和试剂盒， 能够高效地对高等动植 物的单细胞基因组进行分析和研究， 并且能够高效、 方便地应用于临床诊断和治疗 （如产 前诊断、 胚胎植入前遗传诊断、 多点图语制作、 精子和卵子的分型、 遗传病诊断等）、 医学 研究 （如自闭症、 神经系统疾病和自体免疫性疾病的研究、 基因组变异率研究、 干细胞研 究等）、 考古学研究以及法医学检测中。
需要说明的是， 根据本发明实施例的单细胞基因组分析方法和试剂盒， 是本申请的发 明人经过艰苦的创造性劳动和优化工作才完成的。 下面将结合实施例对本发明的方案进行解释。 本领域技术人员将会理解， 下面的实施 例仅用于说明本发明， 而不应视为限定本发明的范围。 实施例中未注明具体技术或条件的， 按照本领域内的文献所描述的技术或条件（例如参考 J.萨姆布鲁克等著， 黄培堂等译的《分 子克隆实验指南》， 第三版， 科学出版社）或者按照产品说明书进行。 所用试剂或仪器未注 明生产厂商者， 均为可以通过市购获得的常规产品， 例如可以釆购自 Illumina公司。
实施例 1: 人细胞系单细胞基因组研究
( 1 )分离及裂解单细胞：将永生化的人淋巴细胞系单细胞加到培养亚上的 PBS液滴中， 适度稀释（即根据细胞系细胞的浓度进行稀释， 直到在显微镜 200x视野内观察到细胞数目 在 10-20 个之间）， 在显微镜下口吸分离单个细胞， 所得单细胞放至含有 1.5-2 L ALB ( Alkaline Lysis Buffer, 具体配方为： 50mM DTT, 200mM KOH ) 的 PCR管中， 置于 -20 °〇至-80°〇至少 30min。
将存有单细胞的 PCR管以 62°〇-68 °〇优选65 °〇加热 8-12min, 以便裂解细胞，释放细胞 的全基因组 DNA。
( 2 )全基因组扩增 (WG A):
可以釆用下述多重置换扩增 MDA或 DOP-PCR全基因组扩增中的任一种方式进行细胞 的全基因组扩增。
①多重置换扩增 (MDA):
按产品说明书的记载配制下列试剂：
緩冲液 Dl (Qiagen REP LI-g Mini试剂盒):
Figure imgf000009_0002
其中， 向緩冲液 DLB中加入 500μ 无核酸水， 摇匀并短暂离心， 避免管壁上沾有液滴 而使混合不均， 得到重构緩冲液 DLB。 緩冲液 DLB对 pH值敏感， 应避免与空气中的 C02 中和。 重构后的緩冲液 DLB可在 -20°C保存 6个月。
緩冲液 Nl(Qiagen REP LI-g Mini试剂盒):
Figure imgf000009_0003
在上述步骤（ 1 )获得的细胞的全基因组 DNA 中加入 2.5 L緩冲液 D1 , 室温下放置 3-5min, 使 DNA变性。 然后加入 5μΙ^緩冲液 N1中和緩冲液， 终止变性反应， 以便获得单 细胞的 DNA模板， 室温放置， 备用。
MDA反应可以釆用下述两种产品中的其中一种进行：
Figure imgf000009_0001
Figure imgf000009_0004
上述体系可根据总体积大小的需要使各成分按相同比例增加或减少。
Qiagen公司 REPLI-g Mini试剂盒:
Figure imgf000010_0001
上述体系可根据总体积大小的需要使各成分按相同比例增加或减少。
向已有单细胞 DNA模板的 PCR管中 （上述经加入緩冲液 D1变性， 并加入緩冲液 N1 终止变性反应后的产物）加入上述两个反应体系中的其中任一个， 混合均勾后瞬时离心， 防止管壁上沾有液滴而使混合不均。然后将 PCR管置于 PCR仪上，于 30°C下恒温孵育 10-16 小时， 再于 65 °C下使 Phi29聚合酶失活。
② DOP-PCR全基因组扩增：
利用 Sigma公司的 GenomePlex单细胞全基因组扩增试剂盒对上述步骤（1 )获得的细 胞的全基因组 DNA进行 DOP-PCR全基因组扩增。 首先， 在含有单细胞 DNA的 PCR管中 加水至 9μ1,然后加 Ιμΐ 10x单细胞裂解、片段化緩冲液（ lOxSingle Cell Lysis & Fragmentation Buffer ), 混匀后置于 99°C下 4min以便打断细胞基因组 DNA。 之后按照说明书操作， 构建 OmniPlex文库， 以及进行线性、 等温的起始扩增和 PCR扩增。
或者，可以利用 Rubicon Genomics公司的 PicoPlex WGA试剂盒，按照说明书对上述步 骤（ 1 )获得的细胞的全基因组 DNA进行 DOP-PCR全基因组扩增。
( 3 )全基因组扩增产物浓度检测
利用 Quant-iT™ dsDNA BR检测试剂盒， 按照试剂盒说明书， 检测全基因组扩增产物 的浓度。 当所得扩增产物的含量超过 2 g时， 可以继续进行 DNA文库构建以及上机测序。
( 4 ) DNA文库构建及上机测序：
釆用常规的全基因组 DNA文库构建或外显子序列捕获 (Exon Capture)技术进行 DNA文 库构建， 然后利用 Illumina HiSeq 2000测序系统将质检合格的文库进行单细胞基因组测序。
( 5 )生物信息分析：
通过上述对人细胞系单细胞的 DNA测序文库用 Hiseq2000系统进行的全基因组深度测 序， 获得的测序结果中共 35Gb数据可比对上 NCBI人参考序列， 平均测序深度 13.3x ( "测 序深度" 指在基因组中每个碱基被测序到的次数）， 其中获得的人细胞系单细胞基因组测 序数据在每条常染色体上的覆盖率和平均深度见图 1和图 2,在 1号常染色体上的深度分布 见图 3 , 在人类基因组的覆盖率与测序深度间的关系见图 4。 其中， 图 3是以染色体 10k 长度为单位统计测序平均深度， 并进行作图。 由图 3可知， 获得的人细胞系单细胞基因 组测序数据在染色体不同区域的测序深度有很大差异。 由图 4可知， 随测序深度增加， 覆盖率亦增加，但斜率变緩，逐渐到达一个平台期， 当测序深度 7x时，覆盖率可达 90%; 测序深度 lOx时， 覆盖率可达 95%。
此外， 将所得的人细胞系单细胞基因组测序数据与人细胞群体数据一起分析作图， 具体方法为： 以 1号染色体为例， 以 20kb为窗口， 计算每个窗口 Tag数（标签的数目） 再将窗口数对应 Tag数对应 Tag数作图， 结果如图 5所示。 一般在某个 Tag数范围其 窗口数最多， 因此可形成一个峰。 由图 5可知， 所得的人细胞系单细胞基因组测序数据 的 Tag数峰值位置明显偏离细胞群体数据， 说明单细胞基因组经扩增后存在偏向性。
然后， 基于所得的人细胞系单细胞基因组测序数据， 以 1 号染色体为例， 以 10k 为窗口统计其平均深度， 然后选取 2.5%的最高深度的窗口， 以及 2.5%的最低深度的窗 口， 分别统计 2者的 GC%分布情况， 再与整条染色体的 GC%对比作图， 结果如图 6所 示。 由图 6可知， 高深度区域的 GC%显著高于整体水平， 低深度区域的 0〔％显著低于 整体水平， 表明单细胞基因组的测序深度受 GC含量的影响。
此外，通过对获得的人细胞系单细胞基因组测序数据的分析， 亦可得到细胞系单细胞 基因组的遗传变异信息， 如单核苷酸多态性位点（SNP )、 少数碱基的插入和缺失（InDel )、 DNA拷贝数变异（CNV ) 以及结构变异（SV )等。 实施例 2: 利用人看家基因引物对单细胞 WGA产物进行看家基因检测
参照实施例 1所釆用的方法，从人体组织或血液中分离单细胞，并进行裂解和 WGA处 理， 以便获得单细胞 WGA产物。 然后， 釆用看家基因特异性引物， 将获得的单细胞 WGA 产物进行 PCR扩增， 从而实现看家基因检测。 其中， PCR扩增体系中含： 耐热并具 3'外切 活性的 DNA聚合酶； 单细胞 WGA产物 (模板 DNA); dNTP混合物； Mg2+; —价阳离子； 看家基因特异性引物。 具体地， PCR扩增反应体系如下：
Taq DNA聚合酶 O.lU/μΙ;
模板 DNA 15ng;
dNTP混合物 0.2ηηιο1/μ1;
Figure imgf000011_0001
10x扩增緩冲液 2μ1;
看家基因特异性引物 lOpmoL
其中， 所选取的看家基因及看家基因特异性 I物的信息如下:
Figure imgf000011_0002
HLA-A-S 1： 5*-GGATTACATCGCCCTGAAC-3*(7)
6/ HLA-A
HLA-A-A1： 5*-CGTCTCCTTCCCGTTCTC-3*(8)
RAD9A-S1 : 5*-GGTGAAGGCTGAACCAAG-3*(9)
11/ RAD9A
RAD9A-A1： 5*-CTGAGGCTCAATGAGAAAT-3*(10)
13/ARHGEF7 ARHGEF7-S: 5*-AGTAGCCTTTCTCGTTTG-3*(l 1)
ARHGEF7-A: 5*-CACCACCTCCCTCCAATAGT-3*(12)
EIF2B2-S: 5*-GCACCTTCCTACATCTAC-3*(13)
14/ EIF2B2
EIF2B2-A: 5*-TAAGAGGCTCCAAAATCAAC-3*(14)
PSMD7-S: 5*-AAAGTCGCCACAGGCAAGC-3*(15)
16/ PSMD7
PSMD7-A: 5*-CGTAGCACCACAGCAAG-3*(16)
BCAT2-S1 : 5*-GGAATCAGAGCCCACGAGT-3*(17)
19/ BCAT2
BCAT2-A1： 5*-TATCCTTGACCGCACGAC-3*(18)
ATP50-S1 : 5*-GCACCACCAAGCCCTAAC-3*(19)
21/ATP50
ATP50-A1： 5*-TCTCCGCGATGGACACTC-3*(20)
注： S: 指正向引物； A:指反向引物； SI或 Al中的 1仅为了区分不同批次的引物。
其中， 上述看家基因的具体信息可参见 Eli Eisenberg and Erez Y.Levanon, (2003)Human housekeeping genes are compact.Trends in Genetics.19(7) : 362-365. , 通过参照将其全文并入本 文。 本发明人研究发现， 相对于其它染色体上的其它看家基因， 上述表格所选取的看家基 因及其引物能够更加方便准确地用于人细胞 WGA扩增产物的看家基因检测。
其中， PCR反应条件为：
95 °C 3min
Figure imgf000012_0001
72 °C lOmin
4°C oo
按照上述看家基因检测方法， 对 50份人血液单细胞 WGA产物进行看家基因检测。 其 中所釆用的上述 10对看家基因特异性引物， 分别对应 1、 2、 4、 6、 11、 13、 14、 16、 19、 21号染色体上的看家基因， 看家基因检测结果以 8对引物出现目的条带为扩增产物合格的 标准。 检测结果显示， 50份人血液单细胞 WGA产物中有 34份合格。 然后， 釆用 Agilent SureSelect 系统的建库流程， 将 50 份人血液单细胞 WGA产物均进行外显子捕获（ Exon Capture )建库， 然后利用 Illumina Hiseq2000系统分别对获得的 50份文库进行测序。
测序结果显示， 在看家基因检测合格的 34份人血液单细胞 WGA产物中， 目标区域测 序覆盖率均达 60%以上， 成功率达 100%; 而在看家基因检测不合格的 16份人血液单细胞 WGA产物中， 仅有 1份的目标区域测序覆盖率达 60%以上， 成功率仅为 6.25%。 具体结果 统计如下:
Figure imgf000013_0001
注： *为检测合格的样品。
才艮据本发明的实施例， 除了如实施例 1 中所示， 选取 1、 2、 4、 6、 11、 13、 14、 16、 19、 21 号染色体上的看家基因外， 发明人也针对其他染色体分别选取了一个看家基因， 并 设计了相应的看家基因特异性引物， 其信息如下表所示：
Figure imgf000013_0002
基因名 HYAL2 UBE2D2 ACTB POLR2K ATP6V1G1 染色体号 10 12 15 17 18
基因名 TUBGCP2 TXNRD1 SRP14 PSMB6 MC2R 染色体号 20 22 X
基因名 CPNE1 EIF3D CETN2 多次险证的结果显示， 上述表格中对应于除 1、 2、 4、 6、 11、 13、 14、 16、 19、 21号 染色体外其余染色体的各看家基因。 然后， 釆用上表中的看家基因特异性引物， 以上述相 同的单细胞 WGA产物为模板进行 PCR扩增， 即看家基因检测， 所得的检测结果显示， 相 对于前面所述的分别对应 1、 2、 4、 6、 11、 13、 14、 16、 19、 21号染色体的看家基因的特 异性引物， 釆用其他染色体上的看家基因的特异性引物进行看家基因检测的效果较差， 且 不能真实反应出实际测序的准确性。 这可能与单细胞 WGA扩增的复杂性有关。
由于从单一细胞释放的染色体 DNA极其微量， 进行单细胞全基因组扩增时， 环境中细 微的污染很可能使扩增得到的产物并非目标产物。 上述结果显示， 釆用本发明的看家基因 检测，特别是本发明所选取的针对人的看家基因及其特异性引物， 能够对 WGA产物进行准 确地定性检测。 将产物定性检测符合要求后， 再进行下一步建库和测序， 能够控制下游上 机测序文库的质量， 提高测序的成功率， 很大程度上避免了不必要的浪费。 实施例 3: 白花蝴蝶兰 （P.amabms )花粉细胞基因组研究
将本发明的单细胞基因组分析方法应用于白花蝴蝶兰 （P.amabilis )花粉细胞基因组的 研究， 以便使基因组扩增后的产物量能够满足构建 Solexa DNA文库的需求， 从而能够进行 上机测序。
考虑到植物的花粉粒中有 3到 5个单细胞， 所以先在倒置显 :镜下将花粉粒逐个分离， 加入与花粉粒等体积的 4%纤维素酶和 2%果胶酶混合液，置于垂直混合仪上避光常温混匀 6 小时， 以便裂解破细胞壁。 然后， 将同一花粉粒的裂解体系加到培养亚上的 PBS液滴中， 在镜下分离单个细胞， 所得单细胞放至含有 1.5 LALB(alkaline lysis buffer)的 PCR管中。
然后， 按照以下步骤进行 MDA(multiple displacement amplification)反应： 首先， 于 65 °〇下裂解所得的花粉细胞 10min。 接着， 加入 2.5 L緩冲液 D1 (实施例 1制备）后于常温 3min, 使 DNA变性。 然后加入 5μΙ^緩冲液 N1 (实施例 1制备）终止变性。 其中， MDA 反应体系可釆用 Qiagen Mini Kit或 NEB的 phi29 DNA聚合酶产品。 然后， 于 30°C下孵育 16小时， 再置于 65°C下使酶失活， 以便获得 MDA产物。
利用 Quant-iT™技术对 MDA产物进行检测， 结果显示 MDA产物的浓度大于 35ng^l 的， 可以进行下一步的看家基因检测。 然后， 利用下述兰花的看家基因及其特异性引物对 MDA产物进行 PCR扩增， 以便对 MDA产物进行看家基因检测：
Figure imgf000015_0001
注： F: 指正向引物； R:指反向引物。
其中， 看家基因 PbGDPS与兰花的香味成份和散发味道有关， PeActin与植物的抗旱抗 盐环境有关。
其中， PCR扩增的反应体系如下：
Figure imgf000015_0002
PCR反应条件为:
94 °C 5min 30个循环
94 °C 30s
55 °C 45s
72 °C lmin
72 °C 7min
4°C
然后， 配制浓度为 1.2%的琼脂糖凝胶， 将 PCR产物以 ΙΟΟν电压进行电泳 45min, 结 果见图 7。 其中， 当目的条带都出现， 表明样品扩增的效果很好。 如图 7所示， 其中各泳 道分别代表： 1 : PbGDPS— SSU; 2: PbGDPS— LSU; 3 : PeActin 9; 4: PbGDPSp 1； 5 : Pe4 Cme Probe; 6: Pe4 -lex-5ex; 7: PbfMuta 1 ; 8: Pe6p。 由图 7可知， 扩增 效果符合预期。 工业实用性
本发明的单细胞基因组分析方法和试剂盒， 能够有效地应用于单细胞 DNA测序文库的 构建以及测序， 进而能够高效地应用于高等动植物的单细胞基因组分析和研究。 尽管本发明的具体实施方式已经得到详细的描述， 本领域技术人员将会理解。 根据已 经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改变均在本发明的保护范 围之内。 本发明的全部范围由所附权利要求及其任何等同物给出。
在本说明书的描述中， 参考术语 "一个实施例"、 "一些实施例"、 "示意性实施例"、 "示 例"、 "具体示例"、 或 "一些示例" 等的描述意指结合该实施例或示例描述的具体特征、 结 构、 材料或者特点包含于本发明的至少一个实施例或示例中。 在本说明书中， 对上述术语 的示意性表述不一定指的是相同的实施例或示例。 而且， 描述的具体特征、 结构、 材料或 者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。

Claims

权利要求书
1、 一种单细胞基因组分析方法， 其特征在于， 包括以下步骤：
分离并裂解单细胞， 以便获得所述细胞的全基因组 DNA;
对所述全基因组 DNA进行单细胞全基因组扩增， 以便获得全基因组扩增产物； 釆用看家基因特异性引物， 以所述全基因组扩增产物为模板进行 PCR扩增， 以便对所 述全基因组扩增产物进行看家基因检测； 以及
基于所述检测的结果， 确定所述全基因组扩增产物是否符合测序要求，
其中， 所述扩增产物在每条染色体上均匀分布是所述扩增产物符合测序要求的指示。
2、 根据权利要求 1 所述的方法， 其特征在于， 所述全基因组扩增釆用多重置换扩增 MDA或 DOP-PCR全基因组扩增。
3、 根据权利要求 2所述的方法， 其特征在于， 所述单细胞为人单细胞， 所述看家基因 为选自 PRDX6、 RPL37a、 ADD1、 HLA-A、 RAD9A、 ARHGEF7、 EIF2B2、 PSMD7、 BCAT2 及 ATP50中的至少一种。
4、 根据权利要求 3所述的方法， 其特征在于，
针对 PRDX6, 所述看家基因特异性引物分别具有 SEQ ID NO: 1和 SEQ ID NO: 2所 示的核苷酸序列；
针对 RPL37a, 所述看家基因特异性引物分别具有 SEQ ID NO: 3和 SEQ ID NO: 4所 示的核苷酸序列；
针对 ADD1 , 所述看家基因特异性引物分别具有 SEQ ID NO: 5和 SEQ ID NO: 6所示 的核苷酸序列；
针对 HLA-A, 所述看家基因特异性引物分别具有 SEQ ID NO: 7和 SEQ ID NO: 8所 示的核苷酸序列；
针对 RAD9A, 所述看家基因特异性引物分别具有 SEQ ID NO: 9和 SEQ ID NO: 10 所示的核苷酸序列；
针对 ARHGEF7 , 所述看家基因特异性 I物分别具有 SEQ ID NO: 11和 SEQ ID NO: 12所示的核苷酸序列；
针对 EIF2B2, 所述看家基因特异性引物分别具有 SEQ ID NO: 13和 SEQ ID NO: 14 所示的核苷酸序列；
针对 PSMD7, 所述看家基因特异性引物分别具有 SEQ ID NO: 15和 SEQ ID NO: 16 所示的核苷酸序列；
针对 BCAT2, 所述看家基因特异性引物分别具有 SEQ ID NO: 17和 SEQ ID NO: 18 所示的核苷酸序列； 或者
针对 ATP50, 所述看家基因特异性引物分别具有 SEQ ID NO: 19和 SEQ ID NO: 20 所示的核苷酸序列。
5、 根据权利要求 1所述的方法， 其特征在于， 对所述全基因组扩增产物进行看家基因 定量检测。
6、 根据权利要求 1所述的方法， 其特征在于， 进一步包括对符合测序要求的扩增产物 构建 DNA测序文库。
7、 根据权利要求 6所述的方法， 其特征在于， 进一步包括对所述 DNA测序文库进行 测序。
8、 一种试剂盒， 其特征在于， 包含看家基因特异性引物。
9、根据权利要求 8所述的试剂盒，其特征在于，所述看家基因为选自 PRDX6、 RPL37a、 ADD1、 HLA-A、 RAD9A、 ARHGEF7、 EIF2B2、 PSMD7、 BCAT2、 及 ATP50中的至少一 种。
10、 根据权利要求 8所述的试剂盒， 其特征在于，
针对 PRDX6, 所述看家基因特异性引物分别具有 SEQ ID NO: 1和 SEQ ID NO: 2所 示的核苷酸序列；
针对 RPL37a, 所述看家基因特异性引物分别具有 SEQ ID NO: 3和 SEQ ID NO: 4所 示的核苷酸序列；
针对 ADD1 , 所述看家基因特异性引物分别具有 SEQ ID NO: 5和 SEQ ID NO: 6所示 的核苷酸序列；
针对 HLA-A, 所述看家基因特异性引物分别具有 SEQ ID NO: 7和 SEQ ID NO: 8所 示的核苷酸序列；
针对 RAD9A, 所述看家基因特异性引物分别具有 SEQ ID NO: 9和 SEQ ID NO: 10 所示的核苷酸序列；
针对 ARHGEF7 , 所述看家基因特异性 I物分别具有 SEQ ID NO: 11和 SEQ ID NO: 12所示的核苷酸序列；
针对 EIF2B2, 所述看家基因特异性引物分别具有 SEQ ID NO: 13和 SEQ ID NO: 14 所示的核苷酸序列；
针对 PSMD7, 所述看家基因特异性引物分别具有 SEQ ID NO: 15和 SEQ ID NO: 16 所示的核苷酸序列；
针对 BCAT2, 所述看家基因特异性引物分别具有 SEQ ID NO: 17和 SEQ ID NO: 18 所示的核苷酸序列； 或者
针对 ATP50, 所述看家基因特异性引物分别具有 SEQ ID NO: 19和 SEQ ID NO: 20 所示的核苷酸序列。
11、 根据权利要求 8-10任一项所述的试剂盒， 其特征在于， 进一步包含适于通过多重 置换扩增 MDA或 DOP-PCR进行单细胞全基因组扩增的试剂。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2895626A4 (en) * 2012-09-12 2016-08-17 Univ California PRECISE GENOME SEQUENCING OF INDIVIDUAL CELLS BY AMPLIFYING AND SEQUENCING A SINGLE STRAND

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013143133A1 (zh) * 2012-03-30 2013-10-03 深圳华大基因科技服务有限公司 全基因组扩增方法及其应用
PT2881739T (pt) * 2013-12-04 2017-08-11 Fraunhofer Ges Forschung Método e estojo para determinação da integridade do genoma e/ou da qualidade de uma biblioteca de sequências de adn obtida através de amplificação determinística do genoma inteiro por locais de restrição
WO2016077750A1 (en) 2014-11-14 2016-05-19 Athena Diagnostics, Inc. Methods to detect a silent carrier genotype
CN104560950A (zh) * 2014-11-28 2015-04-29 深圳市海普洛斯生物科技有限公司 一种基于mda的全基因组扩增的方法
CN104450923A (zh) * 2014-12-15 2015-03-25 赛业健康研究中心（太仓）有限公司 利用囊胚腔液游离dna检测胚胎染色体异常的方法
CN104404034A (zh) * 2014-12-15 2015-03-11 赛业健康研究中心（太仓）有限公司 利用口腔上皮细胞制作单细胞测序参考品的方法
CN105861486A (zh) * 2015-01-19 2016-08-17 深圳华大基因科技服务有限公司 从混合核酸中获得目标核酸的方法
DK3253911T3 (da) * 2015-02-04 2021-04-06 Univ British Columbia Fremgangsmåder og anordninger til analyse af partikler
EP3436605A4 (en) * 2016-03-31 2019-08-21 Perkinelmer Health Sciences (Australia) Pty Ltd AMPLIFICATION OF TARGET SEQUENCES
CN106520997A (zh) * 2016-12-14 2017-03-22 南京诺唯赞医疗科技有限公司 一种单细胞基因表达定量分析的方法
CN110787296B (zh) * 2018-08-01 2024-04-16 复旦大学附属肿瘤医院 一种用于预防或治疗胰腺癌的药物组合物及检测胰腺癌的试剂盒
US10988815B2 (en) * 2018-12-17 2021-04-27 National Cheng Kung University Detective molecule, kit and method for predicting fragrance production in an orchid
CN111379032A (zh) * 2018-12-28 2020-07-07 北京贝瑞和康生物技术有限公司 一种用于构建同时实现基因组拷贝数变异检测和基因突变检测的测序文库的方法和试剂盒
CN112662748B (zh) * 2019-10-15 2023-02-17 骏实生物科技(上海)有限公司 用于单细胞dna测序文库质量鉴定的引物对组合、方法和试剂盒
CN113584138A (zh) * 2021-09-07 2021-11-02 广西壮族自治区妇幼保健院 一种对mda单细胞全基因组扩增产物的质控方法
CN113684266A (zh) * 2021-09-10 2021-11-23 苏州贝康医疗器械有限公司 用于单细胞全基因组dna扩增产物质量评价的核酸组合物及方法
CN117233393B (zh) * 2023-11-15 2024-02-09 四川大学华西医院 双重免疫组化染色试剂盒及其在鉴别良恶性胆管上皮肿瘤中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101137761A (zh) * 2005-03-18 2008-03-05 香港中文大学 产前诊断和监测的标记
CN101333561A (zh) * 2007-06-29 2008-12-31 上海裕隆生物科技有限公司 心脑血管疾病易感基因芯片检测试剂盒
WO2009049889A1 (en) * 2007-10-16 2009-04-23 Roche Diagnostics Gmbh High resolution, high throughput hla genotyping by clonal sequencing
WO2010085498A1 (en) * 2009-01-20 2010-07-29 The Board Of Trustees Of The Leland Stanford Junior University Single cell gene expression for diagnosis, prognosis and identification of drug targets

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2172353T3 (es) * 1998-09-18 2002-09-16 Micromet Ag Amplificacion del adn de una unica celula.
US6617137B2 (en) * 2001-10-15 2003-09-09 Molecular Staging Inc. Method of amplifying whole genomes without subjecting the genome to denaturing conditions
US7297485B2 (en) * 2001-10-15 2007-11-20 Qiagen Gmbh Method for nucleic acid amplification that results in low amplification bias
US20040229233A1 (en) * 2002-10-16 2004-11-18 Ngk Insulators, Ltd. Human housekeeping genes and human-tissue specific genes
WO2004058987A2 (en) * 2002-12-20 2004-07-15 Qiagen Gmbh Nucleic acid amplification
EP1641936B1 (en) * 2003-06-17 2010-08-04 Human Genetic Signatures PTY Ltd. Methods for genome amplification
US7387876B2 (en) * 2004-02-27 2008-06-17 President And Fellows Of Harvard College Amplification of trace amounts of nucleic acids
JP3972106B2 (ja) * 2004-03-03 2007-09-05 大学共同利用機関法人情報・システム研究機構 ゲノムライブラリー作製方法、および同方法により作製されたゲノムライブラリー
US20060172314A1 (en) * 2005-01-31 2006-08-03 Song Min-Sun Quantification of amplified nucleic acids
WO2010065629A2 (en) * 2008-12-02 2010-06-10 Kapabiosystems Nucleic acid amplification

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101137761A (zh) * 2005-03-18 2008-03-05 香港中文大学 产前诊断和监测的标记
CN101333561A (zh) * 2007-06-29 2008-12-31 上海裕隆生物科技有限公司 心脑血管疾病易感基因芯片检测试剂盒
WO2009049889A1 (en) * 2007-10-16 2009-04-23 Roche Diagnostics Gmbh High resolution, high throughput hla genotyping by clonal sequencing
WO2010085498A1 (en) * 2009-01-20 2010-07-29 The Board Of Trustees Of The Leland Stanford Junior University Single cell gene expression for diagnosis, prognosis and identification of drug targets

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DEAN, F. B. ET AL.: "Comprehensive Human Genome Amplification Using Multiple Displacement Amplification", PNAS, vol. 99, no. 8, 16 April 2002 (2002-04-16), pages 5261 - 5266, XP002297504 *
ELI EISENBERG; EREZ YLEVANON: "Human housekeeping genes are compact", TRENDS IN GENETICS, vol. 19, no. 7, 2003, pages 362 - 365, XP004436528, DOI: doi:10.1016/S0168-9525(03)00140-9
J. SAMBROOK; HUANG PT ET AL.: "Molecular Cloning Laboratory Manual", SCIENCE PRESS
MARCY, Y. ET AL.: "Dissecting biological ''dark matter'' with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth", PNAS, vol. 104, no. 29, 17 July 2007 (2007-07-17), pages 11889 - 11894, XP055112670 *
MARCY, Y. ET AL.: "Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells", PLOS GENETICS, vol. 3, no. 9, September 2007 (2007-09-01), pages 1702 - 1708, XP002561035 *
PINARD, R. ET AL.: "Assessment of Whole Genome Amplification-induced Bias through High-throughput, Massively Parallel Whole Genome Sequencing", BMC GENOMICS, vol. 7, no. 216, 23 August 2006 (2006-08-23), pages 1 - 21, XP021014593 *
See also references of EP2660331A4
ZHANG L.; CUI X.; SCHMITT K.; HUBERT R.; NAVIDI W.; AMHEIM N.: "Whole genome amplification from a single cell: Implication for genetic analysis", PROC NATL ACAD SCI USA, 1992, pages 5847 - 5851

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2895626A4 (en) * 2012-09-12 2016-08-17 Univ California PRECISE GENOME SEQUENCING OF INDIVIDUAL CELLS BY AMPLIFYING AND SEQUENCING A SINGLE STRAND
US10752945B2 (en) 2012-09-12 2020-08-25 The Regents Of The University Of California Accurate genome sequencing of single cells by single-stranded amplification and sequencing

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EP2660331A1 (en) 2013-11-06
US20140017683A1 (en) 2014-01-16
CN102533960B (zh) 2014-04-30
EP2660331B1 (en) 2017-10-04
US9238840B2 (en) 2016-01-19
HK1169454A1 (zh) 2013-01-25
EP2660331A4 (en) 2015-04-15

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