WO2021056653A1 - 高通量单细胞转录组与基因突变整合分析编码芯片、方法和装置 - Google Patents

高通量单细胞转录组与基因突变整合分析编码芯片、方法和装置 Download PDF

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WO2021056653A1
WO2021056653A1 PCT/CN2019/112969 CN2019112969W WO2021056653A1 WO 2021056653 A1 WO2021056653 A1 WO 2021056653A1 CN 2019112969 W CN2019112969 W CN 2019112969W WO 2021056653 A1 WO2021056653 A1 WO 2021056653A1
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cell
gene mutation
transcriptome
analysis
microwell
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PCT/CN2019/112969
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English (en)
French (fr)
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周连群
李金泽
姚佳
郭振
张芷齐
张威
李传宇
李超
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中国科学院苏州生物医学工程技术研究所
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Priority claimed from CN201910932328.XA external-priority patent/CN110577983A/zh
Priority claimed from CN201910932203.7A external-priority patent/CN110577982A/zh
Priority claimed from CN201910932615.0A external-priority patent/CN110951580B/zh
Application filed by 中国科学院苏州生物医学工程技术研究所 filed Critical 中国科学院苏州生物医学工程技术研究所
Publication of WO2021056653A1 publication Critical patent/WO2021056653A1/zh

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    • C12M1/00—Apparatus for enzymology or microbiology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Definitions

  • the application relates to the field of biological detection, and in particular to a high-throughput single-cell transcriptome and gene mutation integrated analysis coding chip, an integrated analysis method and an integrated analysis integrated device.
  • Tumor is one of the major diseases that seriously affect human health. There are great differences in tumor cells from genotype to phenotype (high degree of tumor heterogeneity), and this high degree of heterogeneity is related to the degree of malignancy and drug resistance of tumors. Sex, recurrence and metastasis are closely related, which are one of the root causes of difficult early diagnosis of tumors, complicated clinical diagnosis and treatment, drug-resistant recurrence, and poor prognosis. A comprehensive analysis of tumor heterogeneity is the key to achieving precise tumor treatment.
  • Single cell sequencing can obtain the genome variation map and transcriptome expression map of each cell, and accurately divide the clone attribution through the map of a single cell to achieve a comprehensive analysis of the heterogeneous clone population.
  • the Timothy A. Graubert team performed single-cell genotyping of a secondary leukemia sample that has been fully characterized by whole-genome sequencing and targeted deep sequencing, and found that it was previously considered to be one by only 12 cell DNA sequencing data.
  • the subcloning population is actually composed of two mutually exclusive subclones, which fully illustrates the advantages of single-cell sequencing and its necessity in polyclonal research.
  • the author finally used the random forest machine learning algorithm to predict the tumor population, which could not achieve the direct identification of the tumor cell population, nor did the genome and transcriptome heterogeneity correspond well.
  • the technology is complicated to operate, has high sample demand, and is expensive, and is not suitable for comprehensive promotion.
  • the technical problem to be solved by this application is to provide a high-throughput single-cell transcriptome and gene mutation integrated analysis coding chip, method and device in view of the above-mentioned deficiencies in the prior art.
  • this application provides: a high-throughput single-cell transcriptome and gene mutation integrated analysis coding chip, the chip is provided with a plurality of micropores on its substrate, and the micropores A micropore can only accommodate the size and shape of a single cell, each micropore has a unique spatial coordinate code, and the micropores are modified with several known nucleic acid sequences, which in turn include:
  • the universal primer sequence is used as the primer binding region during PCR amplification
  • Cell label used to indicate the cell from which the RNA is derived
  • the modified nucleic acid sequence in each of said micropores is not less than 10 6.
  • the molecular tag is a known random nucleic acid sequence.
  • the cell label of each microwell has a one-to-one correspondence with the spatial coordinate code, all the cell labels in a single microwell have the same sequence, and the sequences of the cell labels in different microwells are not the same, so Identifying the cell from which the RNA is derived by the cell tag;
  • RNA in a single cell is identified by the molecular tags.
  • the micropores have a regular hexagonal shape and are arranged in a honeycomb shape, the number of which is 10 2 -10 6 .
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • a manufacturing process of the above-mentioned high-throughput single-cell transcriptome and gene mutation integration analysis coding chip includes the following steps:
  • the step 1) is specifically: directly forming micro-holes on silicon by photolithography and deep silicon etching, and the micro-holes may be blind holes or through holes.
  • the step 1) is specifically as follows: firstly prepare a positive film by silicon photolithography, then use PDMS casting and demolding to form a soft photolithography pattern, and after combining with a flat glass slide, adopt a capillary micro-molding method to The polyurethane or epoxy resin is cured and the glass slide forms a micro-hole array.
  • the step 2) specifically includes: synthesizing spacers, universal primers, cell tag sequences, and extension linkers in the micropores by means of inkjet printing combined with in-situ chemical synthesis of oligonucleotides;
  • the amplification method uses molecular tags and PolyA as templates to extend the synthesized sequence in situ to form a molecular tag sequence segment to obtain the final nucleic acid sequence.
  • this application provides a high-throughput single-cell transcriptome and gene mutation integration analysis method, which includes the following steps:
  • the chip has a plurality of micropores, each of the micropores has a unique spatial coordinate code, and the micropores are modified with several strips for capturing target RNA
  • the nucleic acid sequence includes a cell tag used to indicate the cell from which the RNA is derived and a molecular tag used to indicate the bound RNA, and the cell tag of each microwell corresponds to the spatial coordinate code on a one-to-one basis;
  • PCR amplification is performed on the cDNA immobilized in the microwells, and the wild-type and mutant types of the target gene are labeled with dual-color fluorescence, and then the ratio of wild-type and mutant-type at each microwell position is calculated through dual-color fluorescence image analysis , Get the gene mutation expression information of a single cell;
  • cDNA For free cDNA, analyze cDNA through gene sequencing to obtain single-cell transcription profile and subtype information, and use the cell tag attached to the transcriptome RNA to know the cell and the position of the micropore from which each transcriptome RNA originated, so that the single cell
  • the gene mutation, transcriptome, and protein expression information of the cells correspond one-to-one to form a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis.
  • the step 2) specifically includes: fluorescently labeling the target gene of the cell in advance, adding the cell to the chip, using the micropore of the chip to capture the single cell, and then performing fluorescence image collection, and The position of the micropores on the fluorescence image is located, and the position of the specific cell containing the target gene is identified by the analysis of the fluorescence image, and the cell protein expression information of each micropore position is obtained.
  • the method for positioning the position of the microwell on the fluorescence image is specifically: establishing an image grid template according to the position of the microwell on the microwell array chip, and then combining the collected fluorescence image It is registered with the image grid template to identify the position of each micro-hole area on the fluorescent image, and locate the micro-hole position on the fluorescent image.
  • the step 4) specifically includes: adding PCR amplification reagents, adding two pre-designed primer probes modified with different fluorescent groups, one of which is used to bind to the wild-type target gene, and the other Used to combine with mutant target genes to perform in situ lysis and amplification of single cells in microwells;
  • Carry out two-color fluorescence image acquisition locate the micro-hole position on the obtained two-color fluorescence image, calculate the intensity value of two types of fluorescence at each micro-hole position, and calculate the ratio of the intensities of the two types of fluorescence at each micro-hole position through a clustering algorithm , So as to obtain the ratio of the wild type to the mutant type of the target gene at each microwell position, and obtain the mutation expression information of the amplified transcriptome RNA sequence.
  • the method for locating the position of the microwell on the two-color fluorescence image is specifically: establishing an image grid template according to the position of the microwell on the microwell array chip, and then combining the collected two-color fluorescence The image is registered with the image grid template, thereby identifying the position of each micro-hole area on the two-color fluorescent image, and positioning the micro-hole position on the two-color fluorescent image.
  • the nucleic acid sequence further includes a Spacer sequence, a universal primer sequence used as a primer binding region during PCR amplification, and Ploy T.
  • all the cell tags in a single microwell have the same sequence, and the sequences of the cell tags in different microwells are all different, so that the cell from which the RNA is derived is identified by the cell tag;
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the micropores have a regular hexagonal shape and are arranged in a honeycomb shape, the number of which is 10 2 -10 6 .
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • the present application provides an integrated device for high-throughput single-cell transcriptome and gene mutation integration analysis, including a high-throughput single-cell coding chip and an integrated analysis device;
  • the high-throughput single cell coding chip has micropores for capturing single cells, each of the micropores has a unique spatial coordinate code, and several nucleic acid sequences for capturing target RNA are modified in the micropores ,
  • the nucleic acid sequence includes a cell tag used to indicate the cell from which the RNA is derived and a molecular tag used to indicate the bound RNA, and the cell tag of each microwell corresponds to the spatial coordinate code in a one-to-one correspondence;
  • the integrated analysis device includes a housing and a temperature-controlled thermal cycle module, a fluorescence imaging module, and a data storage analysis module arranged in the housing.
  • the fluorescence imaging module includes a light source assembly, a microscope objective lens, a fluorescence spectroscopy assembly, and imaging detection.
  • the temperature-controlled thermal cycle component is provided with a hot stage for placing the high-throughput single-cell coding chip, and the temperature-controlled thermal cycle component is used to provide a temperature environment required for the PCR amplification reaction;
  • the excitation light emitted by the light source assembly passes through the fluorescence spectroscopic assembly, it passes through the microscope objective lens and then reaches the high-throughput single-cell encoding chip on the temperature-controlled thermal cycle assembly, and the sample is excited to produce After returning to the original path of fluorescence through the microscope objective lens and the fluorescence spectroscopy module, it then enters the imaging detector for fluorescence imaging;
  • the data storage and analysis module is used to store the fluorescence image information collected by the imaging detector and perform integration analysis of single cell transcriptome and gene mutation.
  • the light source assembly includes a first LED light source, a second LED light source, a third LED light source, a first dichroic mirror, a second dichroic mirror, and a beam expander lens group,
  • the light emitted by the first LED light source sequentially transmits through the first dichroic mirror and the second dichroic mirror to reach the beam expander lens group;
  • the light emitted by the second LED light source is reflected by the first dichroic mirror and transmitted by the second dichroic mirror to reach the beam expander lens group;
  • the light emitted by the third LED light source reaches the beam expander lens group after being reflected by the second dichroic mirror;
  • the first LED light source, the second LED light source, and the third LED light source emit light of three different wavelengths, and the wavelength range of the three lights covers 400nm-700nm.
  • the fluorescence spectroscopy assembly includes a support, a rotating table rotatably arranged on the support, a motor for driving the rotation of the rotating table, and a number of fluorescence spectroscopy modules arranged on the rotating table at even intervals ,
  • the fluorescence spectroscopic module includes an excitation light filter, a sample light filter and a fourth dichroic mirror;
  • the rotating table is used to switch one of the several fluorescence spectroscopic modules into the light path, and the excitation light emitted by the beam expander lens group is reflected by the fourth dichroic mirror after passing through the excitation light filter , And then pass through the microscope objective lens to reach the high-throughput single-cell encoding chip placed on the loading hot stage; the fluorescence generated by the sample in the high-throughput single-cell encoding chip passes through the microscope objective
  • the fourth dichroic mirror is transmitted through the sample light filter and then reaches the imaging detector, and the fluorescence image information collected by the imaging detector is transmitted to the data storage and analysis module.
  • a stakeout window is provided on the housing, and a sliding cover is provided on the stakeout window.
  • the temperature-controlled thermal cycle assembly includes a temperature-controlled box, a radiator arranged in the temperature-controlled box, a Peltier arranged on the radiator, and a side part of the radiator A fan, the hot stage for loading the object is arranged on the Peltier, and the hot stage for loading is provided with a transparent cover.
  • the nucleic acid sequence further includes a Spacer sequence, a universal primer sequence used as a primer binding region during PCR amplification, and Ploy T.
  • all the cell tags in a single microwell have the same sequence, and the sequences of the cell tags in different microwells are all different, so that the cell from which the RNA is derived is identified by the cell tag;
  • RNA in a single cell is identified by the molecular tags.
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the micropores are regular hexagons and arranged in a honeycomb shape, the number of which is 10 2 -10 6 ;
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • the analysis step of the integrated device for high-throughput single-cell transcriptome and gene mutation analysis includes:
  • the imaging module performs fluorescence imaging on the high-throughput single-cell coding chip, and uses the data storage and analysis module to perform single-cell surface protein typing analysis of each microwell position;
  • the fluorescence imaging module collects a two-color fluorescence image, and then analyzes the two-color fluorescence image through the data storage and analysis module, calculates the ratio of the wild type and the mutant type at each microwell position, and obtains the gene mutation expression information of a single cell;
  • this application provides a chip that can be used for high-throughput single-cell transcriptome and gene mutation integration analysis.
  • This application provides a chip that can be used for high-throughput single-cell transcriptome and gene mutation integration analysis.
  • the triple-encoding technology of micropore spatial coordinates, cell nucleic acid tags, and molecular nucleic acid tags By adopting the triple-encoding technology of micropore spatial coordinates, cell nucleic acid tags, and molecular nucleic acid tags, The gene mutation, transcriptome and protein expression information of single cells can be matched one-to-one, which can provide a chip basis for high-throughput single-cell transcriptome and gene mutation integrated analysis.
  • the high-throughput single-cell transcriptome and gene mutation integrated analysis method of the present application combines single-cell surface proteins by designing a high-throughput single-cell coding chip with a triple coding function of micropore spatial coordinates, cell nucleic acid tags and molecular nucleic acid tags
  • Typing, single-cell transcriptome mutation analysis and gene sequencing methods can map single-cell gene mutation, transcriptome and protein expression information one-to-one, forming a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis , To obtain a multi-omics integrated analysis model.
  • the high-throughput single-cell transcriptome and gene mutation integrated analysis device of the present application can integrate single-cell coding chips with triple coding functions of micropore spatial coordinates, cell nucleic acid tags, and molecular nucleic acid tags.
  • the gene mutation, transcriptome, and protein expression information of the cell are one-to-one correspondence; then PCR amplification can be realized through the temperature-controlled thermal cycling module, the fluorescence image of the sample is collected through the fluorescence imaging module, and the fluorescence image is stored in the data storage and analysis module.
  • Analysis the establishment of a database for single-cell surface protein typing and mutation integration analysis, the establishment of a complete database for high-throughput single-cell transcriptome and gene mutation integration analysis, and the realization of single-cell transcriptome and gene mutation integration analysis;
  • this application After clarifying the genomic mutation information carried in a single cell, this application combines single-cell transcriptome and even protein expression information to achieve a comprehensive understanding of tumor cell multi-omics and a comprehensive description of tumor heterogeneous populations, which is the early stage of tumor Provide the basis for diagnosis, drug resistance mechanism, new target exploration and treatment plan optimization.
  • FIG. 1 is a schematic diagram of the manufacturing process of a high-throughput single-cell transcriptome and gene mutation integration analysis coding chip in an embodiment of the application;
  • Figure 2 is a flow chart of the application's high-throughput single-cell transcriptome and gene mutation integration analysis method
  • Figure 3 is a flowchart of nucleic acid sequence modification in an embodiment of the application.
  • Fig. 4 is a schematic block diagram of an integrated device for high-throughput single-cell transcriptome and gene mutation analysis of the application;
  • Figure 5 is a schematic diagram of the internal structure of the integrated device for high-throughput single-cell transcriptome and gene mutation analysis of the application;
  • Figure 6 is a schematic diagram of the external structure of the integrated device for high-throughput single-cell transcriptome and gene mutation analysis of the application;
  • FIG. 7 is a schematic diagram of the structure of the bracket of the fluorescence spectroscopic component of this application.
  • FIG. 8 is a schematic diagram of the structure of the fluorescence spectroscopic module of this application.
  • FIG. 9 is a schematic cross-sectional structure diagram of the fluorescence spectroscopic module of this application.
  • Figure 10 is an exploded view of the temperature control thermal cycle module of the application.
  • Fig. 11 is a light path diagram of the integrated device for high-throughput single-cell transcriptome and gene mutation integration analysis of the present application.
  • a high-throughput single-cell transcriptome and gene mutation integrated analysis coding chip of this embodiment is provided with a plurality of micropores on its substrate.
  • the micropores have a size and shape that can only accommodate a single cell in a micropore.
  • Each microwell has a unique spatial coordinate code, and several known nucleic acid sequences are modified in the microwells, which in turn include:
  • the universal primer sequence is used as the primer binding region during PCR amplification
  • Cell label used to indicate the cell from which the RNA is derived
  • the modified nucleic acid sequence in each pore is not less than 10 6.
  • the molecular tag is a known random nucleic acid sequence.
  • the cell label of each microwell corresponds to the space coordinate code one-to-one.
  • a cell carries the microwell space coordinate code, and this microwell space coordinate corresponds at the same time
  • a known cell tag nucleic acid sequence
  • a set of known molecular tags random sequence.
  • the loaded single cells can be labeled with immunofluorescence, and protein expression information can be obtained through high-throughput multicolor fluorescence imaging.
  • all cell tags in a single microwell have the same sequence, and the sequence of cell tags in different microwells are not the same, so that the cell from which the RNA originated is identified by the cell tag; therefore, the cell tag can be used in the final sequencing data Know which cell the sequence originated from, distinguish which sequences are from the same cell and which are from different cells.
  • RNA in a single cell is identified by the molecular tags.
  • Molecular tagging is only responsible for labeling the RNA in the same cell, regardless of the RNA between different cells.
  • each RNA can be distinguished by molecular tags. Therefore, for the final detection data, different cells are distinguished by cell label, and a cell label corresponds to a unique microwell spatial coordinate code, so that the cell from which the RNA originates and the coordinate position of the microwell are known, and then the molecular label is used to distinguish Every piece of RNA. In this way, the cell and location coordinate information from which each piece of RNA originates can be matched.
  • the micropore spatial coordinates, cell nucleic acid label and molecular nucleic acid label used in the chip of this application can be matched.
  • the triple coding technology can map the gene mutation, transcriptome and protein expression information of a single cell one by one.
  • the released RNA is captured by the nucleic acid sequence in the pore, and the target markers are connected with cell tags and molecular tags through base complementary pairing.
  • cDNA was simultaneously formed on the wall of the well and in the well through amplification.
  • the transcriptome information of a single cell can be obtained, and this omics information will correspond to the microwell spatial coordinate coding of the single cell.
  • in situ fluorescent PCR can be performed to obtain the information of single-cell frontal gene mutations. In this way, the gene mutation, transcriptome, and protein expression information of a single cell can be one-to-one correspondence with the coding technology of the present application.
  • the method of using the chip of the present application to perform high-throughput single-cell transcriptome and gene mutation integration analysis is:
  • PCR amplification reagents to perform PCR amplification on the cDNA immobilized in the microwells, and add two pre-designed primer probes modified with different fluorescent groups, one of which is used to bind to the wild-type target gene, and the other One is used to combine with the mutant target gene to perform in situ lysis and amplification of single cells in the microwell; after amplification, the wild-type target gene and the mutant target gene each carry different fluorescent molecules; then perform dual-color fluorescence Image acquisition, locate the micro-hole position on the obtained two-color fluorescence image, calculate the intensity value of the two types of fluorescence at each micro-hole position, and calculate the ratio of the intensities of the two types of fluorescence at each micro-hole position through a clustering algorithm to obtain The ratio of wild type to mutant type of the target gene in each microwell position, to obtain the gene mutation expression information of a single cell;
  • cDNA is analyzed by gene sequencing to obtain single-cell transcription profile and subtype information. Since the cDNA is attached to the cell label and molecular label, the cell and the position of the microwell of each cDNA source can be known, and the single cell The gene mutation, transcriptome, and protein expression information of the transcriptome corresponded to each other to form a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis, and establish a multi-omics integration analysis model.
  • the micropores are regular hexagons and arranged in a honeycomb shape, and the number is 10 2 to 10 6 .
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • the manufacturing process of a high-throughput single-cell transcriptome and gene mutation integration analysis coding chip includes the following steps:
  • step 1) is specifically: using MEMS technology to directly form micro-holes on silicon by photolithography and deep silicon etching, and the micro-holes may be blind holes or through holes.
  • soft lithography technology referring to Figure 1.
  • the cation film is prepared by silicon photolithography, and then the soft photolithography pattern is formed by PDMS casting and demolding. After combining with the flat glass slide, the capillary micro-molding method is used to The polyurethane or epoxy resin is cured and the glass slide forms a micro-hole array.
  • the coding chip in Figure 1 refers to a chip with modified nucleic acid sequence synthesized in situ on a flat glass slide.
  • step 2) specifically includes: using inkjet printing, combined with in-situ chemical synthesis of oligonucleotides, synthesizing spacers, universal primers, cell tag sequences and extension adapters in the micropores;
  • the nucleic acid amplification method uses molecular tags and PolyA as templates to extend the synthesized sequence in situ to form a molecular tag sequence segment, thereby obtaining the final nucleic acid sequence. Refer to Figure 3 for its flow, where UMI stands for molecular tag.
  • the chip has a plurality of micropores, each of the micropores has a unique spatial coordinate code, and the micropores are modified with several strips for capturing target RNA
  • the nucleic acid sequence includes a cell tag used to indicate the cell from which the RNA is derived and a molecular tag used to indicate the bound RNA, and the cell tag of each microwell corresponds to the spatial coordinate code on a one-to-one basis;
  • PCR amplification is performed on the cDNA immobilized in the microwells, and the wild-type and mutant types of the target gene are labeled with dual-color fluorescence, and then the ratio of wild-type and mutant-type at each microwell position is calculated through dual-color fluorescence image analysis , Get the gene mutation expression information of a single cell;
  • cDNA For free cDNA, analyze cDNA through gene sequencing to obtain single-cell transcription profile and subtype information, and use the cell tag attached to the transcriptome RNA to know the cell and the position of the micropore from which each transcriptome RNA originated, so that the single cell
  • the gene mutation, transcriptome, and protein expression information of the cells correspond one-to-one to form a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis.
  • Example 1 Provides a high-throughput single-cell coding chip
  • the chip is provided with a plurality of micropores on its substrate, each of the micropores has a unique spatial coordinate code, and the micropores are modified with several known nucleic acid sequences for capturing target RNA, so
  • the nucleic acid sequence includes a cell tag used to indicate the cell from which the RNA is derived and a molecular tag used to indicate the bound RNA, and the cell tag of each microwell corresponds to the spatial coordinate code in a one-to-one correspondence.
  • the nucleic acid sequence also includes a Spacer sequence, a universal primer sequence used as a primer binding region during PCR amplification, and Ploy T.
  • the modified nucleic acid sequence within each pore is not less than 10 6.
  • the molecular tag is a known random nucleic acid sequence.
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the micropores are regular hexagons and arranged in a honeycomb shape, and the number is 10 2 to 10 6 .
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • microwell spatial coordinate code When the microwell array is loaded with cells, for each specific microwell, a cell carries the microwell spatial coordinate code.
  • This microwell spatial coordinate corresponds to a known cell tag (nucleic acid sequence) and a set of known Molecular tags (random sequence).
  • the loaded single cells can be labeled with immunofluorescence, and protein expression information can be obtained through high-throughput multicolor fluorescence imaging.
  • all cell tags in a single microwell have the same sequence, and the sequence of cell tags in different microwells are not the same, so that the cell from which the RNA originated is identified by the cell tag; therefore, the cell tag can be used in the final sequencing data Know which cell the sequence originated from, distinguish which sequences are from the same cell and which are from different cells.
  • RNA in a single cell is identified by the molecular tags.
  • Molecular tagging is only responsible for labeling the RNA in the same cell, regardless of the RNA between different cells.
  • each RNA can be distinguished by molecular tags. Therefore, for the final detection data, different cells are distinguished by cell label, and a cell label corresponds to a unique microwell space coordinate code, so that the cell from which the RNA originates and the coordinate position of the microwell are known, and then the molecular label is used to distinguish Every piece of RNA. In this way, the cell and position coordinate information from which each piece of RNA originates can be matched.
  • the micropore space coordinates, cell nucleic acid label and molecular nucleic acid label used in the chip of this application can map the gene mutation, transcriptome and protein expression information of a single cell one by one.
  • the released RNA is captured by the nucleic acid sequence in the pore, and the cell label and molecular label are attached to the detection target marker through the method of base complementary pairing.
  • cDNA was simultaneously formed on the wall of the well and in the well through amplification.
  • the transcriptome information of a single cell can be obtained, and this omics information will correspond to the microwell spatial coordinate coding of the single cell.
  • in situ fluorescent PCR can be performed to obtain the mutation information of single-cell frontal genes.
  • the above-mentioned chip can be obtained through the following manufacturing process:
  • micro-holes are directly formed on silicon through photolithography and deep silicon etching, and the micro-holes can be blind holes or through holes;
  • Example 2 Using the chip of Example 1 to perform single-cell transcriptome and gene mutation integration analysis, referring to Fig. 3, specifically including the following steps:
  • Fluorescently label the target gene of the cells in advance then add the cells to the chip, use the micropores of the chip to capture single cells, incubate, and then perform fluorescence image collection, and locate the micropore positions on the fluorescence image, Use fluorescence image analysis to identify the location of the specific cell containing the target gene, and obtain the cell protein expression information of each microwell location.
  • a three-color fluorescence channel is used for fluorescence imaging, and three different wavelengths of light sources are used to achieve full-wavelength visible light coverage (wavelength 400nm-700nm).
  • the method for positioning the position of the microwell on the fluorescence image is specifically: establishing an image grid template according to the position of the microwell on the microwell array chip, and then registering the collected fluorescence image with the image grid template, thereby Identify the position of each micro-hole area on the fluorescent image, and locate the micro-hole position on the fluorescent image.
  • lysis buffer and amplification reagents to perform in situ lysis and amplification of single cells in the microwells, reverse transcription to synthesize cDNA carrying cell tags and molecular tags, and collect free cDNA for single-cell transcriptome analysis and fix it in the microwells
  • the cDNA sequence inside is used for gene mutation analysis;
  • PCR amplification reagents to perform PCR amplification on the cDNA immobilized in the microwells, and add two pre-designed primer probes modified with different fluorescent groups, one of which is used to bind to the wild-type target gene, and the other One is used to combine with the mutant target gene to perform in situ lysis and amplification of single cells in the microwell; after amplification, the wild-type target gene and the mutant target gene each carry different fluorescent molecules;
  • Carry out two-color fluorescence image acquisition locate the micro-hole position on the obtained two-color fluorescence image, calculate the intensity value of two types of fluorescence at each micro-hole position, and calculate the ratio of the intensities of the two types of fluorescence at each micro-hole position through a clustering algorithm , So as to obtain the ratio of the wild type to the mutant type of the target gene at each microwell position, and obtain the gene mutation expression information of a single cell.
  • the method for locating the positions of the micro-holes on the two-color fluorescent image is specifically: establishing an image grid template according to the positions of the micro-holes on the micro-hole array chip, and then registering the collected two-color fluorescent image with the image grid template, In this way, the position of each micro-hole area on the two-color fluorescent image is recognized, and the position of the micro-hole is located on the two-color fluorescent image.
  • step 2 the single cell surface protein typing information at the same location is combined with the amplified single cell gene mutation expression information to establish a single cell surface protein typing and mutation integration analysis database.
  • cDNA is analyzed by gene sequencing to obtain single-cell transcription profile and subtype information. Since the cDNA is attached to the cell label and molecular label, the cell and the position of the microwell of each cDNA source can be known, and the single cell The gene mutation, transcriptome, and protein expression information of the transcriptome corresponded to each other to form a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis, and establish a multi-omics integration analysis model.
  • an integrated device for high-throughput single-cell transcriptome and gene mutation integration analysis of this embodiment includes a high-throughput single-cell coding chip 1 and an integrated analysis device 2;
  • the high-throughput single cell coding chip 1 has micropores for capturing single cells, each micropore has a unique spatial coordinate code, and several nucleic acid sequences for capturing target RNA are modified in the micropores.
  • the nucleic acid sequences include In order to indicate the cell label of the cell from which the RNA is derived and the molecular label used to indicate the bound RNA, the cell label of each microwell corresponds to the spatial coordinate code one-to-one;
  • the integrated analysis device 2 includes a housing 3, a temperature-controlled thermal cycle module 4, a fluorescence imaging module 5, and a data storage and analysis module arranged in the housing 3.
  • the fluorescence imaging module 5 includes a light source assembly 6, a microscope objective lens 7, and a fluorescence spectroscopic assembly 8 and imaging detector 9;
  • the temperature-controlled thermal cycle component is provided with a hot stage 40 for placing the high-throughput single-cell coding chip 1, and the temperature-controlled thermal cycle component is used to provide the temperature environment required for the PCR amplification reaction;
  • the excitation light emitted by the light source assembly 6 passes through the fluorescence spectroscopic assembly 8, and then passes through the microscope objective lens 7 and then reaches the high-throughput single-cell encoding chip 1 on the temperature-controlled thermal cycle assembly, where the sample is excited and the fluorescence generated by the original path returns through After the microscope objective lens 7 and the fluorescence spectroscopic module 83, it enters the imaging detector 9 for fluorescence imaging;
  • the data storage and analysis module is used to store the fluorescence image information collected by the imaging detector 9 and perform integration analysis of single-cell transcriptome and gene mutation.
  • the light source assembly 6 includes a first LED light source 60, a second LED light source 61, a third LED light source 62, a first dichroic mirror 63, a second dichroic mirror 64, and a beam expander lens group 65.
  • the light emitted by the first LED light source 60 sequentially transmits through the first dichroic mirror 63 and the second dichroic mirror 64 and then reaches the beam expanding lens group 65; the light emitted by the second LED light source 61 passes through the first dichroic mirror 63 is reflected and transmitted by the second dichroic mirror 64 to reach the beam expander lens group 65; the light emitted by the third LED light source 62 is reflected by the second dichroic mirror 64 and then reaches the beam expander lens group 65; the first LED light source 60, The second LED light source 61 and the third LED light source 62 emit light with three different wavelengths, and the wavelength range of the three lights covers 400 nm-700 nm.
  • the fluorescence spectroscopic assembly 8 includes a bracket 80, a rotating table 81 rotatably arranged on the bracket 80, a motor 82 for driving the rotating table 81 to rotate, and a number of fluorescence spectroscopic modules 83 arranged on the rotating table 81 evenly at intervals.
  • the light splitting module 83 includes a lens mounting block 830 and an excitation light filter 831, a sample light filter 832 and a fourth dichroic mirror 833 arranged therein; 3 openings are provided on the lens mounting block 830, and the bottom opening is for excitation light The light directly passes through the sample, the excitation light filter 831 is installed in the left opening, and the sample light filter 832 is installed in the upper opening.
  • the rotating table 81 is used to switch one of the several fluorescence spectroscopic modules 83 into the optical path.
  • the excitation light emitted by the beam expander lens group 65 is reflected by the fourth dichroic mirror 833 after passing through the excitation light filter 831, and then passes through the microscope
  • the objective lens 7 arrives on the high-throughput single-cell encoding chip 1 placed on the hot stage 40; the fluorescence generated by the sample in the high-throughput single-cell encoding chip 1 passes through the microscope objective lens 7 and transmits the fourth dichroic mirror 833 After passing through the sample light filter 832, it reaches the imaging detector 9.
  • the fluorescence image information collected by the imaging detector 9 is transmitted to the data storage and analysis module.
  • five different fluorescence spectroscopic modules 83 are included, which are respectively arranged in five installation grooves 810 opened on the rotating table 81, so as to realize the spectroscopy of a variety of fluorescence.
  • a stakeout window 30 is provided on the housing 3, and a sliding cover 31 is provided on the stakeout window 30.
  • the high-throughput single-cell coding chip 1 can be conveniently placed on the hot stage 40 through the stakeout window 30.
  • the temperature control thermal cycle assembly includes a temperature control box 41, a radiator 42 arranged in the temperature control box 41, a Peltier 43 arranged on the radiator 42, and a fan 44 arranged on the side of the radiator 42.
  • the loading hot stage 40 is arranged on the Peltier 43, and the loading hot stage 40 is provided with a transparent cover plate, which is sealed by the transparent cover plate, and does not affect the fluorescence imaging.
  • the temperature control during the PCR amplification reaction is realized by the temperature-controlled thermal cycle component.
  • the loading hot stage 40 has good thermal conductivity.
  • the Peltier 43 heats the loading hot stage 40.
  • the radiator 42 has a plurality of radiating fins and cooperates with the fan 44 to achieve rapid heat dissipation, thereby achieving temperature rise and fall control.
  • the microscope objective lens 7 is installed on the lifting platform 70, which can be moved up and down for convenient adjustment, and cooperates with a motor to achieve a focusing function.
  • the entire integrated analysis device 2 can be centrally controlled by the host computer.
  • a high-throughput single-cell coding chip 1 is provided.
  • the chip is provided with a plurality of micropores on its substrate, and each micropore has a unique spatial coordinate code, and the micropores are modified with several known nucleic acid sequences for capturing target RNA.
  • the nucleic acid sequences include The cell label indicating the cell from which the RNA originated and the molecular label used to indicate the bound RNA, and the cell label of each microwell corresponds to the spatial coordinate code one-to-one.
  • the nucleic acid sequence also includes the Spacer sequence, the universal primer sequence used as the primer binding region during PCR amplification, and Ploy T.
  • the modified nucleic acid sequence within each pore is not less than 10 6.
  • the molecular tag is a known random nucleic acid sequence.
  • the micropore has a size and shape that can only accommodate a single cell in one micropore.
  • the micropores are regular hexagons and arranged in a honeycomb shape, and the number is 10 2 to 10 6 .
  • the diameter of the circumscribed circle of the micropores is 30-60 ⁇ m, the depth is 20-300 ⁇ m, and the spacing between the holes is 10-30 ⁇ m.
  • microwell spatial coordinate code When the microwell array is loaded with cells, for each specific microwell, a cell carries the microwell spatial coordinate code.
  • This microwell spatial coordinate corresponds to a known cell tag (nucleic acid sequence) and a set of known Molecular tags (random sequence).
  • the loaded single cells can be labeled with immunofluorescence, and protein expression information can be obtained through high-throughput multicolor fluorescence imaging.
  • all cell tags in a single microwell have the same sequence, and the sequence of cell tags in different microwells are not the same, so that the cell from which the RNA originated is identified by the cell tag; therefore, the cell tag can be used in the final sequencing data Know which cell the sequence originated from, distinguish which sequences are from the same cell and which are from different cells.
  • RNA in a single cell is identified by the molecular tags.
  • Molecular tagging is only responsible for labeling the RNA in the same cell, regardless of the RNA between different cells.
  • each RNA can be distinguished by molecular tags. Therefore, for the final detection data, different cells are distinguished by cell label, and a cell label corresponds to a unique microwell space coordinate code, so that the cell from which the RNA originates and the coordinate position of the microwell are known, and then the molecular label is used to distinguish Every piece of RNA. In this way, the cell and location coordinate information from which each piece of RNA originates can be matched.
  • the micropore spatial coordinates, cell nucleic acid label and molecular nucleic acid label used in the chip of this application can be matched.
  • the triple coding technology can map the gene mutation, transcriptome and protein expression information of a single cell one by one.
  • the released RNA is captured by the nucleic acid sequence in the pore, and the cell label and molecular label are attached to the detection target marker through the method of base complementary pairing.
  • cDNA was simultaneously formed on the wall of the well and in the well through amplification.
  • the transcriptome information of a single cell can be obtained, and this omics information will correspond to the microwell spatial coordinate coding of the single cell.
  • in situ fluorescent PCR can be performed to obtain the mutation information of single-cell frontal genes.
  • the above-mentioned chip can be obtained through the following manufacturing process:
  • micro-holes are directly formed on silicon through photolithography and deep silicon etching, and the micro-holes can be blind holes or through holes;
  • a high-throughput single-cell transcriptome and gene mutation integrated analysis device is provided, which is obtained by combining the integrated analysis device 2 of Example 3 and the high-throughput single-cell coding chip 1 of Example 4.
  • the high-throughput single cell encoding chip 1 Fluorescently label the target gene of the cell in advance, and then add the sample to the high-throughput single cell encoding chip 1, capture the single cell through the micropores on it, and place the high-throughput single cell encoding chip 1 on the carrier heat
  • the light source assembly 6, the microscope objective lens 7, the fluorescence spectroscopic assembly 8 and the imaging detector 9 are activated, and the high-throughput single-cell encoding chip 1 is subjected to fluorescence imaging through the fluorescence imaging module 5, and then the data storage and analysis module is used for each Single-cell surface protein typing analysis of the position of the microwell; among them, the high-throughput single-cell coding chip 1 can be taken out from the hot stage 40 for operations such as adding samples and reagents to the high-throughput single-cell coding chip 1 After adding, the adding operation can also be performed directly on the loading hot stage 40 through the sample adding mechanism;
  • the wild-type target gene and the mutant target gene each have different fluorescent molecules
  • the two-color fluorescence image is collected through the fluorescence imaging module 5, and then the two-color fluorescence image is analyzed through the data storage and analysis module, and each is calculated.
  • step 2) For the free cDNA collected in step 2), analyze the cDNA by gene sequencing to obtain the single-cell transcription profile and subtype information. Since the cDNA is attached to the cell label and molecular label, it is possible to know the cell and the source of each cDNA.
  • the location of the micropores can correspond to the gene mutation, transcriptome and protein expression information of single cells, forming a complete database of high-throughput single-cell transcriptome and gene mutation integration analysis, and establish a multi-omics integration analysis model to realize single-cell transcriptome analysis. Analysis of cell transcriptome and gene mutation integration.

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Abstract

本申请公开了一种高通量单细胞转录组与基因突变整合分析编码芯片，芯片在其基板上设置有多个微孔，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，每个微孔具有唯一的空间坐标编码，且微孔内修饰有若干条已知的核酸序列，核酸序列依次包括：Spacer序列；通用引物序列，作为PCR扩增时的引物结合区域；细胞标签，用于标示RNA源自的细胞；分子标签，用于标示结合的RNA；以及Ploy T。还提供一种整合分析方法和整合分析一体化装置。本申请的芯片、方法和装置，通过采用微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码技术，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。

Description

高通量单细胞转录组与基因突变整合分析编码芯片、方法和装置
交叉引用
本申请同时要求在2019年9月29日提交中国专利局、申请号为201910932203.7、申请名称为“高通量单细胞转录组与基因突变整合分析编码芯片”，2019年9月29日提交中国专利局、申请号为201910932328.X、申请名称为“高通量单细胞转录组与基因突变整合分析方法”，2019年9月29日提交中国专利局、申请号为201910932615.0、申请名称为“高通量单细胞转录组与基因突变整合分析一体化装置”三件中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及生物检测领域，特别涉及一种高通量单细胞转录组与基因突变整合分析编码芯片、整合分析方法和整合分析一体化装置。
背景技术
肿瘤是严重影响人类健康的重大疾病之一，肿瘤细胞从基因型到表型上存在极大的差异(肿瘤的高度异质性)，而这种高度异质性与肿瘤的恶性程度、耐药性、复发转移等都密切相关，是造成肿瘤早期诊断困难、临床诊治复杂、耐药复发和预后差的根源之一。全面解析肿瘤异质性是实现肿瘤精准治疗的关键。
高通量测序技术的发展为解析异质性肿瘤群体带来希望。目前各种组学水平的常规高通量测序成为肿瘤异质性群体研究的常用手段，用来发现新的遗传变异或异常通路，探索新的发病或耐药机制等。然而目前基于bulk(混合群体)的常规高通量测序技术无法克服肿瘤细胞高度异质性的难题，仅能通过大样本人群研究发现关键主克隆变异及通路改变，难以实现对单个患者异质性克隆群体的全面解析，成为实现肿瘤精准治疗的瓶颈。近年来新兴的单细胞测序技术为解析肿瘤异质性、鉴别不同功能亚群提供了可能。单细胞测序能够获得每个细胞的基因组变异图谱及转录组表达图谱，通过单个细胞的图谱精确划分克隆归属，实现对异质性克隆群体的全面解析。Timothy A.Graubert团队将一例已通过全基因组测序与靶向深度测序进行全面刻画的继发白血病样本进行了单细胞基因组分型测序，仅通过12个细胞DNA测序数据即发现了之前被认为是一个亚克隆的群体其实是由两个互斥的亚克隆构成，充分说明单细胞测序的优势和其在多克隆研究中的必要性。然而早期的单细胞测序技术往往通量低，成本高，一定程度上限制了精确分析并追踪异质性群体变化的分析。2016年10x Genomics公司推出的10x Chromium Single Cell Gene Expression Solution平台实现了高通量的单细胞转录组测序，具有周期短、成本低、细胞捕获率高等优势，在发育生物学及肿瘤异质性群体研究中应用广泛，在转录组水平实现对异质性肿瘤群体的全面刻画。
然而对于基因组变异驱动的恶性肿瘤群体，仅从转录组水平无法实现对肿瘤群体的鉴定以及功能异质性的解析。研究者开始着眼于基于单细胞水平的多组学研究平台，10x和BD公司分别实现单细胞转录组与单细胞染色质开放性(ATAC-seq)或单细胞蛋白质组的结合。然而，对于肿瘤异质性研究中最需要的单细胞转录组与基因组信息的整合平台，目前尚无成熟技术。对此，来自不同实验室的研究者进行了大量尝试，目前大部分技术仍然依赖同时将单个细胞中的转录组与基因组进行分离而分别测序，操作繁琐且通量较小。对转录组和基因组同时测序的技术又面临扩增效率低下或等位基因扩增偏好等难题，近期新提出的Target-seq技术针对肿瘤群体设计同时检测转录组及特异基因突变的技术，也说明了肿瘤研究中对该技术的需求，但该技术仍处于实验室水平，仍然无法实现一次上千细胞 数的分析。另外，Peter Van Galen等人在Cell发表文献，通过单细胞转录本与三代测序技术相结合，首次实现对白血病患者肿瘤群体(以基因组变异为金标准)中转录组异质性的解析，发现肿瘤群体存在于表达谱不同的多种谱系中，明确了基因组异质性与转录组异质性相互独立又相互影响的关系，也表明在单细胞转录组水平进一步明确细胞的基因组变异的重要性。然而，该研究中使用的三代测序检测突变的技术具有很大局限性，突变检出率受到具体突变位点的限制，单个突变检出率最高仅23％，平均可以检测到突变的细胞不超过5％，作者最终采用随机森林的机器学习算法预测肿瘤群体，无法实现对肿瘤细胞群体的直接鉴定，也没有将基因组与转录组异质性很好的对应。且该技术操作繁琐，样本需求量高，花费大，不适于全面推广。
因此，在肿瘤研究中实现单细胞水平基因组与转录组异质性的整合分析具有重要性与迫切性，而提供一种可用于进行单细胞转录组与基因突变整合分析的芯片显得尤为重要。
申请内容
本申请所要解决的技术问题在于针对上述现有技术中的不足，提供一种高通量单细胞转录组与基因突变整合分析编码芯片、方法和装置。
为解决上述技术问题，一方面，本申请提供：一种高通量单细胞转录组与基因突变整合分析编码芯片，所述芯片在其基板上设置有多个微孔，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条已知的核酸序列，所述核酸序列依次包括：
Spacer序列；
通用引物序列，作为PCR扩增时的引物结合区域；
细胞标签，用于标示RNA源自的细胞；
分子标签，用于标示结合的RNA；
以及Ploy T。
可选地，每个所述微孔内修饰的核酸序列不小于10 6条。
可选地，所述分子标签为一段已知的随机核酸序列。
可选地，每个微孔的细胞标签与空间坐标编码一一对应，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个细胞中的RNA。
可选地，所述微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个。
可选地，所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
一种如上所述的高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，包括以下步骤：
1)制备微孔阵列芯片；
2)在所述微孔内修饰核酸序列。
可选地，所述步骤1)具体为：在硅上通过光刻和深硅刻蚀直接形成微孔，微孔可以是盲孔或通孔。
可选地，所述步骤1)具体为：首先通过硅的光刻制备阳膜，然后通过PDMS浇筑脱模形成软光刻图形，与平面玻片结合后，采用毛细微模塑的方法，将聚氨酯或环氧树脂固化与玻片形成微孔阵列。
可选地，所述步骤2)具体包括：利用喷墨打印的方式，结合寡核苷酸原位化学合成方法，在微孔内合成spacer、通用引物、细胞标签序列和延伸接头；然后通过核酸扩增方 法，以分子标签和PolyA为模板，将原位合成的序列延伸形成分子标签序列段，从而得到最终的核酸序列。
另一方面：本申请提供一种高通量单细胞转录组与基因突变整合分析方法，包括以下步骤：
1)提供一种高通量单细胞编码芯片，所述芯片具有多个微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应；
2)利用所述芯片的微孔捕获单细胞，通过荧光成像进行各个微孔位置的单细胞表面蛋白分型分析；
3)对微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
4)针对固定在微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记，然后通过双色荧光图像分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
5)将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库；
6)针对游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，利用转录组RNA上接上的细胞标签，获知每一条转录组RNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库。
可选地，所述步骤2)具体包括：预先对细胞的目的基因进行荧光标记，再将细胞加入所述芯片中，利用所述芯片的微孔捕获单细胞，然后进行荧光图像采集，并对荧光图像上的微孔位置进行定位，利用荧光图像分析识别含有目的基因的特异细胞的位置，得到各个微孔位置的细胞蛋白表达信息。
可选地，所述步骤2)中，对荧光图像上的微孔位置进行定位的方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的荧光图像与图像网格模板配准，从而识别出荧光图像上的各个微孔区域的位置，将微孔位置定位到荧光图像上。
可选地，所述步骤4)具体包括：加入PCR扩增试剂，加入预先设计的修饰有不同荧光基团的两种引物探针，其中一种用于与野生型目的基因结合，另一种用于与突变型目的基因结合，对微孔中的单细胞进行原位裂解扩增；
进行双色荧光图像采集，对得到的双色荧光图像上的微孔位置进行定位，计算每个微孔位置2种荧光的强度值，通过聚类算法统计每个微孔位置2种荧光的强度的比值，从而得到各微孔位置的目的基因的野生型与突变型的比例，获得扩增后的转录组RNA序列的突变表达信息。
可选地，所述步骤4)中，对双色荧光图像上的微孔位置的定位方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的双色荧光图像与图像网格模板配准，从而识别出双色荧光图像上的各个微孔区域的位置，将微孔位置定位到双色荧光图像上。
可选地，所述核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。
可选地，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个 细胞中的RNA。
可选地，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
可选地，所述微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个。
可选地，所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
又一方面，本申请提供一种高通量单细胞转录组与基因突变整合分析一体化装置，包括高通量单细胞编码芯片和整合分析装置；
所述高通量单细胞编码芯片具有用于捕获单细胞的微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应；
所述整合分析装置包括壳体以及设置在所述壳体内的温控热循环模块、荧光成像模块和数据存储分析模块，所述荧光成像模块包括光源组件、显微物镜、荧光分光组件和成像探测器；
所述温控热循环组件上设置有用于安置所述高通量单细胞编码芯片的载物热台，所述温控热循环组件用于提供PCR扩增反应所需的温度环境；
所述光源组件发出的激发光经所述荧光分光组件后，再经过所述显微物镜后到达所述温控热循环组件上的所述高通量单细胞编码芯片，其中的样品被激发产生的荧光原路返回经过所述显微物镜和荧光分光模块后，再进入所述成像探测器，进行荧光成像；
所述数据存储分析模块用于存储所述成像探测器采集的荧光图像信息，并进行单细胞转录组与基因突变整合分析。
可选地，所述光源组件包括第一LED光源、第二LED光源、第三LED光源、第一二向色镜、第二二向色镜和扩束透镜组，
所述第一LED光源发出的光依次透射所述第一二向色镜、第二二向色镜后到达所述扩束透镜组；
所述第二LED光源发出的光经第一二向色镜反射、第二二向色镜透射后到达所述扩束透镜组；
所述第三LED光源发出的光经第二二向色镜反射后到达所述扩束透镜组；
所述第一LED光源、第二LED光源、第三LED光源发出三种不同波长的光，且三种光的波长范围覆盖400nm-700nm。
可选地，所述荧光分光组件包括支架、可转动设置在所述支架上的旋转台、用于驱动所述旋转台转动的电机以及均匀间隔设置在所述旋转台上的若干个荧光分光模块，
所述荧光分光模块包括激发光滤光片、样品光滤光片和第四二向色镜；
所述旋转台用于将若干个所述荧光分光模块中的一个切换进入光路，所述扩束透镜组出射的激发光经过所述激发光滤光片后被所述第四二向色镜反射，然后经过所述显微物镜到达安置在所述载物热台上的高通量单细胞编码芯片上；所述高通量单细胞编码芯片中的样品产生的荧光经过所述显微物镜后透射所述第四二向色镜，再经过所述样品光滤光片后到达所述成像探测器，所述成像探测器采集的荧光图像信息传输至所述数据存储分析模块。
可选地，所述壳体上设置有放样窗口，所述放样窗口上设置有滑盖。
可选地，所述温控热循环组件包括温控盒体、设置在所述温控盒体内的散热器、设置在所述散热器上的帕尔贴以及设置在所述散热器侧部的风扇，所述载物热台设置在所述帕尔贴上，所述载物热台上设置有透明盖板。
可选地，所述核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。
可选地，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个细胞中的RNA。
可选地，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
可选地，所述微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个；
所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
可选地，所述高通量单细胞转录组与基因突变整合分析一体化装置分析步骤包括：
1)将样品加入所述高通量单细胞编码芯片中，通过其上的微孔捕获单细胞，将所述高通量单细胞编码芯片置于所述载物热台上，通过所述荧光成像模块对所述高通量单细胞编码芯片进行荧光成像，利用所述数据存储分析模块进行各个微孔位置的单细胞表面蛋白分型分析；
2)对所述高通量单细胞编码芯片的微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
3)启动所述温控热循环组件，针对固定在所述高通量单细胞编码芯片的微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记，通过所述荧光成像模块采集双色荧光图像，然后通过所述数据存储分析模块对双色荧光图像进行分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
4)通过所述数据存储分析模块将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库。
本申请的有益效果是：本申请提供了一种能用于高通量单细胞转录组与基因突变整合分析的芯片，通过采用微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码技术，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，能为实现高通量单细胞转录组与基因突变整合分析提供芯片基础。
本申请的高通量单细胞转录组与基因突变整合分析方法，通过设计具有微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码功能的高通量单细胞编码芯片，结合单细胞表面蛋白分型、单细胞转录组突变分析及基因测序的方式，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库，获得多组学整合分析模型。
本申请的高通量单细胞转录组与基因突变整合分析一体化装置，通过设计具有微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码功能的高通量单细胞编码芯片，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来；再通过温控热循环模块可实现PCR扩增，通过荧光成像模块采集样品的荧光图像，通过数据存储分析模块对荧光图像进行存储于分析，能实现单细胞表面蛋白分型与突变整合分析的数据库、高通量单细胞转录组与基因突变整合分析的完整数据库的建立，实现单细胞转录组与基因突变整合分析；
本申请在明确单个细胞中携带的基因组变异信息后，结合单细胞转录组甚至蛋白表达信息，可实现对肿瘤细胞多组学的全面认识和对肿瘤异质性群体的全面刻画，为肿瘤的早期诊断、耐药机制、新靶点探索和治疗方案优化提供基础。
附图说明
图1为本申请的一种实施例中的高通量单细胞转录组与基因突变整合分析编码芯片的制作流程示意图；
图2为本申请的高通量单细胞转录组与基因突变整合分析方法的流程图；
图3为本申请的一种实施例中的核酸序列修饰的流程图；
图4为本申请的高通量单细胞转录组与基因突变整合分析一体化装置的原理框图；
图5为本申请的高通量单细胞转录组与基因突变整合分析一体化装置的内部结构示意图；
图6为本申请的高通量单细胞转录组与基因突变整合分析一体化装置的外部结构示意图；
图7为本申请的荧光分光组件的支架的结构示意图；
图8为本申请的荧光分光模块的结构示意图；
图9为本申请的荧光分光模块的剖视结构示意图；
图10为本申请的温控热循环模块的分解图；
图11为本申请的高通量单细胞转录组与基因突变整合分析一体化装置的光路图。
附图标记说明：
1—高通量单细胞编码芯片；2—整合分析装置；3—壳体；4—温控热循环模块；5—荧光成像模块；6—光源组件；7—显微物镜；8—荧光分光组件；9—成像探测器；30—放样窗口；31—滑盖；40—载物热台；41—温控盒体；42—散热器；43—帕尔贴；44—风扇；60—第一LED光源；61—第二LED光源；62—第三LED光源；63—第一二向色镜；64—第二二向色镜；65—扩束透镜组；70—升降台；80—支架；81—旋转台；82—电机；83—荧光分光模块；810—安装槽；830—镜片安装块；831—激发光滤光片；832—样品光滤光片；833—第四二向色镜。
具体实施方式
下面结合实施例对本申请做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
应当理解，本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。
本实施例的一种高通量单细胞转录组与基因突变整合分析编码芯片，芯片在其基板上设置有多个微孔，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，每个微孔具有唯一的空间坐标编码，且微孔内修饰有若干条已知的核酸序列，核酸序列依次包括：
Spacer序列；
通用引物序列，作为PCR扩增时的引物结合区域；
细胞标签，用于标示RNA源自的细胞；
分子标签，用于标示结合的RNA；
以及Ploy T。
其中，每个微孔内修饰的核酸序列不小于10 6条。分子标签为一段已知的随机核酸序列。
每个微孔的细胞标签与空间坐标编码一一对应，当该微孔阵列装载细胞后，针对每一个特定微孔，一个细胞就携带了该微孔空间坐标编码，这个微孔空间坐标同时对应一个已知的细胞标签(核酸序列)和一组已知的分子标签(随机序列)。装载的单细胞可以进行免疫荧光标记，通过高通量多色荧光成像获取蛋白表达信息。
其中，单个微孔内的所有细胞标签具有相同的序列，不同微孔内的细胞标签的序列均不相同，从而通过细胞标签标识RNA源自的细胞；所以，在最后测序数据中可以通过细胞标签知道序列来源与哪个细胞，区分哪些序列是来自同一个细胞，哪些是来自不同的细胞。
单个微孔内的所有分子标签具有不同的序列，从而通过分子标签标识单个细胞中的RNA。分子标签标识只负责针对同一个细胞内的RNA进行标记，而不管不同细胞之间的RNA。对于单个细胞来说，通过分子标签可区别每一条RNA。所以，对于最后得到的检 测数据，通过细胞标签区分不同的细胞，并且一个细胞标签对应一个唯一的微孔空间坐标编码，从而知道RNA源自的细胞以及微孔坐标位置，然后再通过分子标签区分每一条RNA。从而能将每一条RNA源自的细胞、位置坐标信息对应起来，在单细胞转录组与基因突变整合分析中，通过本申请的芯片所采用的微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码技术，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
当细胞在孔内原位裂解后，释放RNA被孔内的核酸序列捕获，通过碱基互补配对的方式，为检测目标标志物接上了细胞标签和分子标签。并且，通过扩增在孔壁和孔内同时形成了cDNA。针对游离的cDNA通过进行高通量测序，可以获取单细胞的转录组信息，这一组学信息会与单细胞的微孔空间坐标编码进行对应。针对固定在孔壁上的cDNA，进行原位的荧光PCR，可以获取单细胞额基因突变信息。这样通过本申请的编码技术，即可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
例如，在一种实施例中，利用本申请的芯片进行高通量单细胞转录组与基因突变整合分析的方法为：
1)进行单细胞表面蛋白分型分析：预先通过荧光标记目的基因，通过芯片捕获单细胞，细胞孵育，然后进行荧光图像采集，通过微孔位置定位，利用荧光图像分析识别计算含有目的基因的特异细胞的位置，得到各个微孔位置的细胞蛋白表达信息；
2)加入裂解液和扩增试剂，对微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
加入PCR扩增试剂，针对固定在微孔内的cDNA进行PCR扩增，并加入预先设计的修饰有不同荧光基团的两种引物探针，其中一种用于与野生型目的基因结合，另一种用于与突变型目的基因结合，对微孔中的单细胞进行原位裂解扩增；扩增后野生型目的基因和突变型目的基因均分别带有不同的荧光分子；然后进行双色荧光图像采集，对得到的双色荧光图像上的微孔位置进行定位，计算每个微孔位置2种荧光的强度值，通过聚类算法统计每个微孔位置2种荧光的强度的比值，从而得到各微孔位置的目的基因的野生型与突变型的比例，获得单细胞的基因突变表达信息；
针对游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，由于cDNA上接上了细胞标签和分子标签，从而能获知每一条cDNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库，建立多组学整合分析模型。
在优选的实施例中，微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个。微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
在优选的实施例中，高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，包括以下步骤：
1)制备微孔阵列芯片；
2)在微孔内修饰核酸序列，获得高通量单细胞分析芯片。
其中，步骤1)具体为：通过MEMS技术，在硅上通过光刻和深硅刻蚀直接形成微孔，微孔可以是盲孔或通孔。或是利用软光刻技术，参照图1，首先通过硅的光刻制备阳膜，然后通过PDMS浇筑脱模形成软光刻图形，与平面玻片结合后，采用毛细微模塑的方法，将聚氨酯或环氧树脂固化与玻片形成微孔阵列。图1中编码芯片即指先在平面玻片上原位合成的已修饰好核酸序列的芯片。
编码核酸的修饰方法与芯片制备方法对应：可以选择在微孔阵列中原位合成或在平面玻片上原位合成。在优选的实施例中，步骤2)具体包括：利用喷墨打印的方式，结合寡核苷酸原位化学合成方法，在微孔内合成spacer、通用引物、细胞标签序列和延伸接头；然后通过核酸扩增方法，以分子标签和PolyA为模板，将原位合成的序列延伸形成分子标 签序列段，从而得到最终的核酸序列。其流程参照图3，其中UMI表示分子标签。
本实施例的一种高通量单细胞转录组与基因突变整合分析方法，包括以下步骤：
1)提供一种高通量单细胞编码芯片，所述芯片具有多个微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应；
2)利用所述芯片的微孔捕获单细胞，通过荧光成像进行各个微孔位置的单细胞表面蛋白分型分析；
3)对微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
4)针对固定在微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记，然后通过双色荧光图像分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
5)将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库；
6)针对游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，利用转录组RNA上接上的细胞标签，获知每一条转录组RNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库。
实施例1提供一种高通量单细胞编码芯片
所述芯片在其基板上设有多个微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的已知的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应。
其中，所述核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。在进一步优选的实施例中，每个微孔内修饰的核酸序列不小于10 6条。分子标签为一段已知的随机核酸序列。
其中，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。在优选的实施例中，所述微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个。所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
当该微孔阵列装载细胞后，针对每一个特定微孔，一个细胞就携带了该微孔空间坐标编码，这个微孔空间坐标同时对应一个已知的细胞标签(核酸序列)和一组已知的分子标签(随机序列)。装载的单细胞可以进行免疫荧光标记，通过高通量多色荧光成像获取蛋白表达信息。
其中，单个微孔内的所有细胞标签具有相同的序列，不同微孔内的细胞标签的序列均不相同，从而通过细胞标签标识RNA源自的细胞；所以，在最后测序数据中可以通过细胞标签知道序列来源与哪个细胞，区分哪些序列是来自同一个细胞，哪些是来自不同的细胞。
单个微孔内的所有分子标签具有不同的序列，从而通过分子标签标识单个细胞中的RNA。分子标签标识只负责针对同一个细胞内的RNA进行标记，而不管不同细胞之间的RNA。对于单个细胞来说，通过分子标签可区别每一条RNA。所以，对于最后得到的检测数据，通过细胞标签区分不同的细胞，并且一个细胞标签对应一个唯一的微孔空间坐标编码，从而知道RNA源自的细胞以及微孔坐标位置，然后再通过分子标签区分每一条RNA。从而能将每一条RNA源自的细胞、位置坐标信息对应起来，在单细胞转录组与基 因突变整合分析中，通过本申请的芯片所采用的微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码技术，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
当细胞在孔内原位裂解后，释放RNA被孔内的核酸序列捕获，通过碱基互补配对的方式，为检测目标标志物接上了细胞标签和分子标签。并且，通过扩增在孔壁和孔内同时形成了cDNA。针对游离的cDNA通过进行高通量测序，可以获取单细胞的转录组信息，这一组学信息会与单细胞的微孔空间坐标编码进行对应。针对固定在孔壁上的cDNA，进行原位的荧光PCR，可以获取单细胞额基因突变信息。通过这样的三重编码技术，结合本申请的分析方法，即可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
在一种进一步的实施例中，上述芯片可通过以下制作工艺得到：
1)制备微孔阵列芯片：
通过MEMS技术，在硅上通过光刻和深硅刻蚀直接形成微孔，微孔可以是盲孔或通孔；
2)在微孔内修饰核酸序列，获得高通量单细胞分析芯片：
利用喷墨打印的方式，结合寡核苷酸原位化学合成方法，在微孔内合成spacer、通用引物、细胞标签序列和延伸接头；然后通过核酸扩增方法，以分子标签和PolyA为模板，将原位合成的序列延伸形成分子标签序列段，从而得到最终的核酸序列。其流程参照图3，其中UMI表示分子标签。
实施例2利用实施例1的芯片进行单细胞转录组与基因突变整合分析，参照图3，具体包括以下步骤：
一、单细胞表面蛋白分型分析：
预先对细胞的目的基因进行荧光标记，再将细胞加入所述芯片中，利用所述芯片的微孔捕获单细胞，孵育，然后进行荧光图像采集，并对荧光图像上的微孔位置进行定位，利用荧光图像分析识别含有目的基因的特异细胞的位置，得到各个微孔位置的细胞蛋白表达信息。其中，采用三色荧光通道进行荧光成像，采用三种不同波长的光源，实现全波段可见光覆盖(波长400nm-700nm)。
其中，对荧光图像上的微孔位置进行定位的方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的荧光图像与图像网格模板配准，从而识别出荧光图像上的各个微孔区域的位置，将微孔位置定位到荧光图像上。
二、单细胞转录组突变分析：
加入裂解液和扩增试剂，对微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
加入PCR扩增试剂，针对固定在微孔内的cDNA进行PCR扩增，并加入预先设计的修饰有不同荧光基团的两种引物探针，其中一种用于与野生型目的基因结合，另一种用于与突变型目的基因结合，对微孔中的单细胞进行原位裂解扩增；扩增后野生型目的基因和突变型目的基因均分别带有不同的荧光分子；
进行双色荧光图像采集，对得到的双色荧光图像上的微孔位置进行定位，计算每个微孔位置2种荧光的强度值，通过聚类算法统计每个微孔位置2种荧光的强度的比值，从而得到各微孔位置的目的基因的野生型与突变型的比例，获得单细胞的基因突变表达信息。
其中，对双色荧光图像上的微孔位置的定位方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的双色荧光图像与图像网格模板配准，从而识别出双色荧光图像上的各个微孔区域的位置，将微孔位置定位到双色荧光图像上。
三、建立单细胞表面蛋白分型与突变整合分析的数据库：
根据步骤一和步骤二的分析结果，将相同位置的单细胞表面蛋白分型信息与扩增后 的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库。
四、建立高通量单细胞转录组与基因突变整合分析模型：
针对游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，由于cDNA上接上了细胞标签和分子标签，从而能获知每一条cDNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库，建立多组学整合分析模型。
如图4所示，本实施例的一种高通量单细胞转录组与基因突变整合分析一体化装置，包括高通量单细胞编码芯片1和整合分析装置2；
高通量单细胞编码芯片1具有用于捕获单细胞的微孔，每个微孔具有唯一的空间坐标编码，且微孔内修饰有若干条用于捕获目标RNA的核酸序列，核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应；
整合分析装置2包括壳体3以及设置在壳体3内的温控热循环模块4、荧光成像模块5和数据存储分析模块，荧光成像模块5包括光源组件6、显微物镜7、荧光分光组件8和成像探测器9；
温控热循环组件上设置有用于安置高通量单细胞编码芯片1的载物热台40，温控热循环组件用于提供PCR扩增反应所需的温度环境；
光源组件6发出的激发光经荧光分光组件8后，再经过显微物镜7后到达温控热循环组件上的高通量单细胞编码芯片1，其中的样品被激发产生的荧光原路返回经过显微物镜7和荧光分光模块83后，再进入成像探测器9，进行荧光成像；
数据存储分析模块用于存储成像探测器9采集的荧光图像信息，并进行单细胞转录组与基因突变整合分析。
实施例3
在以上基础上，本实施例中提供一种具体的整合分析装置2。
参照图4-11，其中，光源组件6包括第一LED光源60、第二LED光源61、第三LED光源62、第一二向色镜63、第二二向色镜64和扩束透镜组65，第一LED光源60发出的光依次透射第一二向色镜63、第二二向色镜64后到达扩束透镜组65；第二LED光源61发出的光经第一二向色镜63反射、第二二向色镜64透射后到达扩束透镜组65；第三LED光源62发出的光经第二二向色镜64反射后到达扩束透镜组65；第一LED光源60、第二LED光源61、第三LED光源62发出三种不同波长的光，且三种光的波长范围覆盖400nm-700nm。
其中，荧光分光组件8包括支架80、可转动设置在支架80上的旋转台81、用于驱动旋转台81转动的电机82以及均匀间隔设置在旋转台81上的若干个荧光分光模块83，荧光分光模块83包括镜片安装块830以及设置在其中的激发光滤光片831、样品光滤光片832和第四二向色镜833；镜片安装块830上设置3个开口，底部开口供激发光和样品光直接通过，左侧开口安装激发光滤光片831，上部开口安装样品光滤光片832。
旋转台81用于将若干个荧光分光模块83中的一个切换进入光路，扩束透镜组65出射的激发光经过激发光滤光片831后被第四二向色镜833反射，然后经过显微物镜7到达安置在载物热台40上的高通量单细胞编码芯片1上；高通量单细胞编码芯片1中的样品产生的荧光经过显微物镜7后透射第四二向色镜833，再经过样品光滤光片832后到达成像探测器9，成像探测器9采集的荧光图像信息传输至数据存储分析模块。本实施例中包括5个不同的荧光分光模块83，分别设置在旋转台81上开设的5个安装槽810内，从而实现多种荧光的分光。
其中，壳体3上设置有放样窗口30，放样窗口30上设置有滑盖31。通过放样窗口 30方便将高通量单细胞编码芯片1放入到载物热台40上。
其中，温控热循环组件包括温控盒体41、设置在温控盒体41内的散热器42、设置在散热器42上的帕尔贴43以及设置在散热器42侧部的风扇44，载物热台40设置在帕尔贴43上，载物热台40上设置有透明盖板，通过透明盖板密封，且不影响荧光成像。通过温控热循环组件实现PCR扩增反应过程中的温度控制。载物热台40具有很好的导热性能，帕尔贴43对载物热台40进行加热，散热器42具有多个散热鳍片，配合风扇44实现快速散热，从而实现温度升降控制。
其中，显微物镜7安装在升降台70上，可上下移动，方便调节，配合电机实现对焦功能。整个整合分析装置2可通过上位机进行集中控制。
实施例4
在上述基础上，提供一种高通量单细胞编码芯片1。
其中，芯片在其基板上设有多个微孔，每个微孔具有唯一的空间坐标编码，且微孔内修饰有若干条用于捕获目标RNA的已知的核酸序列，核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应。
其中，核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。在进一步优选的实施例中，每个微孔内修饰的核酸序列不小于10 6条。分子标签为一段已知的随机核酸序列。
其中，微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。在优选的实施例中，微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个。微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
当该微孔阵列装载细胞后，针对每一个特定微孔，一个细胞就携带了该微孔空间坐标编码，这个微孔空间坐标同时对应一个已知的细胞标签(核酸序列)和一组已知的分子标签(随机序列)。装载的单细胞可以进行免疫荧光标记，通过高通量多色荧光成像获取蛋白表达信息。
其中，单个微孔内的所有细胞标签具有相同的序列，不同微孔内的细胞标签的序列均不相同，从而通过细胞标签标识RNA源自的细胞；所以，在最后测序数据中可以通过细胞标签知道序列来源与哪个细胞，区分哪些序列是来自同一个细胞，哪些是来自不同的细胞。
单个微孔内的所有分子标签具有不同的序列，从而通过分子标签标识单个细胞中的RNA。分子标签标识只负责针对同一个细胞内的RNA进行标记，而不管不同细胞之间的RNA。对于单个细胞来说，通过分子标签可区别每一条RNA。所以，对于最后得到的检测数据，通过细胞标签区分不同的细胞，并且一个细胞标签对应一个唯一的微孔空间坐标编码，从而知道RNA源自的细胞以及微孔坐标位置，然后再通过分子标签区分每一条RNA。从而能将每一条RNA源自的细胞、位置坐标信息对应起来，在单细胞转录组与基因突变整合分析中，通过本申请的芯片所采用的微孔空间坐标、细胞核酸标签和分子核酸标签的三重编码技术，可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
当细胞在孔内原位裂解后，释放RNA被孔内的核酸序列捕获，通过碱基互补配对的方式，为检测目标标志物接上了细胞标签和分子标签。并且，通过扩增在孔壁和孔内同时形成了cDNA。针对游离的cDNA通过进行高通量测序，可以获取单细胞的转录组信息，这一组学信息会与单细胞的微孔空间坐标编码进行对应。针对固定在孔壁上的cDNA，进行原位的荧光PCR，可以获取单细胞额基因突变信息。通过这样的三重编码技术，结合本申请的分析方法，即可将单细胞的基因突变、转录组和蛋白表达信息一一对应起来。
在一种进一步的实施例中，上述芯片可通过以下制作工艺得到：
1)制备微孔阵列芯片：
通过MEMS技术，在硅上通过光刻和深硅刻蚀直接形成微孔，微孔可以是盲孔或通孔；
2)在微孔内修饰核酸序列，获得高通量单细胞分析芯片：
利用喷墨打印的方式，结合寡核苷酸原位化学合成方法，在微孔内合成spacer、通用引物、细胞标签序列和延伸接头；然后通过核酸扩增方法，以分子标签和PolyA为模板，将原位合成的序列延伸形成分子标签序列段，从而得到最终的核酸序列。
实施例5
提供一种高通量单细胞转录组与基因突变整合分析一体化装置，结合实施例3的整合分析装置2和实施例4的高通量单细胞编码芯片1获得。
本实施例中的高通量单细胞转录组与基因突变整合分析一体化装置的其分析步骤包括：
1)预先对细胞的目的基因进行荧光标记，再将样品加入高通量单细胞编码芯片1中，通过其上的微孔捕获单细胞，将高通量单细胞编码芯片1置于载物热台40上，启动光源组件6、显微物镜7、荧光分光组件8和成像探测器9，通过荧光成像模块5对高通量单细胞编码芯片1进行荧光成像，然后利用数据存储分析模块进行各个微孔位置的单细胞表面蛋白分型分析；其中，向高通量单细胞编码芯片1中加样、加试剂等操作可先将高通量单细胞编码芯片1从载物热台40上取出后加入，也可通过加样机构直接在载物热台40上进行加入操作；
2)向高通量单细胞编码芯片1再加入裂解液和扩增试剂，对高通量单细胞编码芯片1的微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
3)向高通量单细胞编码芯片1中加入PCR扩增试剂，启动温控热循环组件，针对固定在高通量单细胞编码芯片1的微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记(加入预先设计的修饰有不同荧光基团的两种引物探针，其中一种用于与野生型目的基因结合，另一种用于与突变型目的基因结合，扩增后野生型目的基因和突变型目的基因均分别带有不同的荧光分子)，通过荧光成像模块5采集双色荧光图像，然后通过数据存储分析模块对双色荧光图像进行分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
4)通过数据存储分析模块将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库。
5)针对步骤2)中收集的游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，由于cDNA上接上了细胞标签和分子标签，从而能获知每一条cDNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库，建立多组学整合分析模型，实现单细胞转录组与基因突变整合分析。
尽管本申请的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本申请的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本申请并不限于特定的细节。

Claims (30)

  1. 一种高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，所述芯片在其基板上设置有多个微孔，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条已知的核酸序列，所述核酸序列依次包括：
    Spacer序列；
    通用引物序列，作为PCR扩增时的引物结合区域；
    细胞标签，用于标示RNA源自的细胞；
    分子标签，用于标示结合的RNA；
    以及Ploy T。
  2. 根据权利要求1所述的高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，每个所述微孔内修饰的核酸序列不小于10 6条。
  3. 根据权利要求1所述的高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，所述分子标签为一段已知的随机核酸序列。
  4. 根据权利要求1所述的高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，每个微孔的细胞标签与空间坐标编码一一对应，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
    单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个细胞中的RNA。
  5. 根据权利要求1所述的高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，所述微孔呈蜂窝状排列，其数量为10 2-10 6个。
  6. 根据权利要求5所述的高通量单细胞转录组与基因突变整合分析编码芯片，其特征在于，所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
  7. 一种如权利要求1-6中任意一项所述的高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，其特征在于，包括以下步骤：
    1)制备微孔阵列芯片；
    2)在所述微孔内修饰核酸序列。
  8. 根据权利要求7所述的高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，其特征在于，所述步骤1)具体为：在硅上通过光刻和深硅刻蚀直接形成微孔，微孔可以是盲孔或通孔。
  9. 根据权利要求7所述的高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，其特征在于，所述步骤1)具体为：首先通过硅的光刻制备阳膜，然后通过PDMS浇筑脱模形成软光刻图形，与平面玻片结合后，采用毛细微模塑的方法，将聚氨酯或环氧树脂固化与玻片形成微孔阵列。
  10. 根据权利要求7所述的高通量单细胞转录组与基因突变整合分析编码芯片的制作工艺，其特征在于，所述步骤2)具体包括：利用喷墨打印的方式，结合寡核苷酸原位化学合成方法，在微孔内合成spacer、通用引物、细胞标签序列和延伸接头；然后通过核酸扩增方法，以分子标签和PolyA为模板，将原位合成的序列延伸形成分子标签序列段，从而得到最终的核酸序列。
  11. 一种高通量单细胞转录组与基因突变整合分析方法，其特征在于，包括以下步骤：
    1)提供一种高通量单细胞编码芯片，所述芯片具有多个微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每 个微孔的细胞标签与空间坐标编码一一对应；
    2)利用所述芯片的微孔捕获单细胞，通过荧光成像进行各个微孔位置的单细胞表面蛋白分型分析；
    3)对微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
    4)针对固定在微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记，然后通过双色荧光图像分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
    5)将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库；
    6)针对游离cDNA，通过基因测序分析cDNA，获取单细胞转录谱及亚型信息，利用转录组RNA上接上的细胞标签，获知每一条转录组RNA来源的细胞和微孔位置，从而将单细胞的基因突变、转录组和蛋白表达信息一一对应起来，形成高通量单细胞转录组与基因突变整合分析的完整数据库。
  12. 根据权利要求11所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述步骤2)具体包括：预先对细胞的目的基因进行荧光标记，再将细胞加入所述芯片中，利用所述芯片的微孔捕获单细胞，然后进行荧光图像采集，并对荧光图像上的微孔位置进行定位，利用荧光图像分析识别含有目的基因的特异细胞的位置，得到各个微孔位置的细胞蛋白表达信息。
  13. 根据权利要求12所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述步骤2)中，对荧光图像上的微孔位置进行定位的方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的荧光图像与图像网格模板配准，从而识别出荧光图像上的各个微孔区域的位置，将微孔位置定位到荧光图像上。
  14. 根据权利要求13所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述步骤4)具体包括：加入PCR扩增试剂，加入预先设计的修饰有不同荧光基团的两种引物探针，其中一种用于与野生型目的基因结合，另一种用于与突变型目的基因结合，对微孔中的单细胞进行原位裂解扩增；
    进行双色荧光图像采集，对得到的双色荧光图像上的微孔位置进行定位，计算每个微孔位置2种荧光的强度值，通过聚类算法统计每个微孔位置2种荧光的强度的比值，从而得到各微孔位置的目的基因的野生型与突变型的比例，获得扩增后的转录组RNA序列的突变表达信息。
  15. 根据权利要求14所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述步骤4)中，对双色荧光图像上的微孔位置的定位方法具体为：根据所述微孔阵列芯片上的微孔位置建立图像网格模板，然后将采集的双色荧光图像与图像网格模板配准，从而识别出双色荧光图像上的各个微孔区域的位置，将微孔位置定位到双色荧光图像上。
  16. 根据权利要求11所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。
  17. 根据权利要求16所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
    单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个细胞中的RNA。
  18. 根据权利要求17所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
  19. 根据权利要求18所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述微孔呈蜂窝状排列，其数量为10 2-10 6个。
  20. 根据权利要求18所述的高通量单细胞转录组与基因突变整合分析方法，其特征在于，所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
  21. 一种高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，包括高通量单细胞编码芯片和整合分析装置；
    所述高通量单细胞编码芯片具有用于捕获单细胞的微孔，每个所述微孔具有唯一的空间坐标编码，且所述微孔内修饰有若干条用于捕获目标RNA的核酸序列，所述核酸序列包括用于标示RNA源自的细胞的细胞标签和用于标示结合的RNA的分子标签，每个微孔的细胞标签与空间坐标编码一一对应；
    所述整合分析装置包括壳体以及设置在所述壳体内的温控热循环模块、荧光成像模块和数据存储分析模块，所述荧光成像模块包括光源组件、显微物镜、荧光分光组件和成像探测器；
    所述温控热循环组件上设置有用于安置所述高通量单细胞编码芯片的载物热台，所述温控热循环组件用于提供PCR扩增反应所需的温度环境；
    所述光源组件发出的激发光经所述荧光分光组件后，再经过所述显微物镜后到达所述温控热循环组件上的所述高通量单细胞编码芯片，其中的样品被激发产生的荧光原路返回经过所述显微物镜和荧光分光模块后，再进入所述成像探测器，进行荧光成像；
    所述数据存储分析模块用于存储所述成像探测器采集的荧光图像信息，并进行单细胞转录组与基因突变整合分析。
  22. 根据权利要求21所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述光源组件包括第一LED光源、第二LED光源、第三LED光源、第一二向色镜、第二二向色镜和扩束透镜组，
    所述第一LED光源发出的光依次透射所述第一二向色镜、第二二向色镜后到达所述扩束透镜组；
    所述第二LED光源发出的光经第一二向色镜反射、第二二向色镜透射后到达所述扩束透镜组；
    所述第三LED光源发出的光经第二二向色镜反射后到达所述扩束透镜组；
    所述第一LED光源、第二LED光源、第三LED光源发出三种不同波长的光，且三种光的波长范围覆盖400nm-700nm。
  23. 根据权利要求22所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述荧光分光组件包括支架、可转动设置在所述支架上的旋转台、用于驱动所述旋转台转动的电机以及均匀间隔设置在所述旋转台上的若干个荧光分光模块，
    所述荧光分光模块包括激发光滤光片、样品光滤光片和第四二向色镜；
    所述旋转台用于将若干个所述荧光分光模块中的一个切换进入光路，所述扩束透镜组出射的激发光经过所述激发光滤光片后被所述第四二向色镜反射，然后经过所述显微物镜到达安置在所述载物热台上的高通量单细胞编码芯片上；所述高通量单细胞编码芯片中的样品产生的荧光经过所述显微物镜后透射所述第四二向色镜，再经过所述样品光滤光片后到达所述成像探测器，所述成像探测器采集的荧光图像信息传输至所述数据存储分析模块。
  24. 根据权利要求21所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述壳体上设置有放样窗口，所述放样窗口上设置有滑盖。
  25. 根据权利要求21所述的高通量单细胞转录组与基因突变整合分析一体化装置， 其特征在于，所述温控热循环组件包括温控盒体、设置在所述温控盒体内的散热器、设置在所述散热器上的帕尔贴以及设置在所述散热器侧部的风扇，所述载物热台设置在所述帕尔贴上，所述载物热台上设置有透明盖板。
  26. 根据权利要求21所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述核酸序列还包括Spacer序列、作为PCR扩增时的引物结合区域的通用引物序列以及Ploy T。
  27. 根据权利要求26所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，单个所述微孔内的所有细胞标签具有相同的序列，不同所述微孔内的细胞标签的序列均不相同，从而通过所述细胞标签标识RNA源自的细胞；
    单个所述微孔内的所有分子标签具有不同的序列，从而通过所述分子标签标识单个细胞中的RNA。
  28. 根据权利要求27所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述微孔具有在一个微孔中只能容纳单个细胞的尺寸和形状。
  29. 根据权利要求28所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，所述微孔为正六边形，且呈蜂窝状排列，其数量为10 2-10 6个；
    所述微孔的外接圆的直径为30-60μm，深度为20-300μm，孔间的间距为10-30μm。
  30. 根据权利要求27所述的高通量单细胞转录组与基因突变整合分析一体化装置，其特征在于，其分析步骤包括：
    1)将样品加入所述高通量单细胞编码芯片中，通过其上的微孔捕获单细胞，将所述高通量单细胞编码芯片置于所述载物热台上，通过所述荧光成像模块对所述高通量单细胞编码芯片进行荧光成像，利用所述数据存储分析模块进行各个微孔位置的单细胞表面蛋白分型分析；
    2)对所述高通量单细胞编码芯片的微孔中的单细胞进行原位裂解扩增，逆转录合成携带细胞标签、分子标签的cDNA，游离cDNA收集后用于单细胞转录组分析，固定在微孔内的cDNA序列用于基因突变分析；
    3)启动所述温控热循环组件，针对固定在所述高通量单细胞编码芯片的微孔内的cDNA进行PCR扩增，并对目的基因的野生型和突变型进行双色荧光标记，通过所述荧光成像模块采集双色荧光图像，然后通过所述数据存储分析模块对双色荧光图像进行分析，计算各微孔位置的野生型与突变型的比例，得到单细胞的基因突变表达信息；
    4)通过所述数据存储分析模块将相同位置的单细胞表面蛋白分型信息与扩增后的单细胞基因突变表达信息结合，建立单细胞表面蛋白分型与突变整合分析的数据库。
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WO2010142954A1 (en) * 2009-06-09 2010-12-16 Oxford Gene Technology Ip Limited Picowell capture devices for analysing single cells or other particles
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CN108138365A (zh) * 2015-11-17 2018-06-08 深圳华大生命科学研究院 一种高通量的单细胞转录组建库方法
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