WO2020218554A1 - Digital somatic cell variation analysis - Google Patents

Abstract

The present disclosure provides a method for analyzing the composition of variations in a tissue, simply, at low cost, and/or with high precision. The method for analyzing the composition of cells in a tissue according the present disclosure comprises a step for assessing information about the sequence of cells from samples containing amplified nucleic acid derived from single cells in the tissue. This method enables assessment of the cell composition in a tissue simply at low cost when being carried out before whole genome sequencing, and enables improvement in precision since it is possible to conduct comparison of long gene sequences when information regarding the target to be analyzed is narrowed down from digital sequence information after whole genome sequencing is performed.

Description

Digital somatic mutation analysis

This disclosure is available in fields such as biological research, medicine, and healthcare regarding the analysis of cell populations.

Cancer, brain tissue, nerve tissue, etc. are composed of various cell types with different genealogy and function (Non-Patent Document 1 = Woodruff, 1983). In particular, cancer tissues proliferate while acquiring new genomic mutations one after another, so one cancer tissue contains many tumor subclones. These subclones differ in their morphology, proliferation, drug resistance, etc. due to differences in genetic information and epigenetic control. For example, it is known that a mutation in the epidermal growth factor receptor (EGFR) gene causes lung cancer, and that the susceptibility to the EGFR inhibitor gefitinib changes depending on the mutation site (Non-Patent Document 2 = Paezetal. , 2004). In addition to gene mutations, there are also gene regions that are naturally diverse, such as changes in chromosomal polyploidy and copy number variation. Accurate detection of these genetic diversities is essential for a detailed understanding of body tissue.

However, there are many of these genetic features that cannot be detected by simply analyzing the cell population collectively. Since it is indispensable to understand the body tissue at the single cell level for this accurate measurement (Non-Patent Document 3 = Zonget al., 2012), the necessity of single cell analysis is stated in various areas. ..

The present inventors are a method of analyzing the composition of cells in a tissue as a result of diligent invention, and the sequence information of cells (for example, mutation) is obtained from a sample containing an amplified nucleic acid derived from each cell in the tissue. ) Can be cheaply and easily evaluated by the method including the step of evaluating the whole genome sequence before decoding the whole genome sequence. After decoding the whole genome sequence, the information to be analyzed can be narrowed down from the digital sequence information. The present invention has been completed by finding that it becomes possible to compare long gene sequences and the accuracy is improved.

The present disclosure comprehensively reads the gene sequence that identifies the characteristics of each cell from the amplified polynucleotide prepared in parallel for each cell from a cell population (for example, tissue) containing various cells, and selects the cells in the sample. It is possible to digitally count cells one by one and provide information that is digitally counted with the cell composition as an absolute amount.

Examples of embodiments of the present disclosure include:
(Item 1) A method for analyzing mutations in tissues.
A method comprising identifying mutations in a cell or cell-like structure from a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue.
(Item A) A method for analyzing mutations in tissues.
A step of individually obtaining a nucleic acid sequence derived from each cell or cell-like structure in the tissue, and
A method comprising the step of analyzing using the individually obtained set of nucleic acid sequences.
(Item 2) Amplified nucleic acid derived from each cell or cell-like structure
Using a sample containing the tissue, a step of encapsulating cells or cell-like structures in droplets one cell or structure at a time, and
The process of gelling the droplets to form gel capsules,
A step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, wherein the genomic DNA of the cell or a polynucleotide containing a portion thereof is eluted into the gel capsule and the genomic DNA or its portion. The process of holding in the gel capsule with the substance bound to the moiety removed,
The method according to any of the above items, which is produced by a method comprising contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule.
(Item 3) The cells or cell-like structures are encapsulated in the droplets produced by flowing the suspension of the cells or cell-like structures into a microchannel and shearing the suspension with oil. The method according to any one of the above items, characterized in that
(Item 4) The method according to any one of the above items, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
(Item 5) The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform , Guanidin Hydrochloride, Urea, 2-Mercaptoethanol, Dithiotreitol, TCEP-HCl, Sodium Colate, Sodium Deoxycholate, TritonX-100, Triton X-114, NP-40, Brij-35, Brij-58, The item, wherein at least one is selected from the group consisting of Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-hydrochloride, Nonidet P-40, and Zwittergent 3-12. The method described in any of.
(Item 6) The method according to any one of the above items, wherein the gel capsule is a hydrogel capsule.
(Item 7) The method according to any one of the above items, wherein the step of amplifying the polynucleotide in a gel capsule is carried out by a homeothermic chain substitution amplification reaction for 10 to 60 minutes.
(Item 8) The method according to any one of the above items, further comprising a step of selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell or cell-like structure.
(Item 9) The method according to any one of the above items, which comprises a step of detecting a nucleic acid having a specific sequence in a sample containing an amplified nucleic acid derived from each cell or cell-like structure.
(Item 10) The method according to any one of the above items, wherein the step of detecting the nucleic acid having the specific sequence comprises amplifying the nucleic acid having the specific sequence.
(Item 11) The method according to any one of the above items, wherein the mutation comprises a mutation accompanied by a change in the sequence compared to the reference sequence.
(Item 12) The method according to any one of the above items, wherein the mutation comprises a base substitution, insertion or deletion.
(Item 13) The item further comprises a step of obtaining genomic sequence data of each cell or cell-like structure from a sample containing an amplified nucleic acid derived from each cell or cell-like structure. The method described in either.
(Item 14) The method according to any one of the above items, further comprising a step of selecting genomic sequence data to be analyzed from the genomic sequence data of each cell or cell-like structure.
(Item 15) The method according to any one of the above items, wherein the mutation comprises a mutation that does not involve a sequence change compared to a reference sequence.
(Item 16) The method according to any of the above items, wherein the mutation comprises copy number variation (CNV).
(Item 17) The method according to any of the above items, which comprises using the genomic sequence data to identify mutations including base substitutions, insertions or deletions.
(Item 18) The method according to any one of the above items, wherein the tissue is a tissue containing a tumor.
(Item 19) The method according to any one of the above items, wherein the tissue is a human tissue.
(Item 20) A system for analyzing mutations in tissues.
An amplified nucleic acid sample provider that provides a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue.
A system comprising a mutation-identifying part that identifies a mutation in a cell or cell-like structure.
(Item 21) The amplified nucleic acid sample providing unit
Using a sample containing the above-mentioned tissue, a droplet encapsulation portion for encapsulating cells or cell-like structures one cell or a structure unit in a droplet,
A gel capsule generation unit that gels the droplet to generate a gel capsule,
One or more dissolution reagents and
A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, which contains one or more lysis reagents for lysing cells. , The polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted in the gel capsule and retained in the gel capsule with the substance binding to the genomic DNA or the portion removed. There is a cell lysate and
The system according to item 20, which comprises a reagent for amplifying the polynucleotide for amplifying the polynucleotide in a gel capsule.
(Item 22) The item 20 or 21, wherein the composition evaluation unit includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells. system.
(Item 23) The system according to item 22, wherein the detection reagent or detection device includes a nucleic acid amplification sequencing device for amplifying and sequencing a nucleic acid.
(Item 24) The composition evaluation unit further includes a genome sequence data acquisition unit for genome sequence data of each cell from a sample containing an amplified nucleic acid derived from each cell in the microbiota. The system according to any one of 20 to 23.

In the present disclosure, it is intended that the above one or more features may be provided in a further combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.

This disclosure enables easy acquisition of whole genome information of body tissues and efficient detection of gene mutations.

In the case of gene mutation analysis by PCR, it is possible to use a part of the amplified DNA sample, and by performing sequencing after PCR and determining the partial sequence, mutations are made from the information of a specific gene sequence. It is possible to evaluate the composition such as the number of mutated cells and perform a cheap and simple evaluation. When performing mutation analysis based on digital sequence data, it is possible to create composition data by extracting the gene sequence used for gene mutation analysis from the digital sequence data obtained by sequence determination. In this case, not only mutations accompanied by sequence changes, for example, mutations such as nucleotide substitutions and deletions (single-nucleotide variation (SNV)), but also copy number variation (CNV) can be evaluated.

FIG. 1 shows a schematic diagram in which steps are taken to prepare amplified DNA. FIG. 2 shows a schematic diagram of wet sequence screening. FIG. 3 is a schematic diagram of the sequence determination after selection. FIG. 4 is a schematic diagram showing a dry sequence screening step. FIG. 5 is a schematic diagram showing the overall flow of somatic mutation analysis in the present disclosure. FIG. 6 is a schematic diagram showing an example of designing a microfluidic device. The device has two inlets and one outlet. The two inlets are for aqueous solution and oil, respectively. At the cross, the aqueous solution is split with oil to form a large number of droplets. FIG. 7 is a diagram showing encapsulation of a single cell in a gel droplet. The left figure shows the encapsulation of GM12878 cells observed with a high-speed camera, and the right figure shows the GM12878 cells encapsulated in droplets. FIG. 8 shows a fluorescence image of the droplet after MDA (Multiple Displacement Amplification) amplification. The figure on the left is a genome amplification-positive droplet (green fluorescence) showing a spherical morphology. The figure on the right shows the collapse of the shape of the droplet after genome amplification. FIG. 9 shows the circularity of the droplets positive for genome amplification before and after MDA. FIG. 10 shows the quantification of DNA yield of secondary amplification products from genomic amplification positive or negative droplets. FIG. 11 shows a comparison of genomic amplification biases between conventional direct single-cell genome amplification and gel droplet-based single-cell genome amplification. The ΔCt value of the amplification of each single cell genome was measured by comparing the Ct value of each sample with the bulk extracted DNA sample. FIG. 12 shows the circularity of the droplets before the reaction of MDA (0 minutes), reaction times of 30 minutes and 60 minutes. FIG. 13 shows a comparison of genome amplification bias between gel droplet-based mononuclear genome amplification by 10-minute MDA reaction and 30-minute MDA reaction. The ΔCt value of the amplification of each mononuclear genome was measured by comparing the Ct value of each sample with the bulk extracted DNA sample. FIG. 14 shows the quantification of the DNA yield of the second amplification product by FACS sorting. The yield of all selected amplification negative droplets was less than 1000 ng. The yield of 53 selected amplification-positive droplets was 1000 ng or more. FIG. 15 shows the electrophoresis of the EGFR ex19 amplicon obtained from a mononuclear amplicon derived from a gel droplet. FIG. 16 shows the results of Sanger sequence analysis of EGFR ex20 and 21 amplicons obtained from a mononuclear amplicon derived from a gel droplet. (A) EGFR ex20 amplicon array. (B) EGFR ex21 amplicon array. Upper row: Sequences obtained from single-cell genomes known to have wild-type sequences in EGFR ex20 and 21, Lower row: Sequences obtained from single-cell genomes known to have mutant sequences in EGFR ex20 and 21 Array.

Hereinafter, the present disclosure will be described while showing the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of their plural, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

The present disclosure relates to a method for analyzing a somatic mutation composition, which comprises a step of evaluating a somatic mutation from a sample containing an amplified nucleic acid derived from each cell in the somatic cell.

(Definition, etc.)
The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate.

As used herein, the term "cell" refers to a particle that contains a molecule that carries the genetic information and is any particle that can be replicated (whether or not it is possible alone). The term "cell" as used herein includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi and the like.

In the present specification, the "cell-like structure" refers to any particle containing a molecule having genetic information. As used herein, "cell-like structures" include organelles such as mitochondria, cell nuclei, and chloroplasts, and viruses.

In the present specification, the "biomolecule" refers to a molecule possessed by any organism or virus. In vivo molecules can include nucleic acids, proteins, sugar chains, lipids, and the like. As used herein, the term "biomolecular analog" refers to a natural or non-natural variant of a biomolecule. Analogs of in vivo molecules can include modified nucleic acids, modified amino acids, modified lipids or modified sugar chains.

In the present specification, the term "gel" refers to a colloidal solution (sol) in which a polymer substance or colloidal particles interact with each other to form a network structure as a whole and contain a large amount of a liquid phase as a solvent or a dispersion medium. A state in which fluidity is lost. As used herein, "gelling" means changing a solution into a "gel" state.

In the present specification, the "gel capsule" refers to a gel-like fine particle structure capable of holding a cell or a cell-like structure therein.

In the present specification, "gene analysis" means examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample. In one embodiment, the gene analysis can include those that utilize a nucleic acid amplification reaction. Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned.

As used herein, the term "single cell level" means that the genetic information contained in one cell or cell-like structure is processed in a state of being distinguished from the genetic information contained in another cell or cell-like structure. To do. For example, when amplifying a polynucleotide at the "single cell level", the amplification is performed in a state in which the polynucleotide in one cell and the polynucleotide in another cell can be distinguished from each other.

As used herein, "single cell analysis" refers to the analysis of genetic information contained in one cell or cell-like structure in a state of being distinguished from the genetic information contained in another cell or cell-like structure. Point to.

In the present specification, "nucleic acid information" refers to information on nucleic acids contained in one cell or cell-like structure, and includes the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield.

In the present specification, "identity" refers to sequence similarity between two nucleic acid molecules. Identity can be determined by comparing positions in each sequence that can be aligned for comparison.

In the present specification, "tissue" refers to a set of cells or cell-like structures arranged three-dimensionally according to a certain rule. A plurality of tissues are combined to form an organ having a certain function, but in the present disclosure, a "tissue" may be an area that traverses a plurality of tissues. Tissues can contain cells or cell-like structures as well as other components such as extracellular matrix.

In the present specification, the "type" of a cell or cell-like structure is a parameter indicating the classification of a cell or a cell-like structure. A cell or cell-like structure may be classified into multiple types by multiple classifications. For example, from the viewpoint of whether or not a cell has a point mutation A, there are a type having a point mutation A and a type not having a point mutation A. In addition, from another viewpoint, from the viewpoint of whether or not the insertion mutation B is present, there are types having B and types not having B. It is possible to specify which type of each cell is.

As used herein, the term "mutation" refers to a portion of a cell or cell-like structure that differs from another cell or cell-like structure in terms of genetic information. The “mutation” includes, but is not essential, a difference from a sequence (reference sequence) that serves as a reference for genetic information of a cell or a structure for a certain organism.

As used herein, "composition" refers to what type of cell in a tissue is (eg, containing a mutation), or the amount of each type of cell contained. It refers to information and also includes information about whether or not the tissue contains cells of some type.

(Organizational analysis)
In one aspect of the disclosure, a method of analyzing the composition of cells in a tissue, from a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue, the cell or cell-like structure. A method may be provided that comprises the step of identifying sequence information in an object. A tissue is a collection of heterogeneous cells, and by identifying the type of each cell, not only the presence or absence of certain properties (0 or 1) in the tissue, but also the proportion of cells having a certain type. It is possible to obtain information such as (continuous). In the present disclosure, the tissue may be a human tissue.

(Mutation analysis)
In one aspect of the disclosure, a method of analyzing mutations in a tissue from a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue, in a cell or cell-like structure. Methods may be provided that include the step of identifying the mutation. A tissue is a collection of heterogeneous cells, and by identifying mutations in each cell, not only the presence or absence of mutations in the tissue (0 or 1) but also the proportion of cells having mutations (continuous). It is possible to obtain information such as (target). In the present disclosure, the tissue may be a human tissue.

In one aspect of the present disclosure, a method for analyzing mutations in a tissue, the step of individually obtaining a nucleic acid sequence derived from each cell or cell-like structure in the tissue, and the step of individually obtaining the nucleic acid sequence. A method may be provided that comprises the step of analysis using the set of nucleic acid sequences. The amount of nucleic acid per cell is small, and even when analysis is performed in a state where nucleic acids derived from a plurality of cells are mixed, it is common to use an amplification reaction, but nucleic acids such as using a random primer are used. It is known that the degree of amplification is biased for each sequence depending on the GC content and the like even when an attempt is made to amplify the whole. When amplification is biased, it is difficult to measure the relative amount of cells with a particular sequence in the tissue.

(Single cell analysis)
In the present disclosure, nucleic acid sequences derived from individual cells or cell-like structures may be obtained by any method. There are various methods for single-cell analysis, but the genome of one cell has a mass of only a few femtograms for bacteria and a few picograms for animal cells (Gregory, TR, Nicol, JA, Tamm, H., Kullman, B., Kullman, K., Leitch, IJ,… Bennett, MD (2007). Eukaryotic genome size databases. Nucleic Acids Research, 35 (Database issue), D332-8. https://doi.org/10.1093/nar/ gkl828) However, since genome analysis by the next-generation sequencer requires a genome in nanogram units, PCR and whole-genome amplification can be performed to detect multiple genome mutation sites and analyze whole-genome sequences. It is generally necessary to amplify the amount of genome to the nanogram level. The MDA (Multiple Displacement Amplification) method is widely used as a whole-genome amplification method (Non-Patent Document 4 = Zhang et al., 1992). The MDA method is a method for continuously amplifying the genome using phi29 DNA polymerase and a random primer, and is known to be an excellent method for whole-genome amplification. In order to obtain nucleic acid sequences derived from individual cells or cell-like structures, these whole-genome amplification reagents and PCR reagents are allowed to act on individual single cells to amplify the genome, and then perform various analyzes. Can be used.

Due to the evolution of genome amplification methods and the reduction of sequencing costs, gene mutations have been made to tens to hundreds of single cells by whole-genome sequencing or exome sequencing from whole-genome amplification, or by sequencing from specific amplification by PCR. And it is becoming possible to detect copy number variation.

The most basic platform for adding genomic amplification reagents to a single-cell genome is to use a micromanipulator or flow cytometry to dispense one cell into tubes and manually lyse reagents or whole tubes. A method of adding a genome amplification reagent (Babbe, H., Roers, A., Waisman, A., Lassmann, H., Goebels, N., Hohlfeld, R.,… Rajewsky, K. (2000). Clonal. Expansions of Cd8 ⁺ T Cells Dominate the T CellInfiltrate in Active Multiple Sclerosis Lesions as Shown by Micromanipulation and Single Cell Polymerase Chain Reaction. The Journal of ExperimentalMedicine, 192 (3), 393-404. Https://doi.org/10.1084/jem .192.3.393). At this time, it is known that a large amplification bias occurs when whole genome amplification is performed in an excessively large reaction field for a single cell genome (Ning, L., Li, Z., Wang, G. , Hu, W., Hou, Q., Tong, Y.,… He, J. (2015). Quantitative assessment of single-cell whole genome amplification methods for detecting copynumber variation using hippocampal neurons. Scientific Reports, 5,11415.Retrieved from https://doi.org/10.1038/srep11415). It has been clarified that the state of replication in the initial stage of the whole genome amplification reaction has a great influence on the amplification bias, and it is said that the adsorption of the single-cell genome to the tube and the presence of the primer dimer are factors. As a result of the amplification bias, samples of insufficient quality for genome analysis are mass-produced, resulting in a decrease in the number of samples that can be used for analysis and an increase in wasteful use of expensive reagents. Therefore, it may be preferable to adopt a single cell analysis method in which the amplification bias is reduced. Although the applicable samples are limited, it is also possible to perform whole-genome amplification by sorting only cells in the M stage of cell division, which contain a large amount of genome.

It may be preferable to use a method that can efficiently perform cell lysis and whole genome amplification in a small reaction field for a large number of single cells. It is possible to use microfluidics in single cell analysis. Microfluidics is a technology for freely manipulating solutions in micro / nanoliter units in microfluidic devices that require microchannels and microvessels. One example is Droplet Microfluidics. Droplet microfluidics have the advantage of being capable of precise operation and automation compared to conventional tube operations. In the μm size flow path provided in the microfluidic device, the liquid layer is sheared by the oil layer, and pL size droplets are continuously generated in units of 1000 per second. Since each droplet acts as an independent reaction field, by making a solution containing cells into a droplet in a microfluidic device, each cell is encapsulated in the droplet one by one and becomes independent for each cell. The reaction can be carried out. Furthermore, with the development of droplet manipulation technology, it is possible to individually perform complex reactions such as segmentation and sorting of droplets, including fusion of genome-encapsulated droplets and reaction reagent-encapsulated droplets such as PCR. Brouzes, E., Medkova, M., Savenelli, N., Marran, D., Twardowski, M., Hutchison, J. B.,… Samuels, M. L. (2009). Droplet microfluidic technology for single-cell high-throughputscreening. Proceedings of the National Academy of Sciences of the United States of America, 106 (34), 14195-200. Https://doi.org/10.1073/pnas.0903542106). Droplet microfluidics can perform various operations on cells with high throughput by creating droplets and encapsulating the cells, which may be preferable in the present disclosure.

In some embodiments of the present disclosure, it is possible to use a single cell analysis technique using droplet microfluidics, for example, in which droplets are fused without charging and single cells are massively parallel. Techniques for obtaining whole genome amplification products are available. These methods enable high-precision single-cell analysis for a wide variety of samples at high speed, and are extremely useful for single-cell genome acquisition and comprehensive genome mutation analysis of the entire cell population. It can be said that there is.

Further, in the present disclosure, it may be preferable to use a single cell analysis technique that ensures both throughput and convenience at the same time. For example, the device that applies an electrical load to add barcodes and amplification reagents is very fine, and each laboratory uses a different mechanism, so only a limited number of laboratories can be used. Can not. It takes time to learn the technology of the fusion mechanism that does not apply electric charge. In addition, the droplets are produced by shearing with oil and are collected while floating in the oil, so that they cannot be applied to automatic sorting by flow cytometry or the like. In addition, these methods cannot remove the reagent once added from the tube. Some lytic reagents are known to have the effect of inhibiting whole-genome amplification, and because the primer dimer increases the amplification bias, the step of removing the reagent for each reaction and adding the next reagent is high. It may be preferable when performing accurate whole genome amplification.

A commercially available single cell analyzer may be used for the purpose of enabling single cell analysis in many laboratories. When analyzing a genome mutation at only one site, these analyzers can amplify the target site by PCR without performing whole genome amplification. However, in order to obtain a large amount of mutation information and information on the entire genome from one cell, it may be preferable to adopt a method capable of performing whole genome amplification.

In one preferred embodiment of the present disclosure, in order to easily obtain information for each cell-derived nucleic acid from a large number of cells, a sample containing tissue is used, and the cells are encapsulated in droplets one by one. A step of gelling the droplet to generate a gel capsule, and a step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, and the genomic DNA of the cell or the step thereof. The step of eluting the moiety-containing polynucleotide into the gel capsule and retaining it in the gel capsule with the genomic DNA or the substance binding to the moiety removed, and contacting the polynucleotide with an amplification reagent. A single cell-derived nucleic acid can be obtained by a method comprising the step of amplifying the polynucleotide in a gel capsule.

In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..

In one embodiment, droplets can be made by encapsulating one cell by flowing one cell into a microchannel and shearing the suspension with oil. In some embodiments, the gel capsule may be a hydrogel capsule.

The material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like. Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet. The inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.

The gel capsule may be a hydrogel capsule. As used herein, the term "hydrogel" refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.

Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween20, Tween80 , Octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12 can be selected from at least one species.

The cells or cell-like structures that can be targeted in the mutation analysis of the present disclosure are two or more arbitrary numbers, for example, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 5000 or more. It can be 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more. The mutation analysis of the present disclosure may use information on nucleic acids derived from one cell at a time rather than using a conventional single cell reaction system, eg, a 0.2 mL, 1.5 mL microtube reaction system.

In one embodiment of the present disclosure, a gel droplet technique for producing a droplet using an agarose solution can be adopted. This makes it possible to perform analysis in many laboratories without degrading throughput and accuracy. By preparing a droplet using agarose and embedding a single cell inside, the single cell can be isolated and cultured in the droplet, or PCR can be performed. In the present disclosure, a gel droplet is regarded as a reaction field having a network structure in which one cell or one cell nucleus is embedded, and a lysis reagent or a whole genome amplification reagent is allowed to act so as to pass through the network structure to easily make the droplet. It is possible to perform whole genome amplification and reagent removal within. The labor-intensive single-cell genome-embedded droplet fraction may be automated by commercially available flow cytometry.

(Analysis before genome sequencing)
In the present disclosure, nucleic acids or other biomolecules prepared in parallel from various cells or cell-like structures prior to analysis of the total sequence information of individual cells or cell-like structures (eg, genomic sequencing). From the structure or sequence of the above, the structure or sequence may be referred to for individual cell-specific detection and selection. That is, the method may include selecting a sample containing the amplified nucleic acid to be analyzed from a sample containing the amplified nucleic acid derived from each cell or cell-like structure.

In one embodiment, selection can be made based on the presence or absence of a particular gene sequence, the yield of a particular gene or the total nucleic acid yield. In some embodiments, it may be selected when there is a specific gene sequence, or it may be selected when there is no specific gene sequence. In some embodiments, it may be selected if the yield of the particular gene is greater than or equal to the baseline yield. In some embodiments, it may be selected if the total nucleic acid yield is greater than or equal to the reference yield.

In certain embodiments, reagents that specifically detect the presence or absence of a particular gene sequence, agarose gel electrophoresis, microchip electrophoresis, PCR, qPCR, gene sequencing (Sanger sequencing, NGS). Detected by means selected from the group consisting of. In some embodiments, reagents that specifically detect a particular gene sequence include antibodies, probes, DNA-binding fluorescent dyes, fluorescent dye-binding nucleotides.

In a specific embodiment, the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorescence measurement, agarose gel electrophoresis, or microchip electrophoresis. The method may include detecting nucleic acid having a particular sequence in a sample containing amplified nucleic acid derived from each cell. The step of detecting a nucleic acid having a specific sequence may include amplifying and sequencing the nucleic acid having a specific sequence.

Evaluation of the composition of the mutation may include identifying the absolute number of various cells in the tissue. By specifying the type of mutation for each of the amplified nucleic acids derived from each cell, the absolute number of cells having various mutations can be specified. The cell type can be specified, for example, by specifying the presence or absence of a specific gene sequence.

(Analysis with genome sequencing)
The method of the present disclosure may include obtaining genomic sequence data for each cell or cell-like structure from a sample containing amplified nucleic acid from each cell or cell-like structure in a tissue. By obtaining genomic sequence data, it is possible to obtain not only information as a sequence but also information from the viewpoint of the function or characteristic of the sequence for each cell in the tissue.

It is possible to select the genome sequence data to be analyzed from the genome sequence data of each cell. Genome sequence data has a large amount of information, and limiting the amount to be processed leads to a reduction in labor and time.

Selection may include assessing the presence or absence of a particular gene sequence and / or identity with a particular gene sequence. In some embodiments, identity with a particular gene sequence can be assessed by using BLAST or the like. In certain embodiments, the selection may be cell-by-cell selection of nucleic acid information based on the presence or absence of a particular gene sequence. In other embodiments, the selection may be cell-by-cell selection of nucleic acid information based on the identity of a particular gene sequence with nucleic acid information derived from two or more cells. In some embodiments, it may be selected when it has a certain level of identity or more, or it may be selected when it has a certain level of identity or less. In certain embodiments, the identity is 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, It may be 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100%. In other embodiments, the identity is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, It may be 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.

When genomic sequence data is obtained, the composition of mutations may be evaluated by comparing long gene sequences in each cell. The long sequence may be, for example, a gene sequence extracted from de novo assembly data. Comparison of such long gene sequences can not only enhance the evaluation system but also enable evaluation of function in tissues. For example, even if it is known that multiple types of cells exist from sequence information alone, it may not be possible to understand the state of the entire tissue without individual information on the role and state of each cell. Based on this information, it is possible to obtain information about the amount of cells having a specific activity (for example, enzyme activity, drug sensitivity, tumorigenicity) in a tissue.

(Mutation)
In the present disclosure, it is possible to identify mutations that each cell or cell-like structure has in a tissue. Mutations include mutations that involve sequence changes and mutations that do not involve sequence changes. Mutations with sequence changes include substitutions, insertions, deletions, inversions, or translocations. Examples of mutations that do not involve sequence changes include copy number variation. The mutation can be identified by comparing the sequences with each other based on a method known in the art.

In single-cell analysis, in analysis based on partial sequences, information on mutations in the sequences of predetermined parts can be obtained, but when it is unknown in which part the mutations occur, the total sequence information (genome) It is considered possible to identify unknown mutations by obtaining information on.

In addition, mutations that involve sequence changes can be identified based on sequence information, but when identifying mutations that do not involve sequence changes, it is necessary to compare the abundance of the same sequence. In some cases, it may be preferable that the nucleic acid be evenly amplified for each cell or cell-like structure. For such amplification, the features described in the (single cell analysis) column of the present specification can be appropriately adopted.

Tissues that can be analyzed in this disclosure include tumor tissue, nervous tissue, blood, bone marrow fluid, semen, peritoneal lavage fluid, and the like. Even if the tissue is morphologically composed of certain cells, it may be genetically composed of various cells, and individual cells cannot be captured by the data averaged from the cell population. The methods of the present disclosure are useful in understanding the properties of each organization based on its characteristics and functions.

The tissues that can be analyzed in the present disclosure may be derived from any organism, and the animals include, for example, human or non-human mammals (eg, mice, rats, rabbits, sheep, pigs, cows). , Horses, cats, dogs, monkeys, chimpanzees), vertebrates such as birds, reptiles, amphibians, fish, and invertebrates, such as insects and linear animals. Plants include rice, wheat, corn, potato, barley, sweet potato, buckwheat, white indigo, sorghum, tomato, cucumber, cabbage, white vegetable, eggplant, sugar cane, sorghum, apple, orange, banana, peach, poplar, pine, cedar, Examples include angiosperms, grasses, ferns, moss, and algae. In one embodiment, human tissue can be targeted.

In tumor tissue, tumor marker, tumor malignancy index, tumor drug susceptibility index, nerve tissue, neuropsychiatric disease index, peripheral neuropathy index, germ cell, hereditary disease index, blood cell It is possible to identify the amount of cells with mutations that can be used as an index of malignancy and drug susceptibility of acute myeloid leukemia, safety evaluation in regenerative medicine, and the like. It is possible to obtain information such as not only the presence or absence of mutation (0 or 1) in a tissue but also the proportion of cells having a mutation (continuous).

The obtained mutation information for each single cell can be used for development research of new treatment methods and drugs by elucidating genetic heterogeneity in the case of tumor tissues and hematological malignancies, as well as stratification of cancer patients. , Leading to improved treatment selection and pathological monitoring. If the type of mutant cells can be determined and the composition ratio can be known before and after treatment, the drug can be given to the patient under appropriate application conditions. For example, by applying this technology to perform the current cancer panel DNA analysis on a cell-by-cell basis, more precise treatment method selection becomes possible.

The genome in brain nerve cells has brain-specific genome polymorphisms such as chromosome number and retrotransposon transfer, in addition to the effects of mutations that occur during mitosis. It has also been suggested that these genomic polymorphisms are associated with psychiatric disorders.

Conventionally, gene polymorphisms in the entire DNA extracted from tissues have been investigated, but based on the genomic information obtained for each single cell obtained in the present disclosure, the genes in the nerve as cells It is possible to determine the mutant cell ratio and the mutation polymorphism for each cell. Such detailed information leads to elucidation of the mechanism of genomic polymorphism peculiar to nerve cells and understanding and control of the pathophysiology of psychiatric disorders.

When evaluating the safety and quality of transplanted tissues using ES cells and iPS cells for regenerative medicine, genetic polymorphisms in the entire DNA extracted from the tissues have been investigated in the past. Based on the genomic information obtained for each single cell obtained in the present disclosure, it is possible to determine the ratio of mutant cells in the tissue for transplantation as cells and the mutation polymorphism for each cell. Such detailed information will lead to elucidation of the mechanism of genome polymorphism development in transplant tissues, development of control methods for maintaining safety, and quality evaluation.

(system)
In another aspect of the disclosure, a system for analyzing mutations in tissues may be provided. The system may be provided with a method or means for implementing a process thereof that comprises any of the features described in the other items herein.

The system may include a device for amplifying polynucleotides in cells. The device can be, among other things, capable of amplifying polynucleotides in cells at the single cell level. The device is a droplet preparation unit that encapsulates cells or cell-like structures in droplets one cell or structure at a time; a gel capsule generation unit that gels droplets to generate gel capsules; a reagent for dissolving gel capsules. It may be provided with a dissolving reagent dipping part to be immersed in; a removing part for removing contaminants from the gel capsule; and / or an amplification reagent dipping part for immersing the gel capsule in the amplification reagent. The system or device may further comprise a sorting unit that sorts the gel capsules and houses the gel capsules in a storage container.

The system or device may optionally include media for encapsulating cells or cell-like structures, gel capsule materials, lysis reagents, amplification reagents, reagents used for sequencing nucleic acids (eg, polymerases, primer sets (eg, polymerases, primer sets). Barcode sequences may be included), etc.) and other reagents. As the reagent, in addition to those described elsewhere in the present specification, reagents known in the art may be used.

The device or system may further include a sequencing section for sequencing the nucleic acid sequence in the amplified polynucleotide in the amplification reagent immersion section. The sequencing unit may be provided integrally with the above device or as another device in the system. The sequencing unit includes the Sanger method, the Maxam-Gilbird method, single molecule real-time sequencing (for example, Pacific Biosciences, Menlo Park, California), and ion semiconductor sequencing (for example, Ion Torrent, South San Francisco, California). Bisynthesis, pyrosequencing (eg, 454, Brandored, Connecticut), ligation sequencing (eg, Life Technologies, Carlsbad, California SOLiD sequencing), synthetic and reversible terminator sequencing (eg, Illumina) , California), nucleic acid imaging techniques such as transmission electron microscopy, nanopore sequencing, and the like.

The system or device may be equipped with means for detecting and measuring the amplified gene. For example, a flow cytometry device suitable for handling the shape of a gelul capsule may be provided integrally with the above device or as another device in the system. Means for detecting and measuring the amplified gene include means for performing a detection reaction (eg, thermal cycler and suitable reagents) and / or means for detecting signals (optical sensors, cameras, and suitable means for analysis). Can be included.

The system or device may include a calculator that may be configured to perform any information processing described elsewhere herein. The calculation unit may be provided integrally with the above-mentioned device, or may be provided as another device (computer) in the system. In another aspect of the present disclosure, a calculation unit may also provide a program for performing information processing described elsewhere in the specification to implement the method of the present disclosure and a storage medium on which it is recorded. The calculator may optionally include such a program and / or a storage medium on which it is recorded.

(kit)
One aspect of the disclosure provides a kit that can be used in the methods of the disclosure. In the present disclosure, kits for analyzing mutations in tissues may be provided. The kit may include the material of the gel capsule, and the use of the gel capsule is advantageous for amplifying nucleic acids in cells or cell-like structures at the single cell level, as described elsewhere herein. And can be used as described herein for the analysis of mutations in tissues. The kit may include, for example, the material of the gel capsule and, optionally, one or more reagents. As the reagent, in addition to those described elsewhere in the present specification, reagents known in the art may be used.

Kits for analyzing mutations in tissues may include reagents for lysis. Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, It may include at least one selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, Zwittergent 3-12. Dissolving reagents are useful for obtaining amplified polynucleotides at the single cell level, especially genomic-wide amplification products.

The kit may include reagents for amplifying nucleic acids. Amplification reagents include, for example, polymerases, primer sets (which may include barcode sequences), base mixes, suitable buffers and the like. The kit may include reagents such as reagents used for sequencing nucleic acids (eg, polymerases, primer sets (which may include barcode sequences), etc.). For example, reagents (polymerase, primer set (which may include a barcode sequence), etc.) for amplifying / decoding a specific gene by a Sanger sequence or NGS may be included. The kit may also include reagents that can be used to detect and measure specific sequences, such as nucleic acid binding dyes, fluorescently labeled probes, and the like. Using these reagents, the presence or absence of a specific sequence can be measured by an instrument that detects and measures the amplified gene (flow cytometry, etc.).

(Preferable embodiment)
In this disclosure, we focused on gel droplets prepared using agarose, and developed a simple, highly accurate, and massively parallel whole-genome amplification method by using a highly permeable microreaction field. By encapsulating animal cells in the gel droplet and adding a reaction reagent, whole genome amplification of a single cell was performed in the gel droplet. As a result, a highly accurate whole-genome amplification product with little amplification bias could be obtained at high speed. In addition, we were able to detect genomic mutations at multiple locations with high accuracy at the single cell level by actually using amplification products. By acquiring tens of thousands of single-cell whole genome amplification products and detecting genome mutations by this method, it is possible to detect genome mutations in a cell population and select cells with target mutations at high speed and at low cost. It became. This method is expected to be useful for comprehensive understanding of genomic mutations in body tissues, detailed understanding of cells with target mutations, invention of cell lineage / disease acquisition mechanism, and clinical diagnosis.

(system)
A tissue comprising an amplified nucleic acid sample donor that provides a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue and a mutation-identifying portion that identifies a mutation in the cell or cell-like structure. A system that analyzes mutations in is capable of single-cell whole-genome amplification within gel droplets. In this case, the amplified nucleic acid sample provider uses the sample containing the tissue to encapsulate the cells or cell-like structures in the droplets one cell or each structure, and gels the droplets. A gel capsule generator that produces a gel capsule, one or more lysis reagents, and one or more lysis reagents for lysing cells are stored in the gel capsule. A cell lysate that lyses the cell by immersing in the cell, in which the genomic DNA of the cell or a polynucleotide containing a portion thereof elutes into the gel capsule and binds to the genomic DNA or portion thereof. Includes a cell lysate, which is configured to be retained in the gel capsule with the substance to be removed, and the polynucleotide amplification reagent for amplifying the polynucleotide in the gel capsule. Is preferable. Further, the composition evaluation unit includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells, and the detection reagent or the detection device amplifies the nucleic acid and By including a nucleic acid amplification sequencing device for sequencing, a highly accurate whole-genome amplification product with little amplification bias could be obtained at high speed. In addition, this system enables highly accurate detection of genomic mutations at multiple locations at the single cell level.

References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as they are specifically described.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in the present specification, and is limited only by the scope of claims.

Hereinafter, examples of the present disclosure will be described.
Specifically, the reagents described in the examples were used, but equivalent products of other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R & D Systems, USCN Life Science INC, etc.) can be substituted.

(Example 1: Preparation of agarose gel droplet)
(Manufacturing of microfluidic device)
Using the drafting software AUTOCAD, we designed a microfluidic device in which the aqueous solution is sheared by oil to produce droplets in a cross structure in the flow path (Fig. 6). The width of the flow path inside the device was designed to be 100 μm, the carrier oil side of the cross structure was 17 μm, and the aqueous solution side was 8.5 or 17 μm. A mold for device fabrication was produced by transferring the flow path to a glass substrate coated with a negative resist (SU-8 3050, MicroChem Corp., Newton, MA) using a photomask. A spin coater (MS-B150, Mikasa, Japan) was used to coat the resist on the glass substrate. The height of the flow path of the device having a cross-structured aqueous solution side flow path width of 17 μm was 50 μm, and the height of the flow path of the device having 8.5 μm was 30 μm. Polydimethylsiloxane (PDMS; Sylgard 184: Dow Corning Corp., Midland, MI) was used to fabricate the microfluidic device. The prepared microchannel made of polydimethylsiloxane was used to prepare the droplet.

(Preparation of agarose gel droplet using microfluidic device)
A 1.5% agarose solution was prepared using agarose (SIGMA-ALDRICH, St. Louis, MO). Pico-Surf1 (2% in Novec 7500) (hereinafter referred to as "oil") was used as the oil for producing the droplets. The oil was installed in a pressure controlled pump (Mitos P-pump, Dolomite, Royston, UK) and connected to the inlet of the microchannel. The oil and agarose solution were pumped and introduced into the flow path of the device. A high-speed camera (FHS-33, Flovel, Tokyo, Japan) was used to observe how the agarose solution was sheared by oil at the crossroads to form droplets (Fig. 7, left figure). The prepared droplets were collected in 1.5 mL tubes. A part of the collected droplets was observed using a microscope (BZ-X710, KEYENCE, Osaka, Japan) (Fig. 7, right figure). The collected gel droplets were allowed to stand on ice for 15 minutes to gel the gel droplets.

(Aqueous layer replacement and shape observation of gel droplet)
The prepared gel droplet is in a state of floating in oil, and it is necessary to replace the oil layer with an aqueous layer in order to add a dissolving reagent or a whole genome amplification reagent. First, the oil in the tube was removed as much as possible to replace the droplets with an aqueous layer. Subsequently, 200 μL of a 1: 9 mixed solution of 1H, 1H, 2H, 2H-Perfluoro-1-octanol (SIGMA-ALDRICH, St. Louis, MO) and Novec 7500 was added, mixed with the droplet by tapping, and adsorbed on the droplet. The oil that had been used was removed. Centrifuge in a tabletop centrifuge (Petit Change, WAKENBTECH CO., Kyoto, Japan) for 1 second to remove the supernatant, and then mix 1H, 1H, 2H, 2H-Perfluoro-1-octanol and Novec 7500 1: 9 in the same manner. The solution was mixed and centrifuged in a tabletop centrifuge for 1 second to remove the supernatant. Subsequently, 500 μL of acetone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added, mixed by a vortex mixer (Nissinrika Co., Tokyo, Japan), and then centrifuged in a tabletop centrifuge for 30 seconds to remove the supernatant. After that, 2-propanol (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added, and the mixing and removal operations were carried out in the same manner, and finally suspended in 500 μL of DPBS. The gel droplets were replaced with an aqueous layer by mixing the droplet suspension and removing it by centrifugation three times.

(Example 2: Whole genome amplification and amplification bias evaluation in gel droplet)
(Encapsulation of model animal cells in gel droplet)
GM12878, a human lymphoblastic cell, was used as a model cell. A mixture of FBS (Thermo Fisher Scientific, Waltham, MA) and Penicillin-Streptomycin Solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was added to Advanced RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) and a flask (NIPPON Genetics Co) ., Ltd, Tokyo, Japan) liquid culture. After 48 hours after culturing, 90% confluent animal cells were collected by centrifugation at 250 × g for 3 minutes at 4 ° C., suspended in DPBS, and the cell concentration was measured. Cells were then stained with SYBR Green (invitrogen, Carlsbad, CA). According to the measurement results, the concentration of the cell suspension was adjusted to 3,000 cells / μL, and then mixed with a 3% agarose solution prepared in advance at a ratio of 1: 1. The agarose concentration of the mixed solution was 1.5%, and the cell concentration was When a droplet having a particle size of 1,500 cells / μL and a particle size of 50 μm was prepared, the encapsulation ratio was 1 cell / drop. Droplets were made using a microfluidic device using the prepared solution. The collected gel droplets were allowed to stand on ice for 15 minutes to gel the gel droplets.

(Whole genome amplification in gel droplet by MDA method)
GM12878 was encapsulated by the above method to prepare droplets having two types of particle sizes, 50 μm and 70 μm. The conditions are an agarose concentration of 1.5% and an encapsulation ratio of 0.1 cell / drop. After preparation, the aqueous layer was replaced by the method described above. After that, Buffer D2 (1M DTT and Buffer DLB) contained in Repli-g Single Cell Kit (QIAGEN, Valencia, CA) is prepared in a droplet suspension and reacted at 40 ° C for 10 minutes to produce one piece of DNA. Chaining was performed. The reagent included in the kit was similarly mixed with the tube after the reaction, and the whole genome was amplified by reacting at 30 ° C. for 3 hours. After whole-genome amplification, 100 μL of DPBS was added to the droplet and centrifuged for 1 minute, and the operation of removing the supernatant was repeated twice to wash. After that, the DNA was stained with SYBR Green and washed again with 100 μL of DPBS three times. Since a part of the DNA amplification positive droplet was deformed by the genome amplification, the circularity of the fluorescent droplet was measured by ImageJ using a fluorescent image (FIGS. 8 and 9).

(Example 3: Evaluation of amplification bias of single-cell whole genome amplification product)
The 70 μm diameter whole genome amplification product-encapsulated droplets prepared in Example 2 were aspirated one by one under a microscope using a micromanipulator (Microdispenser, Drummond Scientific Company, Broomall, PA) and transferred to a PCR tube (hereinafter, this). Work is called a pick). Pick 20 DNA amplification positive and 20 negative droplets each, add Buffer D2 and react at 40 ° C for 10 minutes, then add reagents other than alkaline reagents and react at 30 ° C for 2 hours. A second whole-genome amplification using DNA as a template was performed (secondary amplification). After the amplification reaction was stopped by a reaction of 65 degrees 3 minutes after the secondary amplification, the amplified genome was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA) (Fig. 10). Next, with reference to previous studies (Leung et al., 2015) (Hosono et al., 2003), primers (Table 1) targeting loci on chromosomes 1 to 22 were designed.

Using 9 single-cell genomes after quantification, using a 10 ng genome as a template, and using Power Up SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) as an enzyme, qPCR was performed with the composition shown in Table 2. The conditions for qPCR were 50 ° C for 2 minutes, 95 ° C for 2 minutes, and 40 cycles (95 ° C for 3 seconds, 60 ° C for 30 seconds), and a StepOnePlus real-time PCR system (Applied Byosystems, City of Foster City, CA) was used.

In addition, as a control for the sample in which the whole genome was amplified in the droplet, 9 GM12878 cells were directly picked without being encapsulated in the droplet, and the whole genome was amplified in the PCR tube to evaluate the amplification bias. Furthermore, a non-amplified genome was obtained from the GM12878 cell population by using a genome extraction kit (DNeasy, QIAGEN, Valencia, CA), and qPCR was performed in the same manner as a reference. By calculating the absolute value of the difference between the Ct value of each obtained single-cell genome and the Ct value of the extracted genome and creating a box plot, the single-cell genome obtained by MDA in the gel droplet and the single-cell genome, The amplification bias of the genomes subjected to MDA without encapsulation in the droplet was compared (Fig. 11).

(Example 4: Gel droplet whole genome amplification of cell nucleus and fractionation by FACS)
(Preparation of cell nuclei from cultured animal cells and encapsulation in gel droplets)
From the GM12878 cells used in Example 2, cell enucleation treatment was performed. First, the cultured cells were collected and washed 3 times with 1 mL of DPBS. After washing, 500 μL of a lysing reagent was added to the cell pellet and reacted on ice for 5 minutes to dissolve the cell membrane. The composition of the dissolving reagent is 10 mM Tris-HCl (invitrogen, Carlsbad, CA), 10 mM NaCl (SIGMA-ALDRICH, St.Louis, MO), 3 mM MgCl ₂ (SIGMA-ALDRICH, St.Louis, MO), 0.1% Nonidet. ^TM P40 (Thermo Fisher Scientific, Waltham, MA). Then, 500 μL of DPBS containing 1% amount of Bovine Serum Albumin (SIGMA-ALDRICH, St.Louis, MO) was added, and only the cell nucleus was recovered by centrifugation at 500 × g for 5 minutes at 4 ° C. The cells were similarly washed 3 times with DPBS containing 1% BSA, and the cell concentration was measured by an animal cell counter. The cell nucleus concentration was adjusted to 14,000 cells / μL and mixed with a 3% agarose solution at a ratio of 1: 1 to obtain a concentration of 0.1 cells / drop when a droplet having a particle size of 30 μm was prepared. From the prepared solution, a 30 μm gel droplet was prepared and cells were encapsulated.

(Examination of whole genome amplification time of cell nucleus embedding droplets)
In order to confirm whether the deformation of the droplet in MDA in the gel droplet changes depending on the degree of cell lysis, whole genome amplification was performed on the prepared nuclear-embedded droplet. First, add 0.1 mg / mL Proteinase K (Promega, Madison, WI) and 0.05% Sodium Dodecyl Sulfate (Thermo Fisher Scientific, Waltham, MA) to remove as much nuclear components as possible, and add at 40 ° C for 10 minutes. After the reaction, MDA was performed after washing 5 times with 500 μL of DPBS. Four DNA amplification positive droplets of the samples reacted for 10 minutes and 30 minutes were picked, and after the secondary amplification, the amplification bias was measured by qPCR in the same manner as in Example 3 (FIG. 13).

(Animal cell whole genome amplification product embedding gel droplets sorted by FACS)
Gel droplets prepared by the method described above and subjected to the MDA reaction for 30 minutes were introduced into FACS Melody. Then, 58 DNA amplification positive droplets and 14 DNA amplification negative droplets were collected so that one droplet was collected in one tube. After sorting, secondary amplification was performed, and the genome was quantified using Qubit.

(Gel droplet whole genome amplification and genome mutation detection of human lung cancer cells)
Three types of human lung cancer cells NCI-H358, H1975, and H1650 having gene mutations in the ex19-21 region of the EGFR gene were used as model cells (Table 3). After culturing in the same manner as in Example 2, 48 hours after culturing, 500 μL of Gibco ^TM TrypLE Express Enzyme (Thermo Fisher Scientific, Waltham, MA) was added to 90% confluent animal cells after removing the medium, and at 37 ° C. The cells adhering to the flask were peeled off by incubating for 5 minutes. After the washing operation and cell counting, the concentrations of the three types of cells were made uniform and mixed 1: 1: 1 to prepare a cell suspension containing equal amounts of the three types of cells. A 3% agarose solution was prepared, mixed 1: 1 with a mixed cell suspension, and encapsulated in a 70 μm diameter droplet at 0.1 cell / drop. MDA was performed after aqueous layer replacement and 102 DNA amplification positive droplets were picked after washing and staining. Three types of primers were designed to include the three gene mutations shown in Table 3 (Table 4), and PCR was performed on the single-cell whole genome amplification products with the three types of primers. The enzyme used was PyroMark PCR Master Mix (QIAGEN, Valencia, CA). The PCR conditions are 95 ° C for 15 minutes, 40 cycles (94 ° C for 30 seconds, 62 ° C for 30 seconds, 72 ° C for 30 seconds), and 72 ° C for 10 minutes. The presence or absence of deletion mutations in each single cell genome was detected by performing agarose gel electrophoresis on the PCR products targeting the deletion mutations of ex19. In addition, PCR products targeting SNPs of ex20 and 21 were sent to fasmac Co., Ltd., and the nucleotide sequences of the PCR products were obtained by the Sanger sequencing method to detect the presence or absence of SNPs at two locations in the single cell genome. ..

(Animal cell whole genome amplification in gel droplet)
The alkaline reagent and the MDA reagent were sequentially reacted with the GM12878 cells encapsulated in the gel droplet, and it was verified that the single cell lysis and the whole genome amplification reaction were performed in the gel droplet. As a result, DNA amplification positive gel droplets (green fluorescence) were observed at the same ratio (10%) as the encapsulation ratio (Fig. 8, left figure). The DNA amplification-positive gel droplets showed clear brightness, suggesting that the amplified DNA was retained inside the droplets even after repeated centrifugation and washing with vortex. However, in some of the DNA amplification-positive gel droplets, the contour of the droplet was distorted, and it was observed that the internal DNA or cellular components were leaking (Fig. 8, right figure). Therefore, a DNA amplification reaction was carried out at two types of particle sizes (50 μm and 70 μm), and the circularity after the DNA amplification reaction was measured (FIG. 9). It was shown that when the droplet size was large (70 μm), the circularity fluctuated little and the shape was relatively stable, but when the original size was small (50 μm), the shape collapsed significantly. Since this decrease in droplet contour collapse was observed for the first time after the genome amplification reaction, the influence of the amplified DNA component and the like was considered. As described above, since the presence or absence of amplification can be easily distinguished by fluorescence observation, it is possible to manually select amplification-positive droplets under a microscope even if the droplet shape is deformed. However, when attempting FACS sorting, it is expected that uneven droplet size will affect sorting, so it is necessary to reduce the collapse of the droplet shape as much as possible.

Next, in order to evaluate the cross-contamination of amplified DNA due to the shape collapse of the gel droplets, 20 DNA amplification positive and 20 negative droplets were manually picked, secondary amplification was performed, and the amount of amplified DNA was quantified. (Fig. 10). As a result, 5945 ± 1623 ng of final DNA amplification product was obtained from the DNA amplification positive droplet. On the other hand, the final DNA amplification product of 11.85 ng ± 10.7 ng was obtained from the DNA amplification negative droplet. The difference in final DNA yield was clear and the cross-contamination of amplified DNA was considered to be negligible. In addition, it was shown that the single-cell genome amplification product can be reliably recovered by recovering the DNA amplification-positive droplet using fluorescence as an index.

(Example 5: Evaluation of amplification bias of gel droplet whole genome amplification product)
In order to confirm whether the single-cell genome obtained from the gel droplet has sufficient quality as a gene analysis sample, qPCR targeting the loci on each chromosome 1 to 22 was performed, and amplification bias was performed. The degree of was evaluated. Using a non-amplified genome extracted from a cell population as a comparison target, the difference in Ct value during qPCR of a single-cell genome amplified product was evaluated as the magnitude of amplification bias (Fig. 11). In the sample in which the MDA reaction was performed directly from one cell in one tube without encapsulation in a gel droplet as in the conventional method, the amplification bias was large, and the difference between the maximum and minimum Ct values was 3 or more at all chromosomal sites. It was 6 or more in 15 places. This means that there was a difference of 10 times or more in the degree of amplification between single-cell genomes at all sites, and a variation of 100 times or more at 15 sites. On the other hand, in the single-cell genome amplification product sample prepared using the gel droplet, the difference in Ct value exceeded 3 in only 7 places, and the difference in Ct value exceeded 6 in 0 places. From this, it was confirmed that the variation in genome amplification was 10 times or less at 15 locations. This result suggests that temporary encapsulation of a single cell in a gel droplet environment and temporary amplification reaction in a microenvironment significantly suppresses bias on the final amplification product. .. From existing studies, it is known that in the whole genome amplification reaction, the priming and replication conditions at the initial stage of the reaction have a great influence on the subsequent bias scale. In particular, in the conventional PCR tube reaction, since one cell is treated in a large volume of reaction solution, the influence of adsorption on the tube and the primer dimer becomes large. On the other hand, in the gel droplet, DNA was retained in the gel and diffusion was restricted, and components derived from the primer dimer were mainly present outside the gel, and these were washed and removed. It is considered that these influences have eliminated the bias factor that has been a problem in the conventional reaction. Therefore, the effect of suppressing bias was obtained by performing the secondary amplification after the initial genome amplification reaction was completed in the gel and reached a certain volume.

(Example 6: Whole-genome amplification and amplification bias evaluation of nuclear-encapsulated gel droplet)
Since it was confirmed in Example 4 that a part of the gel droplet was deformed so as to be distorted by the MDA reaction, the MDA reaction time was evaluated from the two viewpoints of amplification bias and the degree of droplet deformation in order to apply it to sorting by FACS. did. First, we considered that the cause was diffusion due to lysis of intracellular components, and examined a method of removing cytoplasmic components in advance and encapsulating them in droplets as cell nuclei to proceed with the subsequent reaction.

After encapsulation, in order to remove components other than genomic DNA as much as possible, cell lysis with Proteinase K and SDS was performed before alkaline treatment, and MDA reactions were performed for 10 minutes, 30 minutes, and 60 minutes. As a result of measuring the circularity of the droplets having a reaction time of 30 minutes and 60 minutes before the reaction, it was confirmed that the deformation progressed as the reaction time was extended (Fig. 12). Subsequently, four DNA amplification-positive droplets that had undergone the MDA reaction for 10 minutes and 30 minutes were picked and bias checked by qPCR (FIG. 13). A large amplification bias was confirmed in the droplets with an MDA reaction time of 10 minutes, but in the droplets with an MDA reaction time of 30 minutes, the amplification bias was similar to that of the gel droplet MDA product measured in Example 5. Yes, it was confirmed that the amplification was homogeneous. Based on the above, it was decided that the subsequent nuclear-encapsulated gel droplet MDA should be carried out in a reaction time of 30 minutes under the condition that the amplification bias is suppressed and the degree of deformation of the gel droplet is reduced.

(Example 7: FACS preparative verification of gel droplet whole genome amplification product)
(FACS preparative verification of gel droplet)
(FACS sorting of nuclear-encapsulated gel droplet MDA products)
A 30 μm nuclear-encapsulated gel droplet that had undergone the MDA reaction for 30 minutes was introduced into FACS, and DNA amplification positive droplets and DNA amplification negative droplets were separated into tubes one by one, and quantification was performed after secondary amplification. (Fig. 14). The yields of all the tubes sorted with DNA amplification negative droplets were 1000 ng or less. When the sorting efficiency of DNA amplification positive droplets was calculated based on 1000ng, 53 tubes were confirmed to have genome amplification of 1000ng or more, and the sorting efficiency was 91%. These experiments have shown that FACS sorting of cell nucleus gel droplet MDA products is possible with an efficiency of 90% or higher.

(Detection of somatic genome mutations at the single cell level using model cells)
Using gel droplet MDA, we attempted to detect multiple types of genome mutations in about 100 single-cell genomes. Three types of lung cancer cells, H358, H1975, and H1650, were encapsulated in a gel droplet at a ratio of 1: 1: 1, and an MDA reaction was performed. Was done. As a result of quantification of 102 single-cell genomes, more than 1000 ng of genomes were confirmed in 96 of them. It is probable that the DNA amplification positive droplets were not picked in the 6 tubes due to the problem of the picking procedure, and sufficient amplification was not applied. PCR targeting the mutation sites ex19, 20, and 21 was performed on each of the 96 single-cell genomes with a yield of 1000 ng or more. A part of the result of agarose gel electrophoresis after PCR is shown in FIG. Previous studies (Kawada et al., 2008) have confirmed that two bands are detected by electrophoresis of the genome containing a heterozygous deletion in EGFR ex19. As a result of agarose gel electrophoresis, two bands were detected in 24 amplification products, and one PCR band was detected in 72 amplification products. From this result, it can be seen that the 24 genomes were obtained from cells containing a deletion mutation in EGFR ex19.

Subsequently, the nucleotide sequences of EGFR ex20 and 21 region amplification products were obtained by the Sanger sequencing method. As a result of the experiment, we were able to obtain 191 base sequences out of 192 regions. Combined with the amplification results of the Ex19 region, only one of the three regions (288 locations in total) of the 96 single-cell genome that could be obtained could not be amplified. This result means that the deletion of genetic information due to the amplification bias of the gel droplet MDA product occurred only at a frequency of 0.4% or less, and it proved again that the amplification bias was sufficiently small. Subsequently, SNP mutations were detected from the obtained nucleotide sequence waveforms. In the T790M mutation of Ex20, one guanine was replaced with adenine, and in the L858R mutation of ex21, one adenine was replaced with cytosine. When analyzing heterogeneous SNPs by the Sanger sequencing method, it has been confirmed from previous studies that a diagram in which two waveforms of a wild-type base and a mutant-type base overlap can be obtained (Pao et al., 2005). When the obtained nucleotide sequence was confirmed, mutations of T790M and L858R were observed simultaneously from 34 single-cell genomes, and a wild-type genome was observed from 61 single-cell genomes. A part of the base sequence is shown in FIG. The 34 single-cell genomes with mutations in ex20 and 21 were different samples from the 24 single-cell genomes with mutations in ex19 by agarose gel electrophoresis. In summary, the proportions of each mutation detected were classified into three types as expected (Table 5). From the fact that there was no genome in which three mutations of Ex19-21 were observed at the same time, it was found that there were no gel droplets containing the genomes of H1650 and H1975 at the same time among the 96. It was. This means that the doublets were present in a sufficiently small proportion (1/20, the H1650 and H1975 doublets were 1/120), as theoretically. From the above experiments, it is possible to perform genome mutation analysis at the single cell level with high efficiency after enclosing a cell population in which multiple cell types exist in a gel droplet and performing simple and highly accurate whole genome amplification. Shown.

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(Example 8: Diagnosis of cancer)
(1) Collection of tumor sample A tumor tissue sample is collected by biopsy. The tissue is enzymatically treated and the cell nucleus is removed. If necessary, the target cell group is concentrated by flow cytometry or the like. In the case of hematological malignancies, blood is collected, the peripheral blood mononuclear cell fraction is purified, and the target cell group is concentrated by flow cytometry or the like as necessary.
(2) Obtaining Single Cell Genome Information The single cell is encapsulated in a gel capsule according to the procedure described in Examples 1 and 2 of the present specification. Prepare the amplified polynucleotide in a gel capsule. Collect the gel capsule containing the amplified polynucleotide. Single-cell genomic information is obtained by genetic analysis of amplified polynucleotides.
(3) Analysis of information Evaluate the gene region related to drug resistance of molecular-targeted drugs and select an appropriate drug prescription. In this method, since the genomic polymorphism of cells can be determined for each cell, mutant cells with low abundance in tumors can be detected, and how mutant cells are collected depending on the place and time of collection in the tissue. It is also possible to monitor whether the ratio is different and changes. This method makes it possible to adjust the treatment method at any time according to the state of the tumor composition of each patient.

The single-cell technology disclosed in this disclosure enables non-invasive monitoring and early detection of cancer. For example, circulating tumor cells can be collected from blood samples, but only 1 to 50 cells can be obtained from one patient. For example, in the case of gastric cancer patients, biopsy itself is life-threatening. Single cell technology can be used as a non-invasive monitoring method. Moreover, since the degree of heterogeneity between cancer cells constituting cancer and non-cancerous cells can be determined, it is also possible to find a correlation with the survival rate.

Furthermore, since the single cell technology of the present disclosure can analyze a very small amount of cancer cells, it is possible to detect a relationship with metastasis and a rare mutation to chemotherapy.

(Example 9: Safety and quality assurance in regenerative medicine)
(1) Collecting a transplanted tissue sample A part is collected from a transplanted tissue sample prepared from ES cells or iPS cells. The tissue is enzymatically treated and the cell nucleus is removed. If necessary, the target cell group is concentrated by flow cytometry or the like.
(2) Obtaining Single Cell Genome Information The single cell is encapsulated in a gel capsule according to the procedure described in Examples 1 and 2 of the present specification. Prepare the amplified polynucleotide in a gel capsule. Collect the gel capsule containing the amplified polynucleotide. Single-cell genomic information is obtained by genetic analysis of amplified polynucleotides.
(3) Analysis of information SNP and CNV are evaluated as genomic changes related to tumorigenicity. In this method, since the genomic polymorphism of cells can be determined for each cell, mutant cells with low abundance in tissues can be detected, and how the mutant cell ratio can be determined by induction of differentiation from stem cells or purification methods. Unlike, it is possible to monitor how it changes. This method enables the safety and quality assurance of the tissue used for transplantation.

(Example 10: Detection of chromosomal abnormalities in germline cells, preimplantation genetic diagnosis, prenatal diagnosis)
(1) Collect embryo tissue sample A part of the early embryo blastocyst is collected. When targeting fetal nucleated red blood cells, fetal nucleated red blood cells are collected from maternal blood.
(2) Obtaining Single Cell Genome Information The single cell is encapsulated in a gel capsule according to the procedure described in Examples 1 and 2 of the present specification. Prepare the amplified polynucleotide in a gel capsule. Collect the gel capsule containing the amplified polynucleotide. Single-cell genomic information is obtained by genetic analysis of amplified polynucleotides.
(3) Analysis of information It is used for pre-implantation genetic diagnosis, etc., and is used to select cells without serious mutations. Evaluate SNPs and CNVs as genomic changes associated with serious genetic diseases. In this method, since the genomic polymorphism of cells can be determined for each cell, mutant cells having a low abundance can be detected. This method enables preimplantation genome screening and prenatal diagnosis of embryos.

(Example 11: Analysis using various tissues)
Leung, ML, Wang, Y., Waters, J., & Navin, NE (2015). SNES: Singlenucleus exome sequencing. Genome Biology, 16 (1). Disperse in cell units. Using the sample prepared in this procedure, nucleated cells are prepared by the method described in Example 4, gel droplets are prepared, and the cells are encapsulated.

Using cells derived from various tissues enclosed in gel droplets, whole genome amplification and genome mutation detection can be performed in the same manner as in Example 4.

(Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to this embodiment. It is understood that the present disclosure should be construed only by the claims. It will be understood from those skilled in the art that the description of the specific preferred embodiments of the present disclosure will enable equivalent scope to be implemented based on the description of the present disclosure and common general technical knowledge. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood. This application claims priority to patent application 2019-85837 filed with the Japan Patent Office on April 26, 2019, and the content of the application itself is specifically described in the present specification. It is understood that the content should be incorporated as a reference to this specification as well as.

This disclosure is available in fields such as biological research, medicine, and healthcare.

SEQ ID NOs: 1-44: Primers listed in Table 1 SEQ ID NOs: 45-50: Primers listed in Table 4.

Claims (24)

1. A method for analyzing mutations in tissues
   A method comprising identifying mutations in a cell or cell-like structure from a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue.
2. Amplified nucleic acids derived from each of the cells or cell-like structures
   Using a sample containing the tissue, a step of encapsulating cells or cell-like structures in droplets one cell or structure at a time, and
   The process of gelling the droplets to form gel capsules,
   A step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, wherein the genomic DNA of the cell or a polynucleotide containing a portion thereof is eluted into the gel capsule and the genomic DNA or its portion. The process of holding in the gel capsule with the substance bound to the moiety removed,
   The method of claim 1, wherein the method is produced by a method comprising contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule.
3. A suspension of the cell or cell-like structure is allowed to flow into a microchannel and sheared with oil to produce the droplet encapsulating the cell or cell-like structure. The method according to claim 2, which is characterized.
4. The method according to any one of claims 2 to 3, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
5. The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride , Urea, 2-mercaptoethanol, dithioreagent, TCEP-HCl, sodium cholate, sodium deoxycholate, TritonX-100, TritonX-114, NP-40, Brij-35, Brij-58, Tween20, Tween80 , Octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12, characterized in that at least one is selected from the group. The method according to any one item.
6. The method according to any one of claims 2 to 5, wherein the gel capsule is a hydrogel capsule.
7. The method according to any one of claims 2 to 6, wherein the step of amplifying the polynucleotide in a gel capsule is carried out by a homeothermic chain substitution amplification reaction for 10 to 60 minutes.
8. The method according to any one of claims 1 to 7, further comprising a step of selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell or cell-like structure. ..
9. The method according to any one of claims 1 to 8, which comprises a step of detecting a nucleic acid having a specific sequence in a sample containing an amplified nucleic acid derived from each of the cells or cell-like structures.
10. The method according to claim 9, wherein the step of detecting the nucleic acid having the specific sequence comprises amplifying the nucleic acid having the specific sequence.
11. The method according to any one of claims 1 to 10, wherein the mutation includes a mutation accompanied by a change in the sequence compared with the reference sequence.
12. The method of claim 11, wherein the mutation comprises base substitution, insertion or deletion.
13. Any of claims 1 to 12, further comprising the step of obtaining genomic sequence data of each cell or cell-like structure from a sample containing an amplified nucleic acid derived from each cell or cell-like structure. The method according to item 1.
14. The method according to claim 13, further comprising a step of selecting genomic sequence data to be analyzed from the genomic sequence data of each cell or cell-like structure.
15. The method of claim 13 or 14, wherein the mutation comprises a mutation that does not involve a sequence change compared to a reference sequence.
16. The method of claim 15, wherein the mutation comprises copy number variation (CNV).
17. The method according to any one of claims 13 to 16, which comprises identifying a mutation including a base substitution, insertion or deletion using the genomic sequence data.
18. The method according to any one of claims 1 to 17, wherein the tissue is a tissue containing a tumor.
19. The method according to any one of claims 1 to 18, wherein the tissue is a human tissue.
20. A system for analyzing mutations in tissues
    An amplified nucleic acid sample provider that provides a sample containing an amplified nucleic acid derived from each cell or cell-like structure in the tissue.
    A system comprising a mutation-identifying part that identifies a mutation in a cell or cell-like structure.
21. The amplified nucleic acid sample providing unit
    Using a sample containing the above-mentioned tissue, a droplet encapsulation portion for encapsulating cells or cell-like structures one cell or a structure unit in a droplet,
    A gel capsule generation unit that gels the droplet to generate a gel capsule,
    One or more dissolution reagents and
    A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, which contains one or more lysis reagents for lysing cells. , The polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted in the gel capsule and retained in the gel capsule with the substance binding to the genomic DNA or the portion removed. There is a cell lysate and
    The system of claim 20, comprising the polynucleotide amplification reagent for amplifying the polynucleotide in a gel capsule.
22. The system according to claim 20 or 21, wherein the composition evaluation unit includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells.
23. 22. The system of claim 22, wherein the detection reagent or device comprises a nucleic acid amplification sequencing device for amplifying and sequencing nucleic acids.
24. The composition evaluation unit further includes a genome sequence data acquisition unit for collecting genome sequence data of each cell from a sample containing an amplified nucleic acid derived from each cell in the microbiota, claims 20 to 23. The system according to any one of the above.

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