阅读说明：本技术 整体性检测单个细胞的非破坏性测量信息和基因组相关信息的方法 (Method for detecting non-destructive measurement information and genome-related information of single cells in integrity ) 是由 谷内江望 太田祯生 于 2018-05-02 设计创作，主要内容包括：本发明提供整体性检测单个细胞的非破坏性测量信息和基因组相关信息的方法。更具体而言,本发明使用包括下述工序的方法：准备多个隔室,每1个隔室包含单个细胞或来源于其的物质、第一微珠、和第二微珠；在准备各隔室前或在各隔室中,对单个细胞的非破坏性测量信息和第一微珠的成像信息一同进行检测,将单个细胞的非破坏性测量信息与第一微珠的成像信息进行关联；得到杂交复合物；制造来源于该杂交复合物的扩增产物；整体性检测单个细胞的非破坏性测量信息和基因组相关信息。(The present invention provides methods for the integrity of non-destructive measurements and genomic related information from individual cells. More specifically, the present invention uses a method comprising the following steps: preparing a plurality of compartments, each 1 compartment comprising a single cell or a substance derived therefrom, a first microbead, and a second microbead; before preparing each compartment or in each compartment, detecting the nondestructive measurement information of the single cell and the imaging information of the first microbead together, and correlating the nondestructive measurement information of the single cell and the imaging information of the first microbead; obtaining a hybrid compound; producing an amplification product derived from the hybridization complex; integrity testing individual cells for non-destructive measurement information and genome-related information.)

1. A method for integrity testing individual cells for non-destructive measurement information and genomic related information, said method comprising the steps of:

preparing a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead,

wherein the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms each containing the first barcode nucleic acid corresponding to each piece of imaging information,

and, the imaging information of the first microbeads in each compartment can be distinguished from each other,

the second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof;

before preparing each compartment or in each compartment, detecting the nondestructive measurement information of the single cell and the imaging information of the first microbead together, and correlating the nondestructive measurement information of the single cell and the imaging information of the first microbead;

cleaving the first barcode nucleic acid from the associated first microbead, and hybridizing the genome-associated nucleic acid and the first barcode nucleic acid with a second barcode nucleic acid, respectively, to obtain a hybridization complex;

producing amplification products derived from the hybridization complex;

and using the expression pattern of the amplification product as an index to integrally detect the nondestructive measurement information and genome-related information of the single cell.

2. The method of claim 1, wherein non-destructive measurement information of a single cell is detected using, as an indicator, an expression pattern of a first amplification product derived from a hybridization complex formed by a first barcode nucleic acid and a second barcode nucleic acid,

detecting genome-associated information of the individual cell using as an indicator an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-associated nucleic acid and a second barcode nucleic acid.

3. The method according to claim 1 or 2, having at least any one of the features (a) to (c):

(a) the number of the first microbeads per 1 compartment is plural,

(b) the number of second microbeads per 1 compartment was 1,

(c) the compartments are in the form of wells, droplets or gel particles.

4. The method according to any one of claims 1 to 3, wherein the genome-associated nucleic acid is the single-cell genomic DNA, RNA derived from the genome of the single cell or cDNA thereof, or a nucleic acid probe specific for a protein expressed in the single cell.

5. The method according to any one of claims 1 to 4, wherein the nondestructive measurement information is nondestructive measurement information based on at least 1 measurement information selected from color, fluorescence, size, shape, electromagnetic wave, transmission, phase, scattering, reflection, coherent Raman, and absorption spectrum.

6. The method of any one of claims 1-5, wherein the first barcode nucleic acids of the first beads each comprise a first common barcode region common among the first beads corresponding to the same imaging information, and a first hybridization region capable of hybridizing to the second barcode nucleic acids.

7. The method of any one of claims 1 to 6, wherein the sequence information of the first common barcode region is an index for determining non-destructive measurement information of a single cell.

8. The method of any one of claims 1-7, wherein the plurality of second barcode nucleic acids attached to the second microbead each comprise a second common barcode region that is common to each other, a second native barcode region that is distinguishable from each other, and a second hybridization region that is hybridizable to the genome-associated nucleic acid or the first barcode nucleic acid.

9. The method of claim 8, wherein the sequence information of the second common barcode region is indicative of a determination of an individual cell present in the compartment or a substance derived therefrom.

10. The method according to claim 8 or 9, wherein the sequence information of the second inherent barcode region is an index for determining a genome-associated nucleic acid.

11. The method of any one of claims 1 to 10, wherein the second barcode nucleic acid further comprises a PCR primer region.

12. The method of any one of claims 8 to 11, wherein the second hybridizing region comprises a nucleic acid complementary to the first hybridizing region or the genome-associated nucleic acid.

13. A system for integrity testing individual cells for non-destructive measurement information and genome-associated information, comprising:

a compartment preparing section that prepares a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead, wherein,

the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each imaging information, or organisms each containing the first barcode nucleic acid corresponding to each imaging information,

and, the imaging information of the first microbeads in each compartment can be distinguished from each other,

the second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof;

a measurement unit for measuring nondestructive measurement information and imaging information on a single cell together with the imaging information on the first bead before or in each compartment, and correlating the nondestructive measurement information on the single cell with the imaging information on the first bead;

a hybridization complex formation unit that cleaves a first barcode nucleic acid corresponding to each piece of imaging information from the associated first bead, and hybridizes the genome-associated nucleic acid and the first barcode nucleic acid to a second barcode nucleic acid, respectively, to obtain a hybridization complex;

an amplification product producing unit that produces an amplification product derived from the hybridization complex; and

and a detection unit for detecting nondestructive measurement information and genome-related information on a single cell as a whole using the expression pattern of the amplification product as an index.

14. The system of claim 13, wherein the non-destructive measurement information and imaging information measurement unit comprises at least 1 selected from a microscope and a flow cytometer device.

15. A combination of a first microbead and a second microbead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the composition is capable of detecting non-destructive measurement information of a single cell using, as an indicator, an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid, and detecting genome-related information of a single cell using, as an indicator, an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid.

16. A detection agent comprising a first microbead for use with a second microbead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the detection agent is capable of detecting non-destructive measurement information of a single cell with an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid as an index, and detecting genome-related information of a single cell with an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid as an index.

17. A detection agent comprising a second bead for use with the first bead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the detection agent is capable of detecting non-destructive measurement information of a single cell with an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid as an index, and detecting genome-related information of a single cell with an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid as an index.

18. A method for classifying a test cell based on nondestructive measurement information of the test cell using a classification model obtained based on nondestructive measurement information and genome-related information detected by the method according to any one of claims 1 to 12.

19. A method for obtaining nondestructive measurement information and genome-related information on a single cell of a test substance, which comprises the step of detecting the nondestructive measurement information and genome-related information on a single cell as defined in any one of claims 1 to 12,

the method comprises a step of allowing single cells or a substance derived therefrom, the first beads, and the second beads to coexist.

20. A method for screening a test substance using the information on nondestructive measurement of a single cell of the test substance obtained by the method according to claim 19 and the information on genome-associated therewith.

Technical Field

The present invention relates to a method for the integrated detection of non-destructive measurement information and genome-related information of individual cells.

Background

A cell is the smallest unit that constitutes an organism. Conventionally, attempts have been made to elucidate the functions, structures, forms, and the like of organisms only for cell groups. However, recent studies have found that gene expression varies and varies among similar cell types such as cancer tissues, and the like, and thus, there is a need to elucidate gene expression and the like of each cell.

Therefore, currently, as a method for detecting a transcription product derived from one cell, the following methods are known: data of genetic information of cells was obtained using sequencing technology using microbeads conjugated with oligonucleotides containing barcode (barcode) sequences. (patent document 1, non-patent document 1)

On the other hand, it is known to use an imaging technique typified by a microscope for determining morphological information of cells. By using an imaging technique such as a microscope or an imaging cytometer, nondestructive measurement information of each cell can be obtained.

However, the data of cells measured using the above-described imaging and sequencing techniques can only be correlated by selective and physical cell sorting. For example, in order to obtain information on the genome of a cell to be imaged, it is necessary to perform a process of physically capturing individual cells, compartmentalizing the cells, lysing the cells, and amplifying nucleic acids independently for each cell. Such a method is low in throughput and high in cost. Therefore, it is difficult to correlate non-destructive measurement information of a single cell with genetic information.

In addition, conventionally, morphological information of cells photographed by an imaging technique is evaluated by visual observation. Even if the discrimination is advanced by machine learning techniques in the future, it is still necessary to evaluate the adequacy of the discrimination. In the present case, only a single cell observed by visual observation or the like can be physically sorted, and comparison with a gene or other diagnostic technique is carried out.

Under such a technical background, means for detecting non-destructive measurement information and genome-related information of a single cell as a whole is required to facilitate mutual utilization of the non-destructive measurement information and genome-related information of a single cell and to improve the value between the respective information.

Disclosure of Invention

The aim of the invention is to detect non-destructive measurement information and genome-related information of individual cells in a global manner.

The present inventors have found that, when 1 compartment (component) contains a plurality of beads linked to a barcode nucleic acid together with a single cell or a substance derived therefrom, and genome-related information is measured and correlated while correlating nondestructive measurement information of a single cell with imaging information of the beads, nondestructive measurement information and genome-related information of a single cell can be detected in a whole and efficient manner. In addition, the correlation between the information on the non-destructive measurement of a single cell and the imaging information of a microbead can also be made between a single cell before being contained in a compartment and the microbead. The present invention has been completed based on the above findings.

The present invention includes the following inventions.

(1) A method for integrity testing individual cells for non-destructive measurement information and genomic related information, said method comprising the steps of:

preparing a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead,

wherein the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms each containing the first barcode nucleic acid corresponding to each piece of imaging information,

and, the imaging information of the first microbeads in each compartment can be distinguished from each other,

the second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof;

before preparing each compartment or in each compartment, detecting the nondestructive measurement information of the single cell and the imaging information of the first microbead together, and correlating the nondestructive measurement information of the single cell and the imaging information of the first microbead;

cleaving the first barcode nucleic acid from the associated first microbead, and hybridizing the genome-associated nucleic acid and the first barcode nucleic acid with a second barcode nucleic acid, respectively, to obtain a hybridization complex;

producing amplification products derived from the hybridization complex;

and using the expression pattern of the amplification product as an index to integrally detect the nondestructive measurement information and genome-related information of the single cell.

(2) The method according to (1), wherein the plurality of compartments are obtained by subjecting a cell population or a substance derived therefrom, a plurality of first microbeads, and a plurality of second microbeads to a distribution treatment.

(3) The method according to (1) or (2), wherein non-destructive measurement information of a single cell is detected using, as an index, an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid,

detecting genome-associated information of the individual cell using as an indicator an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-associated nucleic acid and a second barcode nucleic acid.

(4) The method according to any one of (1) to (3), wherein the number of the first microbeads per 1 compartment is plural.

(5) The method according to any one of (1) to (4), wherein the number of the second microbeads per 1 compartment is 1.

(6) The method according to any one of (1) to (5), wherein the compartment is in the form of a well, a droplet or a gel particle.

(7) The method according to any one of (1) to (6), wherein the genome-associated nucleic acid is the single-cell genomic DNA, RNA derived from the genome of the single cell, or cDNA thereof, or a nucleic acid probe specific for a protein expressed in the single cell.

(8) The method according to any one of (1) to (7), wherein the nondestructive measurement information is nondestructive measurement information based on at least 1 measurement information selected from color, fluorescence, size, shape, electromagnetic wave, transmission, phase, scattering, reflection, coherent raman, and absorption spectrum.

(9) The method according to any one of (1) to (8), wherein the first barcode nucleic acids of the first microbeads each contain a first common barcode region common to the first microbeads corresponding to the same imaging information, and a first hybridization region capable of hybridizing with the second barcode nucleic acids.

(10) The method according to any one of (1) to (9), wherein the sequence information of the first common barcode region is an index for determining nondestructive measurement information of a single cell.

(11) The method according to any one of (1) to (10), wherein each of the plurality of second barcode nucleic acids linked to the second bead comprises a second common barcode region common to each other, a second inherent barcode region distinguishable from each other, and a second hybridization region hybridizable with the genome-associated nucleic acid or the first barcode nucleic acid.

(12) The method of (11), wherein the sequence information of the second common barcode region is an indicator for determining an individual cell present in the compartment or a substance derived therefrom.

(13) The method according to (11) or (12), wherein the sequence information of the second inherent barcode region is an index for determining a genome-associated nucleic acid.

(14) The method of any one of (1) to (13), wherein the second barcode nucleic acid further comprises a PCR primer region.

(15) The method according to (14), wherein the second barcode nucleic acid comprises a PCR primer region, a second common barcode region, a second inherent barcode region and a second hybridization region in this order from the second bead side.

(16) The method according to any one of (11) to (15), wherein the second hybridization region comprises a nucleic acid complementary to the first hybridization region or the genome-associated nucleic acid.

(17) The method according to any one of (1) to (16), wherein the first barcode nucleic acid and the second barcode nucleic acid are RNA, DNA or a combination thereof.

(18) The method according to any one of (1) to (17), wherein the nondestructive measurement information is measured by a flow cytometer or a microscope.

(19) The method of any one of (1) to (18), wherein the non-destructive measurement information is imaging information.

(20) The method according to any one of (1) to (18), wherein the nondestructive measurement information is morphological information of a cell.

(21) A system for integrity testing individual cells for non-destructive measurement information and genome-associated information, comprising:

a compartment preparing section that prepares a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead, wherein,

the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each imaging information, or organisms each containing the first barcode nucleic acid corresponding to each imaging information,

and, the imaging information of the first microbeads in each compartment can be distinguished from each other,

the second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof;

a nondestructive measurement information measurement unit that measures nondestructive measurement information on a single cell and imaging information on the first bead together before or in each compartment, and associates the nondestructive measurement information on the single cell with the imaging information on the first bead;

a hybridization complex formation unit that cleaves a first barcode nucleic acid corresponding to each piece of imaging information from the associated first bead, and hybridizes the genome-associated nucleic acid and the first barcode nucleic acid to a second barcode nucleic acid, respectively, to obtain a hybridization complex;

an amplification product producing unit that produces an amplification product derived from the hybridization complex; and

and a detection unit for detecting nondestructive measurement information and genome-related information on a single cell as a whole using the expression pattern of the amplification product as an index.

(22) The system according to (21), wherein the measurement unit of the nondestructive measurement information and the imaging information includes at least 1 selected from a microscope and a flow cytometer.

(23) A combination of a first microbead and a second microbead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the composition is capable of detecting non-destructive measurement information of a single cell using, as an indicator, an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid, and detecting genome-related information of a single cell using, as an indicator, an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid.

(24) A detection agent comprising a first microbead for use with a second microbead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the detection agent is capable of detecting non-destructive measurement information of a single cell with an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid as an index, and detecting genome-related information of a single cell with an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid as an index.

(25) A detection agent comprising a second bead for use with the first bead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein,

the first beads are particles that are attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms containing the first barcode nucleic acid corresponding to each piece of imaging information,

the second microbeads are linked to a plurality of second barcode nucleic acids capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof,

the detection agent is capable of detecting non-destructive measurement information of a single cell with an expression pattern of a first amplification product derived from a hybridization complex formed between a first barcode nucleic acid and a second barcode nucleic acid as an index, and detecting genome-related information of a single cell with an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid as an index.

(26) A method for classifying a cell to be tested, which comprises classifying the cell to be tested based on nondestructive measurement information of the cell to be tested, using a classification model obtained based on the nondestructive measurement information and genomic information obtained by the overall detection by the method according to any one of (1) to (20).

(27) A method for obtaining nondestructive measurement information and genome-related information on a single cell of a test substance, which comprises using the method for detecting nondestructive measurement information and genome-related information on a single cell as described in any one of (1) to (20),

the method comprises a step of allowing the analyte to coexist with the single cell or a substance derived therefrom, the first bead, and the second bead.

(28) A method for screening a test substance using the information on nondestructive measurement of a single cell of the test substance obtained by the method of (27) and the information on genome-associated protein, to screen the test substance.

According to the present invention, nondestructive measurement information and genome-related information of a single cell can be detected integrally and efficiently.

Drawings

FIG. 1 is a schematic view of the detection method according to the present invention.

FIG. 2 is a schematic view of a first microbead cleavable linked to a first barcode nucleic acid according to the present invention.

FIG. 3 is a schematic diagram of a second microbead linked with a second barcode nucleic acid according to the present invention.

FIG. 4 is a schematic diagram showing another embodiment of the first bead linked to a first barcode nucleic acid and the second bead linked to a second barcode nucleic acid of the present invention.

FIG. 5 is a schematic view showing another embodiment of the first bead linked to a first barcode nucleic acid and the second bead linked to a second barcode nucleic acid of the present invention.

FIG. 6 is a schematic diagram showing another embodiment of the first bead linked to the first barcode nucleic acid of the present invention.

FIG. 7 is a schematic diagram showing another embodiment of the first bead linked to a first barcode nucleic acid and the second bead linked to a second barcode nucleic acid of the present invention.

FIG. 8 is a schematic view showing a method of manufacturing a compartment according to the present invention.

FIG. 9 is a schematic view showing another embodiment of the method for manufacturing a cell according to the present invention.

FIG. 10 is a schematic view showing another embodiment of the method for manufacturing a cell according to the present invention.

FIG. 11 is a schematic view showing another embodiment of the method for manufacturing a cell according to the present invention.

FIG. 12 is a flow chart of the system of the present invention.

FIG. 13 is a photograph of a water-in-oil compartment containing first microbeads, second microbeads, and NIH3T3 cells.

FIG. 14A is a composite photograph of NIH3T3 cells carrying green fluorescent beads, taken by a fluorescence microscope and a bright field microscope. B is a composite photograph of K562 cells carrying red fluorescent beads, which was taken with a fluorescence microscope and a bright field microscope. C is a composite photograph of MIA-PaCa2 cells carrying green fluorescent beads and red fluorescent beads, which was taken by a fluorescence microscope and a bright field microscope.

FIG. 15A is a phase contrast micrograph of first beads containing cells. B is a composite photograph of the first beads including cells, which was taken by a fluorescence microscope and a bright field microscope.

FIG. 16 shows the results of measuring the number of gene transcripts in a compartment.

FIG. 17A is a composite photograph of a mixture of bacteria containing a first barcode nucleic acid, which was taken by a fluorescence microscope and a bright field microscope. And B is a sequencing result of the sequence of the plasmid with the EGFP protein gene region and the first common barcode region. C is a photograph of a water-in-oil compartment containing bacteria and cells containing a first barcode nucleic acid. D is an electrophoretogram of an amplification product comprising the sequence of the third inherent barcode region and the first common barcode region corresponding to EGFP.

Detailed Description

Definition of

The term "genome-related information" as used herein refers to nucleic acid sequence information derived from a genome-related nucleic acid corresponding to the genome of a cell or an expression product thereof. Here, the "genome-related nucleic acid" preferably means a genomic DNA of a single cell, an RNA such as mRNA derived from the genome of a single cell, or a cDNA thereof, or a nucleic acid probe specific to a molecule such as a protein expressed in a single cell. The aforementioned nucleic acid probe preferably contains barcode nucleic acids which are cleavable linked to a molecule capable of specifically binding to a molecule such as a protein to be a target (hereinafter also referred to as a binding molecule) and are distinguishable from each other. When the nucleic acid is genomic DNA, the DNA may be a fragment obtained by cleavage with a restriction enzyme or the like, or a DNA tag may be introduced.

The term "barcode region" as used herein refers to a region comprising a random nucleotide sequence of T (thymine), U (uracil), A (adenine), G (guanine), and C (cytosine). In addition, barcode nucleic acid is nucleic acid comprising barcode region, and can identify genome-related information of cells, imaging information from microbeads.

As the barcode region, there are both a common barcode region and an inherent barcode region.

The common barcode region is a barcode region common to the same identification target. The identification of genomic-related information regarding a cell as an object is a barcode region that differs for each cell, i.e., a barcode region that is common to one cell. By labeling at the common barcode region, genome-related information derived from the same cell can be identified. In the case of recognizing the imaging information of the beads as the objects, the barcode regions are different for the beads having the same imaging information, that is, the barcode regions are common to the beads having the same imaging information. By marking at the common barcode region, imaging information from microbeads having the same imaging information can be identified.

In addition, in a nucleic acid probe having specificity for a molecule such as a protein expressed in a single cell, the nucleic acid probe contains a molecule (binding molecule) capable of specifically binding to the molecule such as the protein. Among them, the barcode regions different from each other in the binding molecule in the nucleic acid probe, that is, the barcode regions common to the same binding molecule are also regarded as common barcode regions.

The unique barcode region is a region that can be distinguished from each barcode nucleic acid by labeling barcode regions different from barcode nucleic acid to each barcode nucleic acid, and allows identification of beads linked to each barcode nucleic acid and genome-related nucleic acids hybridized to each barcode nucleic acid. The length of the barcode region is not particularly limited, but is preferably 10 to 40 bases in length, and for example, in the case where the barcode region is 12 bases in length, the length of the barcode region may be 4¹²The various barcode sequences can be amplified at one time to produce 4¹²And (4) planting the micro-beads.

The term "hybridization" as used herein means that a hybridization region of a barcode nucleic acid forms a double-stranded complex with a genome-associated nucleic acid or other barcode nucleic acid corresponding to the genome of a cell or an expression product thereof under stringent conditions. Here, the stringent conditions are conditions under which a so-called specific complex is formed and a non-specific complex is not formed. Such stringent conditions are well known to those skilled in the art and can be set, for example, by reference to molecular cloning (third edition, Cold spring harbor laboratory Press, New York) and Current protocols in molecular biology (Current protocols in molecular biology, eds. (Frederick M. Ausubel et al, 1987).

Thus, the term "hybridizing region" refers to a region that binds to (hybridizes to) a genome-associated nucleic acid or other barcode nucleic acid corresponding to the genome of a cell or its expression product.

For example, where the genome-associated nucleic acid is an mRNA, the second hybridizing region in the second barcode nucleic acid is preferably a polythymine composed of T. The length of the poly-thymine may be such that it anneals (hybridizes) to the tail of poly-adenine (a) of mRNA. In the above case, the first hybridization region is preferably a sequence complementary to poly-thymine, for example, poly-adenine (poly-A).

When the genome-associated nucleic acid is a DNA such as a genomic DNA, the second hybridization region in the second barcode nucleic acid preferably includes a sequence complementary to a specific sequence of the DNA or a sequence of a DNA tag introduced into the DNA. In the above case, the first hybridization region is preferably a sequence complementary to the second hybridization region.

In addition, when the nucleic acid probe specific for a molecule such as a protein expressed in a single cell includes a barcode nucleic acid and the barcode nucleic acid includes a hybridization region, the second hybridization region in the second barcode nucleic acid preferably includes a sequence complementary to the hybridization region. In the above case, the hybridization region in the barcode nucleic acid to which the nucleic acid probe is ligated is preferably a sequence complementary to the second hybridization region.

The term "compartment" as used herein means a space isolated from other liquid or a surrounding medium. Preferably a volume of liquid or gel held in the space. In addition, the above-mentioned compartments are also referred to as micro-compartments. Isolation of the compartment from the surroundings can be achieved by a solid barrier around the compartment, or by phase separation. For example, an aqueous droplet suspended in a hydrophobic carrier fluid may constitute a compartment. The cells may be in the form of, for example, pores, droplets, or gel particles, and specific examples thereof include aqueous droplets, oil droplets, gel particles of hydrogel (e.g., agarose, collagen, or alginic acid), water-oil structures in which a plurality of non-mixed interfaces are superposed, pores of a multi-well plate, and the like.

Detection method

The method for detecting non-destructive measurement information and genome-related information of a single cell in its entirety according to the present invention is characterized by comprising the steps of:

preparing a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead,

wherein the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each piece of imaging information, or are organisms each containing the first barcode nucleic acid corresponding to each piece of imaging information,

and, the imaging information of the first microbeads in each compartment can be distinguished from each other,

the second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof;

before preparing each compartment or in each compartment, detecting the nondestructive measurement information of the single cell and the imaging information of the first microbead together, and correlating the nondestructive measurement information of the single cell and the imaging information of the first microbead;

cleaving the first barcode nucleic acid from the associated first microbead, and hybridizing the genome-associated nucleic acid and the first barcode nucleic acid with a second barcode nucleic acid, respectively, to obtain a hybridization complex;

producing amplification products derived from the hybridization complex;

and using the expression pattern of the amplification product as an index to integrally detect the nondestructive measurement information and genome-related information of the single cell.

The detection method of the present invention will be described below based on preferred embodiments, but the present invention is not particularly limited.

Compartment preparation procedure

The compartment preparation step of the present invention is a step of: a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead, are prepared. Wherein the first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each imaging information or are organisms each containing the first barcode nucleic acid corresponding to each imaging information, and the imaging information of the first microbeads in each compartment are distinguishable from each other. The second microbeads are linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof.

Here, the positional relationship between the cells and the first beads in each compartment is not particularly limited as long as it does not interfere with the acquisition of nondestructive measurement information of the cells and imaging information of the first beads, and may be appropriately set according to the type, size, and properties of the cells and the first beads. That is, in the present invention, the cell and the first bead may be present in the same compartment with or without contact with the cell, as long as imaging information can be obtained together, and the first bead may be introduced into the cell. Preferred examples of the mode of contacting the first beads with the cells include a mode in which the cell surface and the first beads are directly bonded to each other, or a mode in which the first beads are carried on the cells by using a commercially available cell membrane modifier. Preferred examples of the method in which the first bead is not in contact with the cell include a method in which the first bead is present in the same compartment without being bound to the cell, and a method in which a sub-compartment not containing the cell but containing the first bead is bound to the cell to support the cell as a sub-compartment. Further, as a preferable example of introducing the first beads into the interior of the cell, there is a mode in which the first beads are engulfed by the cell.

The type and form of the cell to be detected are not particularly limited as long as the effect of the present invention is not impaired, and the cell can be selected according to the purpose. The cells contained in the above-mentioned compartments may be cell-derived substances. Examples of the cell-derived material include a disrupted cell material, a cell content, a cell lysate, and a cell composed of cells (for example, a cell mass, a cell pellet, and an organoid).

The cell-derived substance can be obtained by using a known method such as allowing cells to coexist with a cell lysis buffer or the like. The step of obtaining a substance derived from the cells may be performed after non-destructive measurement information of the cells and image information of the first beads are obtained and correlated with each other and before the cells are compartmentalized together with the second beads, or the substance may be generated in the compartments by enclosing the cells, the first beads and the second beads together with a cell lysis buffer.

One embodiment of the compartment preparation process of the present invention will be described below with reference to fig. 1a, but the present invention is not particularly limited.

First, a cell group 101, a plurality of first microbeads 102, and a plurality of second microbeads 103, which are detection targets such as tissues and a plurality of cells, are prepared. In FIG. 1a, the first barcode nucleic acid is RNA, includes a first common barcode region and poly-adenine, and is linked to the first bead via an ultraviolet-cleavable linker. The second barcode nucleic acid is DNA, and comprises a PCR primer region, a second common barcode region, a second inherent barcode region, and poly-thymine.

Then, the cell population 101, the plurality of first microbeads 102, and the plurality of second microbeads 103 are subjected to a distribution process to obtain a plurality of compartments 104. By the distribution process, a combination of the single cell 105, the first bead 102, and the second bead 103 in the cell population 101 is distributed to the plurality of compartments 104.

The method of the dispensing process is described later.

The number of the first beads per 1 compartment is not particularly limited, and may be 1, but in order to increase the number of types of compartments that can be distinguished from each other, it is preferably a plurality of the first beads, more preferably 2 to 100, and further preferably 2, 3, 4, 5, 6, 7, 8, 9, and 10. By simultaneously enclosing a plurality of first microbeads in the compartments, the number of variations (variations) in the combination of imaging information of the first microbeads in the compartments can be drastically increased based on the combination of imaging information of a smaller number of kinds of first microbeads, and a large number of compartments can be distinguished from each other. Here, the first microbeads also have microbeads having the same imaging information, but it is preferable that a plurality of first microbeads having different kinds of imaging information are present in the compartment.

For example, the size of the bead, the color of fluorescence, and the concentration of fluorescence are selected as examples of the imaging information of the first bead as follows. The size of the beads was 3 (3, 7, 11 μm), the color of the fluorescent dye was 3 (blue, green, red), and the intensity level based on the fluorescent dye concentration was 6 (0, 1, 2, 3, 4, 5). In this case, the kind of bead is (intensity level)^{The size category}-1) × size category ═ 6³-1)×3＝645。

Here, in the case where 3 first microbeads are present in the compartment, the kind of each combination of the first microbeads can be obtained₆₄₅C₃＞10⁷Such a very wide variety of combinations.

The number of the second beads in each 1 compartment is not particularly limited, and is preferably 1 in each 1 compartment from the viewpoint of identifying genome-related information derived from the same cell.

In addition, reagents required in the subsequent steps, for example, reagents for PCR such as cell lysis buffer and PCR reaction mixture (reaction mix) may be sealed at the time of the dispensing treatment.

Correlation procedure of nondestructive measurement information of single cell and imaging information of microbead

The step of correlating the information on nondestructive measurement of single cells with the information on imaging of microbeads of the present invention comprises the steps of: before or in each compartment, nondestructive measurement information of the single cell and imaging information of the first microbead are detected together, and the nondestructive measurement information of the single cell and the imaging information of the first microbead are correlated.

One embodiment of the process for correlating nondestructive measurement information according to the present invention will be described below with reference to fig. 1b, but the present invention is not particularly limited thereto.

Information on non-destructive measurements of cells 105 in compartment 104 is determined along with imaging information of first bead 102. Non-destructive measurement information of the cells 105 in the compartment 104 is then correlated with imaging information of the first microbeads 102 based on the resulting assay results.

Examples of a method for detecting or measuring the nondestructive measurement information of the single cell and the imaging information of the first bead together include flow cytometry (for example, an imaging flow cytometry method for observing a cell flowing in a channel), microscopic measurement (a cell observation method in a microwell using a common optical microscope, or the like), and the like.

In addition, according to another aspect of the present invention, since the compartment 104 can be identified using the imaging information of the beads, for example, the nondestructive measurement information of the cells may be measured again after a predetermined time, for example, 10 minutes after the incubation, the chemical analysis, the acquisition of the imaging information or the nondestructive measurement information of the cells.

Here, the imaging information of the first beads is not particularly limited as long as the imaging information of the first beads in each compartment is distinguishable from each other, and may be the imaging information of the beads themselves or the imaging information given by labeling. Here, "imaging" includes a method capable of performing separation measurement of measurement information of objects to be measured such as microbeads temporally overlapping on the basis of spatial information. The measurement information of the measured object obtained by the above-described imaging is referred to as imaging information. Examples of the imaging information include at least 1 measurement information selected from color (color), fluorescence, size, shape, electromagnetic wave, transmission, phase, scattering, reflection, coherent raman, infrared spectroscopy, raman spectroscopy, absorption spectrum, and the number of first beads. As the imaging information, infrared spectroscopic imaging, raman spectroscopic imaging, color imaging, and fluorescence imaging are preferable.

The fluorescence can be obtained using organic fluorescent molecules, biological fluorescent molecules, quantum dots, inorganic substances such as heavy metals, or a combination thereof.

The measurement information such as transmission, phase, scattering, reflection, and the like can be obtained using an organic substance, an inorganic substance, or a combination thereof, the refractive index and color of which vary depending on the concentration. These pieces of information can be obtained by a bright field observation method or the like.

The absorption spectrum and raman spectrum can be obtained using organic molecules or inorganic substances having different absorption wavelength bands (molecular imprinting) of absorption and raman scattering spectra, and combinations thereof, and examples thereof include acetylene-based compounds having a wavelength band that does not overlap with a cell signal.

In addition, the coherent raman can be measured by, for example, a coherent anti-stokes raman scattering (CARS) method or a Stimulated Raman Scattering (SRS) method.

The size, color, and shape of the bead also become imaging information of the first bead, and the size, color, and shape can be varied by forming the bead using, for example, flow lithography (flow lithography). These pieces of information can be obtained by a bright field observation method or the like.

In addition, since the imaging information of the beads is spatially separated from the cells, the separation can be performed without interfering with the non-destructive measurement information of the cells.

The nondestructive measurement information of the cell is not particularly limited as long as the characteristic of the cell can be recognized, and examples thereof include imaging information, morphological information obtained from the cell, measurement information of a physical wave (e.g., sound or ultrasonic wave) obtained from the cell, and measurement information of an electromagnetic wave (e.g., light or terahertz) obtained from the cell. Here, the above-mentioned imaging information can be obtained similarly to the imaging information of the first beads. Examples of the nondestructive measurement information include imaging information based on measurement information such as color, fluorescence, size, shape, electromagnetic wave, transmission, phase, scattering, reflection, coherent raman, infrared spectroscopy, raman, or absorption spectrum, and morphological information of cells such as size of nucleus and cytoplasm, density of cytoskeleton, feature amount of internal structure, uniformity of membrane, fluorescence intensity of each structure, local presence of molecules, and positional relationship of molecules or objects to be observed, and morphological information obtained from cells is preferable.

Fig. 1f and 1g illustrate the flow of analysis of imaging information more specifically as an example.

Fig. 1f is a schematic diagram of a database of first microbeads having imaging information of n kinds (ID) of microbeads prepared by controlling size, RGB, fluorescence brightness, and other optical characteristics as indices. In FIG. 1f, the imaging information of the first microbead is measured and the resulting imaging information of the first microbead is correlated with the optical microbead ID.

In fig. 1g, the combination of the cell and the first bead is measured, and information on nondestructive measurement of the cell is correlated with information on imaging of the first bead. First, the optical barcode ID of each first microbead is read (optical calibration is performed here). Further, a nucleic acid barcode ID is given to each first bead based on the first common barcode region of the first barcode nucleic acid linked to the first bead. Therefore, the combination of the nucleic acid barcode IDs of the first beads and the combination of the optical bead IDs are compared to identify the cells. The non-destructive measurement information of the cell can then be correlated with genomic-related information of the cell.

The step of correlating the information on the nondestructive measurement of the single cell with the imaging information on the first bead according to the present invention may be performed before or after the preparation of each compartment. In particular, in the method of detecting and correlating nondestructive measurement information of a single cell and imaging information of the first bead together before preparing each compartment, it is preferable that a cell-derived substance is obtained by lysing/crushing the cell after the correlation, the cell-derived substance is enclosed in the compartment together with the mixture of the first bead and the second bead, and the subsequent hybridization complex obtaining step is performed.

Here, the positional relationship between the cell and the first bead in the mode of detecting and correlating the nondestructive measurement information of the single cell and the imaging information of the first bead together before preparing each compartment is the same as the positional relationship between the cell and the first bead in each compartment described above, and is not particularly limited as long as the nondestructive measurement information of the cell and the imaging information of the first bead are not obtained, and may be appropriately set according to the type, size, and properties of the cell and the first bead. That is, in the present invention, the first beads may be present in the same compartment with or without contact with the cells, or the first beads may be introduced into the cells, as long as the nondestructive measurement information of the cells and the imaging information of the first beads can be obtained together. Preferable examples thereof include a method in which the first beads are carried by a cell, a method in which a sub-compartment not containing a cell but containing the first beads is bonded to a cell to carry the sub-compartment by the cell, and a method in which the first beads are swallowed by a cell.

Hybridization Complex obtaining procedure

The hybridization complex obtaining step includes the following steps: after the first barcode nucleic acid is cleaved from the associated first bead, the genome-associated nucleic acid and the first barcode nucleic acid are hybridized with a second barcode nucleic acid, respectively, to obtain a hybridization complex.

The above-described steps can be carried out by known methods. For example, the method can be carried out by the method described in high hly parallel genome-side expression Profiling of industrial Cells Using NanoliterilterDroplets, cell.161,1202-1214(2015), E.Z.Macosko et al.

One embodiment of the hybridization complex obtaining step is described below with reference to FIG. 1c, but the method is not particularly limited.

Following the above-described correlation process of the nondestructive measurement information of a single cell with the imaging information of a microbead, the cleavable linker is cleaved (for example, the ultraviolet-cleavable linker is cleaved by ultraviolet irradiation), the first barcode nucleic acid 106 is cleaved, and then the cell is lysed. Then, in the compartment, mRNA107 from the cell and the first barcode nucleic acid 106 from the first bead are hybridized with the second barcode nucleic acid 108, respectively, to obtain a hybrid complex 120. Then, the droplets are broken.

Process for producing amplification product derived from hybridization complex

The process for producing an amplification product derived from a hybridization complex includes the steps of: an amplification product derived from the hybridization complex obtained in the hybridization complex obtaining step is produced.

The above-described steps can be carried out by known methods. For example, the method can be carried out by the method described in high hly parallel genome-wide Expression Profiling of industrial Cells Using NanoliterilterDroplets, cell.161,1202-1214(2015), E.Z.Macosko et al.

One embodiment of the process for producing an amplification product derived from a hybridization complex will be described below with reference to FIG. 1d, but the process is not particularly limited.

Reverse transcription reaction is performed on the hybrid complex obtained in the hybrid complex obtaining step. By this reverse transcription reaction, cDNA110 for mRNA107 from the cell and cDNA109 for the first barcode nucleic acid 106 can be synthesized. Then, template switching (template switching) may be performed.

Then, PCR reaction was performed. This PCR reaction produces 2 amplification products 111, i.e., a first amplification product 112 derived from a hybridization complex formed between the first barcode nucleic acid 106 and the second barcode nucleic acid 108, and a second amplification product 113 derived from a hybridization complex formed between the cell-derived mRNA107 and the second barcode nucleic acid 108. When the genome-related nucleic acid is DNA, an extension PCR method can be performed as the PCR reaction.

Then, based on the obtained amplification products, a library of amplification products derived from each of the 1 to n compartments and including the first amplification product and the second amplification product is prepared.

Process for detecting integrity of information on nondestructive measurement and genome-related information of individual cell

The step of detecting non-destructive measurement information and genome-related information of a single cell as a whole includes the steps of: the nondestructive measurement information and genome-related information of the individual cells are detected as a whole using, as an index, the expression pattern of the amplification product obtained in the step of producing the amplification product derived from the hybridization complex. The expression pattern of the amplification product includes, for example, sequence information of the amplification product obtained by sequencing, or sequence information of the first barcode nucleic acid, sequence information of the first common barcode region, sequence information of the first unique barcode region, sequence information of the second barcode nucleic acid, sequence information of the second common barcode region, or sequence information of the second unique barcode region in the sequence information.

Hereinafter, one embodiment of the step of detecting non-destructive measurement information and genome-related information of a single cell in a whole body will be described with reference to fig. 1e, but the step is not particularly limited.

The sequence of the amplification product (first amplification product, second amplification product) obtained in the step of producing the amplification product derived from the hybridization complex is determined by a sequencer, and the sequence information of the amplification product is analyzed. In the analysis of the second amplification product, the cells from which the respective amplification products are derived are divided using the sequence information of the second common barcode region as an index. Further, since each mRNA molecule can be identified based on the sequence information of the second unique barcode region, information such as the sequence of mRNA of each cell and the expression level thereof can be obtained using the sequence information as an index. Based on the information obtained in the analysis of the second amplification product, the transcriptome information 115 of each cell can be obtained.

The non-destructive measurement information 114 of the cells is then analyzed. Here, as described above, the nondestructive measurement information of the single cell is correlated with the imaging information of the first microbead to which the first barcode nucleic acid corresponding to each imaging information is attached. Therefore, in the above analysis, the nondestructive measurement information 114 of the cell from which the first amplification product is derived can be classified for each first amplification product based on the sequence information of the first common barcode region of the first barcode nucleic acid.

Next, the non-destructive measurement information 114 of the cell is matched with the transcriptome information 115. Thus, the genome-related information of individual cells in each compartment can be combined with non-destructive measurement information in a 1-to-1 manner.

Further, there can be provided a method of analyzing, discriminating, or classifying a test cell based on nondestructive measurement information of the test cell using a classification model obtained based on the nondestructive measurement information of a single cell and genomic-related information obtained as described above.

In the above method, first, a database of nondestructive measurement information and genome-related information of a single cell can be prepared. Then, by using the database, the genome-related information is clustered 116 based on the matching result between the nondestructive measurement information of the single cell and the genome-related information such as the transcriptome of the cell, and thereby each cell can be classified into a plurality of types 117(A, B, C, … … N-type cells). Further, the obtained nondestructive measurement information of a single cell and the cell classification result are used as training data, and a classification model can be obtained by machine learning the nondestructive measurement information of a single cell (supervised machine learning).

Further, the classification model obtained in the above can be used to analyze, discriminate, or classify a cell to be measured based on nondestructive measurement information of the cell to be measured. For example, a cell type clustered based on genome-related information can be identified for the cell based on the non-destructive measurement information of the above-mentioned test cell.

In addition, as another method of analyzing, discriminating, or classifying the cells to be measured, the following methods can be mentioned.

First, a database of non-destructive measurement information and genome-related information for individual cells can be prepared. By using the database, the nondestructive measurement information is clustered based on the matching result between the nondestructive measurement information of a single cell and genome-related information such as a transcriptome of the cell, and thereby each cell can be classified into a plurality of types (A, B, C, … … N-type cells). Further, the obtained nondestructive measurement information of a single cell and the cell classification result are used as training data, and a classification model can be obtained by machine learning the nondestructive measurement information of a single cell (supervised machine learning).

The classification model obtained as described above can be used to analyze, discriminate, or classify a cell to be tested based on genomic-related information of the cell.

Hereinafter, the first beads, the second beads, and the like used in the present specification will be described.

First micro-molecules cleavably linked to first barcode nucleic acids and having mutually distinguishable imaging information Bead

Fig. 2 shows a mode in which the first barcode nucleic acid is RNA as one mode of the first microbead (hereinafter, also referred to as a first microbead linked to the first barcode nucleic acid) which is linked to the first barcode nucleic acid in a cleavable manner and has imaging information distinguishable from each other.

The first barcode nucleic acid 202 is attached to the first microbead 201 having imaging information distinguishable from each other. The first barcode nucleic acid 202 comprises a first common barcode region 203 and a first hybridization region 204. The first barcode nucleic acid 202 includes a first common barcode region 203 and a first hybridization region 204 in this order from the first bead side. Among them, poly-adenine is given as the first hybridization region 204. Further, the first barcode nucleic acid 202 is linked to the first bead 201 so as to be cleavable via a cleavable linker 205.

Organisms containing a first barcode nucleic acid

The first microbead of the present invention may be a living organism containing first barcode nucleic acids and having imaging information distinguishable from each other (hereinafter, also referred to as a living organism containing first barcode nucleic acids).

The organism preferably contains a plasmid having a first barcode nucleic acid. The imaging information of the living body is not particularly limited as long as it is the imaging information of the present invention, and the number and fluorescence of the living body are preferable, and the fluorescence includes not only the spectrum of each color but also the luminance information thereof. Further, the fluorescence is preferably obtained by using a fluorescent protein expressed by a fluorescent protein gene present in a plasmid. Thus, the organisms of the invention preferably contain, for example, a plasmid with a first barcode nucleic acid and a fluorescent protein gene. Here, the first barcode nucleic acid includes a first common barcode region (for example, a common barcode region common to fluorescent proteins) and a first hybridization region, and the first hybridization region may be poly-adenine. Further, the first barcode nucleic acid may comprise a third intrinsic barcode region. This third inherent barcode region enables individual clones of an organism to be distinguished. Accordingly, the plasmid in the organism of the present invention is constituted, for example, by a fluorescent protein gene region, a third inherent barcode region, a first common barcode region, and a first hybridizing region in this order.

First microbead

The first microbeads of the present invention are microbeads distinguishable from each other based on imaging information.

In the present specification, the beads are not particularly limited as long as they are particles to which barcode nucleic acids can be linked or organisms that can contain barcode nucleic acids, and the shape is not limited.

When the first beads are particles, the material thereof is not particularly limited, and examples thereof include semiconductors such as quantum dots (semiconductor nanoparticles) made of semiconductor materials such as cadmium selenide (CdSe), zinc sulfide (ZnS), cadmium sulfide (CdS), zinc selenide (ZnSe), and zinc oxide (ZnO), inorganic substances such as heavy metals such as gold, hydrogels such as acrylamide, agarose, collagen, alginic acid, and PEG-based, polystyrene, polypropylene, and hydrophilic vinyl polymers (for example, resins such as Toyopearl HW-65s (tosohcorporation)), or hydrophilic vinyl polymers in which PEG or derivatives thereof are bonded, and hydrogel is preferable, and acrylamide and alginic acid are more preferable.

When the first beads are living organisms, the type and form of the living organisms are not particularly limited as long as the effect of the present invention is not impaired, and the living organisms may be selected according to the purpose. Examples of the organism include eukaryotes, prokaryotes, and cells thereof, for example, microorganisms, specifically, bacteria such as Escherichia coli, and fungi such as yeast. The organism is preferably capable of amplifying a plasmid having a first barcode nucleic acid.

First barcode nucleic acid

The first barcode nucleic acid of the present invention is not particularly limited as long as it contains a barcode region corresponding to each imaging information, and the nucleic acid is, for example, RNA, DNA, or a combination thereof.

The first barcode nucleic acid of the present invention preferably includes a first common barcode region common to the first microbeads each corresponding to the same imaging information, and a first hybridization region capable of hybridizing with the second barcode nucleic acid. Here, by using the sequence information of the first common barcode region, it is possible to correspond one-to-one with the imaging information of the first microbeads having the same imaging information in each compartment. Thus, by performing the correlation, it becomes possible to be an index for determining nondestructive measurement information of a single cell existing in the same compartment.

The first barcode nucleic acid is preferably attached to a plurality of beads per 1 bead.

In addition, the first microbead of the present invention is preferably linked with a first barcode nucleic acid.

The first barcode nucleic acid may be attached to the first microbead, directly or indirectly. The first barcode nucleic acid is preferably attached to the first microbead in a cleavable manner, e.g., via a cleavable linker. In the present invention, the cleavable linker includes a linker capable of chemical cleavage, a linker capable of cleavage by light such as ultraviolet light, a linker capable of thermal cleavage, a linker capable of enzymatic cleavage, and the like. It was shown that by using the above linker, the linked nucleic acid can be cleaved from the bead, isolated or liberated. Examples of such a linker include a linker cleavable by light, such as PC-biotin and isspc, and examples of a linker cleavable chemically include a disulfide bond.

In another preferred embodiment of the first microbead according to the present invention, the first microbead may further include a first unique barcode region and a primer region, which are distinguishable from each other. As another preferable embodiment of the first beads of the present invention, an acrylamide moiety such as an acryl phosphoramidite moiety (Acrydite (trademark)) may be provided via a cleavable linker.

First micro-molecules cleavably linked to first barcode nucleic acids and having mutually distinguishable imaging information Method for producing beads

The first beads to which the first barcode nucleic acid is linked can be produced by a known method. For example, the compound can be produced by the method described in A.M. Klein et al, Droplet decoding for Single-Cell transduction specific to electromagnetic Stem cells, cell.161,1187-1201 (2015).

As an example of the above method, acrylamide containing a first barcode nucleic acid is: an aqueous solution of bisacrylamide was made into an acrylamide polymer in an organic solvent layer to be used as the first microbeads. Here, the bead-linking side of the cleavable linker bound to the first barcode nucleic acid may be modified with an acrylamide moiety such as an acryl phosphoramidite moiety (Acrydite (trademark)). By the modification, the first barcode nucleic acid is also polymerized together with the acrylamide polymer at the time of acrylamide polymerization. Prior to the emulsion formation, for example, first microbeads having various colors and various fluorescence intensities can be prepared by dissolving a fluorescence-labeled acrylamide monomer in the aqueous solution. Specifically, the first beads can be produced by a droplet production method using a microfluidic technology such as a flow focusing device (flow focusing device) or a micro well. The size of the beads can be controlled by varying the fluid conditions in the fluid focusing device and the size of the individual chambers in the microwells. After polymerization, the resulting first microbeads were taken out from the droplets, washed several times, and used as the first microbeads.

The first beads to which the first barcode nucleic acid is linked can also be produced by a known method. For example, the production can be carried out by the methods described in Japanese patent application laid-open Nos. 2009-513948 and 2017-506877.

An example of the above method will be described. First, a specific nucleic acid sequence is prepared for the first barcode nucleic acid by a solid phase synthesis method or an enzymatic synthesis method. Then, the first beads are bound via a cleavable linker. When the barcode nucleic acid is RNA, a DNA template that is a complementary strand of a single-stranded barcode nucleic acid is synthesized, and then synthesized by a linear amplification reaction using an RNA polymerase such as T7 (which binds to a promoter sequence on the DNA template to synthesize RNA including a single-stranded barcode region).

Second microbeads linked to a plurality of second barcode nucleic acids

Fig. 3 shows one embodiment of second microbeads linked to a plurality of second barcode nucleic acids that can hybridize to the genome-associated nucleic acids and the first barcode nucleic acids (hereinafter, also referred to as second microbeads linked to the second barcode nucleic acids).

In FIG. 3, a second barcode nucleic acid 302 is attached to a second microbead 301. The second barcode nucleic acid 302 comprises a second common barcode region 303, a second intrinsic barcode region 304, and a second hybridization region 305. The second barcode nucleic acid 302 includes a PCR primer region 306, a second common barcode region 303, a second unique barcode region 304, and a second hybridization region 305 in this order from the second bead side. The second hybridization region 305 is poly-thymine.

The second microbeads are preferably linked with 1,000 to 100,000 second barcode nucleic acids from the viewpoint of being able to hybridize with many pieces of genome-associated nucleic acids.

Second microbead

The material of the second beads of the present invention may be the same as that of the first bead particles.

The material of the second bead of the present invention is preferably hydrogel or resin, and more preferably acrylamide, polystyrene, a hydrophilic vinyl polymer, or a hydrophilic vinyl polymer in which PEG or a derivative thereof is bonded.

Second barcode nucleic acid

The second barcode nucleic acid of the present invention is not particularly limited as long as it contains a barcode region, and for example, the nucleic acid is RNA, DNA, or a combination thereof. The second barcode nucleic acid can be linked directly or indirectly to a second microbead.

The second barcode nucleic acid of the present invention preferably comprises a second common barcode region common to each other, a second inherent barcode region distinguishable from each other, and a second hybridization region. Here, the sequence information of the second common barcode region described above can be used as an index for determining the cells present with the second barcode nucleic acid in the compartment. Furthermore, the sequence information of the second inherent barcode region described above can be used as an index for determining a genome-associated nucleic acid. In addition, the second hybridization region may hybridize to the genome-associated nucleic acid and the first barcode nucleic acid, respectively. Thus, the second hybridization region preferably comprises poly-thymine, or a nucleic acid complementary to a genome-associated nucleic acid.

As described above, the sequence information of the genome-associated nucleic acid and the second common barcode region is correlated, and thus the cell from which the genome-associated nucleic acid is derived can be determined. Further, since the second barcode nucleic acid is hybridized with the first barcode nucleic acid, sequence information such as the first common barcode region of the first bead is also associated with sequence information of the second common barcode region. It should be noted that the sequence information of the first common barcode region is associated with the imaging information of each first microbead. In addition, information on nondestructive measurement of the cells is also correlated with the imaging information of each first microbead. Thus, the genome-associated nucleic acid information can correspond one-to-one to the non-destructive measurement information of the cell.

FIG. 4 shows another embodiment of the first bead linked to the first barcode nucleic acid and the second bead linked to the second barcode nucleic acid. FIG. 4 shows another embodiment in which the first barcode nucleic acid and the second barcode nucleic acid are each DNA.

In the first microbead 400 attached with a first barcode nucleic acid of fig. 4A, a first barcode nucleic acid 402 is attached with a first microbead 401 having imaging information distinguishable from each other. The first barcode nucleic acid 402 includes a first common barcode region 403 and a first hybridization region 404. The first barcode nucleic acid 402 includes a first common barcode region 403 and a first hybridization region 404 in this order from the first bead side. Further, the first barcode nucleic acid 402 is connected to the first microbead 401 via a cleavable linker 405 in a cleavable manner. Furthermore, the first hybridization region 404 is a sequence complementary to a second hybridization region 415 described below.

In addition, in the second microbead 410 of fig. 4A to which a second barcode nucleic acid is attached, a second barcode nucleic acid 412 is attached to a second microbead 411. The second barcode nucleic acid 412 comprises a second common barcode region 413, a second intrinsic barcode region 414, and a second hybridization region 415. The second barcode nucleic acid 412 includes a PCR primer region 416, a second common barcode region 413, a second inherent barcode region 414, and a second hybridization region 415 in this order from the second bead side. The second hybridization region 415 is a sequence complementary to a specific sequence of the genome-associated nucleic acid 422, which hybridizes to the sequence.

In addition, in FIG. 4A, a compartment 430 containing a cell 420 is shown along with a first microbead 400 attached with a first barcode nucleic acid and a second microbead 410 attached with a second barcode nucleic acid.

In addition, hybridization complexes are shown in FIG. 4B. Here, the second barcode nucleic acid 412 hybridizes to the first hybridization region 404 of the first barcode nucleic acid 402 at a second hybridization region 415. In addition, the second barcode nucleic acid 412 hybridizes to a specific sequence 421 of the genome-associated nucleic acid 422 at a second hybridization region 415.

FIG. 5 shows another embodiment of a first bead linked to a first barcode nucleic acid and a second bead linked to a second barcode nucleic acid. Fig. 5 shows an embodiment in which the first barcode nucleic acid and the second barcode nucleic acid are each DNA and include a nucleic acid probe 520 (hereinafter, also referred to as a nucleic acid probe 520) specific to a molecule such as a protein expressed in a single cell.

In the first microbead 500 attached with a first barcode nucleic acid of fig. 5A, a first barcode nucleic acid 502 is attached with a first microbead 501 having imaging information distinguishable from each other. The first barcode nucleic acid 502 comprises a first common barcode region 503 and a first hybridization region 504. The first barcode nucleic acid 502 includes a first common barcode region 503 and a first hybridization region 504 in this order from the first bead side. Further, the first barcode nucleic acid 502 is connected to the first microbead 501 via a cleavable linker 505 in a cleavable manner. In addition, the first hybridization region 504 is a sequence complementary to the second hybridization region 515 described below.

In addition, in the second microbead 510 attached with a second barcode nucleic acid of fig. 5A, a second barcode nucleic acid 512 is attached to a second microbead 511. The second barcode nucleic acid 512 comprises a second common barcode region 513, a second intrinsic barcode region 514, and a second hybridization region 515. The second barcode nucleic acid 512 includes a PCR primer region 516, a second common barcode region 513, a second inherent barcode region 514, and a second hybridization region 515 in this order from the second bead side. The second hybridization region 515 is a sequence complementary to the hybridization region 524 of the nucleic acid probe 520.

In FIG. 5B (1), a nucleic acid probe 520 is shown. FIG. 5B (2) shows the manner in which the nucleic acid probe 520 binds to a protein expressed in the cell 530, which is achieved by mixing and washing the nucleic acid probe 520 with the cell 530. In addition, in FIG. 5B (3), a compartment 540 containing a cell 530 to which the nucleic acid probe 520 is bound is shown together with a first microbead 500 linked to a first barcode nucleic acid and a second microbead 510 linked to a second barcode nucleic acid.

In the nucleic acid probe 520 of fig. 5B (1), the third barcode nucleic acid 522 is linked to a molecule 521 (hereinafter, also referred to as a binding molecule 521) that can specifically bind to a molecule such as a target protein expressed in a single cell. Third barcode nucleic acid 522 comprises third common barcode region 523 and third hybridization region 524. The third barcode nucleic acid 522 comprises, in order from the binding molecule 521, a third common barcode region 523 and a third hybridization region 524. In addition, the third barcode nucleic acid 522 is cleavable linked to the binding molecule 521 via a cleavable linker 525. In addition, the third hybridization region 524 is a sequence complementary to the second hybridization region 515. The nucleic acid probe 520 may be a plurality of probes including a third barcode nucleic acid 522 different for each binding molecule 521.

In addition, fig. 5C shows a hybridization complex. The second barcode nucleic acid 512 hybridizes to the first hybridization region 504 of the first barcode nucleic acid 502 at a second hybridization region 515. In addition, the second barcode nucleic acid 512 hybridizes to a third hybridization region 524 of a third barcode nucleic acid 522 at a second hybridization region 515.

By analyzing the sequence information of the amplification product derived from the sequence of the third common barcode region 523 of the nucleic acid probe 520, the presence or absence and the expression level of the target molecule in the cell can be confirmed.

Therefore, by using a nucleic acid probe specific to a molecule expressed in a single cell, a large number of proteomes can be corresponded one-to-one with nondestructive measurement information of the cell.

In addition, other modes of the first beads to which the first barcode nucleic acid is linked are shown in FIG. 6. FIG. 6 shows, as another embodiment, a mode in which the first barcode nucleic acid is DNA and contains a specific sequence.

In the first microbead 600 attached with a first barcode nucleic acid of fig. 6A, a first barcode nucleic acid 602 is attached with a first microbead 601 having imaging information distinguishable from each other. The first barcode nucleic acid 602 comprises a first common barcode region 603 and a first hybridization region 604. The first barcode nucleic acid 602 includes a first common barcode region 603 and a first hybridization region 604 in this order from the first bead side. Further, the first barcode nucleic acid 602 is connected to the first microbead 601 in a cleavable manner via a cleavable linker 605. In addition, between the first barcode nucleic acid 603 and the cleavable linker 605, a specific sequence 606 is included. In addition, the first hybridization region 604 is a sequence complementary to the second hybridization region 615.

In addition, the hybridization complex is shown in FIG. 6B (1). 610 represents a second microbead linked to a second barcode nucleic acid. Wherein the second barcode nucleic acid 612 hybridizes to the first hybridization region 604 of the first barcode nucleic acid 602 at the second hybridization region 615. Further, as shown in fig. 6B (2), by using a primer 607 having a sequence complementary to a specific sequence 606, DNA complementary to the first barcode nucleic acid 602 and the second barcode nucleic acid 612 can be synthesized, and further, DNA complementary to the second barcode nucleic acid 612 can be synthesized by template conversion.

FIG. 7 shows another embodiment of the first bead linked to the first barcode nucleic acid and the second bead linked to the second barcode nucleic acid. FIG. 7 shows another embodiment in which the first barcode nucleic acid and the second barcode nucleic acid are each DNA.

Fig. 7A shows a compartment preparation process. In the first microbead 700 attached with a first barcode nucleic acid of fig. 7A, a first barcode nucleic acid 702 is attached with a first microbead 701 having imaging information distinguishable from each other. The first barcode nucleic acid 702 comprises a first common barcode region 703 and a first hybridization region 705. The first barcode nucleic acid 702 includes a PCR primer region 708, a first common barcode region 703, a first inherent barcode region 704, and a first hybridization region 705 in this order from the first bead side. The first barcode nucleic acid 702 is cleavable through a cleavable linker 706 and is linked to an acrylamide moiety 707 such as an acryl phosphoramidite moiety (Acrydite (trademark)) bound to the first bead 701. In addition, the first hybridization region 705 is poly adenine.

In addition, in the second microbead 710 attached with a second barcode nucleic acid of fig. 7A, a second barcode nucleic acid 712 is attached to a second microbead 711. The second barcode nucleic acid 712 comprises a second common barcode region 713, a second intrinsic barcode region 714, and a second hybridization region 715. The second barcode nucleic acid 712 includes a PCR primer region 416, a second common barcode region 713, a second inherent barcode region 714, and a second hybridization region 715 in this order from the second bead side. The second hybridizing region 715 is poly-thymine. 720 denotes a cell.

FIGS. 7B to E show a process of obtaining a hybridization complex after a process of correlating nondestructive measurement information of a single cell with imaging information of a microbead.

In FIG. 7B, the photocleavable linker is cleaved to lyse the cells. In FIG. 7B, a first microbead 701 after the first barcode nucleic acid 702 has been cleaved, a second microbead 710 linked to a second barcode nucleic acid, and mRNA721 from a cell are shown.

In addition, hybridization complexes are shown in fig. 7C. Here, the second barcode nucleic acid 712 hybridizes to the first hybridization region 705 of the first barcode nucleic acid 702 at a second hybridization region 715. Further, a complementary strand DNA723 is synthesized using a DNA polymerase 722. In addition, the second barcode nucleic acid 712 hybridizes to the poly-adenine of the mRNA721 from the cell at a second hybridization region 715.

In FIG. 7D, in the hybridization complex, a reverse transcription reaction was performed to synthesize cDNA724 for mRNA721 derived from the cell.

In FIG. 7E, the 3' end of the synthesized cDNA is labeled with a DNA tag 725. The DNA tag can be used as a primer site for PCR, and examples thereof include a transposon chimeric End (ME) sequence.

Method for producing second microbeads linked to second barcode nucleic acids

The second beads to which the plurality of second barcode nucleic acids are linked can be produced by a known method. For example, they can be produced by the method described in high hly Parallel Genome-side expression profiling of industrial Cells Using nanoliters draglines, cell.161,1202-1214(2015) by E.Z. Macosko et al.

To briefly describe the above method, the second common barcode region in the second barcode nucleic acid can be produced by split-and-pool synthesis (split-and-pool synthesis). For example, the production can be performed by performing n (round) division-confluence synthesis steps. Each wheel includes: i) dividing the bead group into 4 pieces; ii) separately synthesizing A, G, C, T each of the bead groups; iii) a step of combining 4 bead groups and collecting them. The number of rounds n can be set as appropriate according to the length of the barcode sequence to be manufactured. For example, n is 6 to 40.

After the second common barcode is manufactured, a second inherent barcode region is synthesized. For all microbeads linked to the second common barcode region, m rounds of synthesis were performed in the presence of A, T, G, C total bases. The number of rounds m can be set as appropriate according to the length of the barcode sequence to be manufactured. For example, m is 3 to 15.

Distribution process

One embodiment of the dispensing process, i.e., the compartment manufacturing method, will be specifically described with reference to fig. 8 and 9.

Fig. 8a, b show a method of manufacturing a compartment in a single step and detecting and/or determining non-destructive measurement information of a single cell together with imaging information of a first microbead. Specifically, a solution containing a cell 801, a first microbead 802 linked to a first barcode nucleic acid, and a second microbead 803 linked to a second barcode nucleic acid is added to an oil 806 to make droplets as compartments 807, which are released at an outlet 808. In fig. 8a, the non-destructive measurement information is measured before and after the formation of the compartment, and in fig. 8b, the non-destructive measurement information is measured after the formation of the compartment. In fig. 8a, various 1 assays, such as fluorescence imaging assay 804, bright field imaging 805, can be performed before and after formation of the compartment. Here, accurate imaging information of the cell and the first bead can be obtained in the measurement before the compartment is made, and the combination of the cell and the first bead in the compartment can be confirmed in the measurement after the compartment is made. In addition, according to the methods of FIGS. 8a and b, the natural states of the cells and the first beads can be imaged. In the above method, as a specific apparatus to be used, for the production of the cell, there are mentioned apparatuses used in a droplet production method using a microfluidic technology, such as a fluid focusing device; the measurement of nondestructive measurement information includes an imaging flow cytometer and the like. As the above-mentioned device, a known device may be used as long as it does not interfere with the present invention. Examples of the apparatus used in a method for producing droplets Using a microfluidic technique, such as a fluid focusing device, include apparatuses described in E.Z. Macosko et al, high strain Genome-with Expression Profiling of Industrial cells Using Nanoliter dispersions, cell.161,1202-1214(2015), microfluidic Nanofluid (2008)5: 585-. With the apparatus used in the droplet production method using the microfluidic technology such as the fluid focusing device described above, microbeads having imaging information represented by color, shape, and size can be produced efficiently and in large quantities.

In addition, in fig. 9, a method of manufacturing the compartment in 2 steps is shown. First, in step a, a solution containing a cell 901, a first microbead 902 linked to a first barcode nucleic acid, and a second microbead 903 linked to a second barcode nucleic acid is added to an oil 904 to form a compartment 905, and then released at an outlet 906 to gel 907. Then, in step B, the following method is shown: the gelled particles 907 are released into an aqueous solvent containing a cell lysis buffer 908, subjected to fluorescence imaging measurement 909, and enclosed in an oil 910 to further form a compartment 911. With the method of fig. 9, although rotation may occur when imaging cells in a compartment such as a droplet is planned, cells or microbeads enclosed in gel particles present in an aqueous phase can be stably imaged without rotation.

In addition, in fig. 10, another way of manufacturing the compartment in 2 steps is shown. First, in step a, a solution containing a first bead 1002 attached to a first barcode nucleic acid and a second bead 1003 attached to a second barcode nucleic acid is added to an oil 1004 to form a compartment 1005, and then released at an outlet 1006 to gel 1007. Then, in step B, the following method is shown: the gelled particles 1007 and cells 1001 are released into an aqueous solvent containing a cell lysis buffer 1008, subjected to fluorescence imaging measurement 1009, and enclosed in oil 1004 to further form a compartment 1010. Finally, the cell and the gelled particles are dissolved in the lysis buffer in the compartment in which the cell, the cell lysis buffer, the first bead and the second bead are enclosed, and the hybrid complex can be obtained.

In addition, in fig. 11, another other way of manufacturing the compartment in 2 steps is shown. First, in step a, a solution containing the first beads 1102 linked to the first barcode nucleic acid and the cell 1101 is prepared and mixed. Then, for example, cells carrying the first beads can be obtained in the channel. Fluorescence imaging measurement 1103 of the obtained cells was performed. Then, in step B, a solution containing the cells 1104 having the first beads supported thereon, the second beads 1105 linked to the second barcode nucleic acid, and an aqueous solvent containing a cell lysis buffer 1106 are mixed in a channel. The resulting mixed liquid is discharged into a channel filled with oil 1108, and droplets are produced as compartments 1107 and discharged at an outlet 1109. The above method can also be used in a manner to include a substance derived from the cell in the compartment.

System for controlling a power supply

The system for integrity testing non-destructive measurement information and genome-related information of individual cells according to the present invention is characterized in that it comprises: a compartment preparation section that prepares a plurality of compartments, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead that is a particle each attached in a cleavable manner to a first barcode nucleic acid corresponding to each imaging information or an organism each containing a first barcode nucleic acid corresponding to each imaging information, and the imaging information of the first microbead in each compartment is distinguishable from each other, and a second microbead that is attached to a plurality of second barcode nucleic acids that can hybridize to a genome-associated nucleic acid corresponding to a genome of a cell or an expression product thereof or the first barcode nucleic acid; a measurement unit for nondestructive measurement information, which measures nondestructive measurement information of a single cell together with imaging information of the first bead before or in each compartment, and correlates the nondestructive measurement information of the single cell with the imaging information of the first bead; a hybridization complex formation unit that cleaves a first barcode nucleic acid corresponding to each piece of imaging information from the associated first bead, and hybridizes the genome-associated nucleic acid and the first barcode nucleic acid to a second barcode nucleic acid, respectively, to obtain a hybridization complex; an amplification product producing unit that produces an amplification product derived from the hybridization complex; and a detection unit for detecting the nondestructive measurement information and the genome-related information of the individual cell as a whole using the expression pattern of the amplification product as an index.

The present system is explained in accordance with the flowchart of fig. 12.

(A) In the compartment preparation section, a plurality of compartments are prepared, each 1 compartment containing a single cell or a substance derived therefrom, a first microbead, and a second microbead. The plurality of compartments are preferably obtained by distributing a cell population, a plurality of first microbeads, and a plurality of second microbeads. Here, the above-mentioned first microbeads are particles each attached in a cleavable manner to the first barcode nucleic acid corresponding to each imaging information, or organisms each containing the first barcode nucleic acid corresponding to each imaging information, and the imaging information of the first microbeads in each compartment can be distinguished from each other. In addition, the second beads are linked to a plurality of second barcode nucleic acids capable of hybridizing with the first barcode nucleic acid or a genome-associated nucleic acid corresponding to the genome of the cell or an expression product thereof. Examples of the device used as the cell preparation unit include devices used in a droplet production method using a microfluidic technology, such as a fluid focusing device.

(B) The nondestructive measurement information and imaging information measuring unit measures nondestructive measurement information of a single cell and imaging information of the first bead together before preparation of each compartment or in each compartment, and correlates the nondestructive measurement information of the single cell with the imaging information of the first bead. Examples of devices for measuring nondestructive measurement information include a flow cytometer and a microscope. In the case where the cell is in a form such as a droplet or gel particle that is not destroyed by the measurement of the nondestructive measurement information and the imaging information, the nondestructive measurement information of the cell and the imaging information of the first bead may be measured again after one measurement. The re-determination is advantageous in terms of being able to obtain the change in the non-destructive measurement information of the cells in the form of a time series.

When the cell-derived substance is contained in the compartment, it is preferable that nondestructive measurement information on a single cell and imaging information on the first beads be measured and correlated before the compartment is prepared. Therefore, the nondestructive measurement information of the single cell and the imaging information of the first bead can be measured and correlated in the measurement unit of the nondestructive measurement information and the imaging information (B) before the preparation unit of the compartment (a) prepares the compartment.

(C) The hybridization complex forming unit cleaves a first barcode nucleic acid specific to the first bead from the first bead, and hybridizes the genome-associated nucleic acid and the first barcode nucleic acid to a second barcode nucleic acid to obtain a hybridization complex. Examples of the apparatus and the reagent for forming a hybridization complex include a cleavage device for a cleavable linker connected to the first barcode nucleic acid, and a reagent and a device for forming a hybridization complex. The cleavage device for the cleavable linker may be appropriately selected depending on the type of the cleavable linker, and for example, in the case where the cleavable linker is a linker cleavable by ultraviolet light (for example, iSpPC (Integrated DNA Technologies)), an ultraviolet irradiation device (for example, a device equipped with a BlackRay xenon lamp) may be mentioned. Further, as a reagent for forming a hybridization complex, a reagent used for hybridization of a general nucleic acid can be mentioned, and as an apparatus, a water bath or the like can be mentioned.

(D) The amplification product producing section can produce an amplification product derived from the hybridization complex. Examples of the production of amplified products include reverse transcription and PCR when the genome-related nucleic acid is RNA, and elongation PCR when the genome-related nucleic acid is DNA. The reagents for producing an amplification product include reagents used in a general reverse transcription or PCR reaction, and the apparatus includes a PCR device.

(E) The nondestructive measurement information and genome-related information detecting unit detects the nondestructive measurement information and genome-related information of a single cell as a whole using the expression pattern of the amplification product as an index. The genome-associated information can be obtained by determining sequence information of the genome-associated nucleic acid using a sequencer. The obtained genome-related information can be correlated with non-destructive measurement information of the individual cells using a computer.

The system can be implemented according to the description of the detection method of the present invention.

Composition comprising a metal oxide and a metal oxide

As another embodiment of the present invention, there is a composition of a first microbead and a second microbead for detecting non-destructive measurement information and genome-related information of a single cell as a whole, wherein the first microbead is a particle that is cleavably linked to a first barcode nucleic acid corresponding to each imaging information or an organism each containing the first barcode nucleic acid corresponding to each imaging information, the second microbead is linked to a plurality of second barcode nucleic acids that are capable of hybridizing to the genome-related nucleic acid corresponding to the genome of the cell or an expression product thereof or the first barcode nucleic acid, the composition is capable of detecting non-destructive measurement information of a single cell using, as an index, an expression pattern of a first amplification product derived from a hybridization complex formed by the first barcode nucleic acid and the second barcode nucleic acid, and the composition is capable of detecting non-destructive measurement information of a single cell using, as an index, a second amplification product derived from a hybridization complex formed by the genome-related nucleic acid and the second barcode nucleic acid The expression pattern of the substance is used as an index to detect genome-related information of a single cell. The composition of the present invention is not particularly limited as long as it is used for the total detection of information on nondestructive measurement and information on genome of a single cell, and may be in the form of 1 composition or a pharmaceutical agent such as a reagent, or may be composed of a combination of a plurality of compositions or pharmaceutical agents such as reagents. In addition, the above-mentioned composition may be constituted in the form of a whole or an independent individual. Examples of combinations of the plurality of or independent compositions include a combination of a composition containing the first beads and a composition containing the second beads. Here, the above-mentioned composition may also be a kit for use in a method for detecting non-destructive measurement information and genome-related information of a single cell as a whole. The kit may contain a buffer, a reagent required for a reverse transcription reaction or a PCR reaction, a cell lysis buffer, and other necessary reagents, instructions for use, and the like.

Detection agent comprising first bead

In another embodiment of the present invention, there is provided a detection agent comprising first microbeads for use together with second microbeads for collectively detecting nondestructive measurement information and genome-related information on a single cell, wherein the first microbeads are particles which are cleavably linked to first barcode nucleic acids corresponding to respective pieces of imaging information or organisms each containing the first barcode nucleic acids corresponding to respective pieces of imaging information, the second microbeads are linked to a plurality of second barcode nucleic acids which are capable of hybridizing with genome-related nucleic acids corresponding to a genome of a cell or an expression product thereof or the first barcode nucleic acids, and the detection agent is capable of detecting nondestructive measurement information on a single cell using as an index an expression pattern of a first amplification product derived from a hybrid complex formed between the first barcode nucleic acids and the second barcode nucleic acids, and detecting genome-related information of the individual cell using, as an indicator, an expression pattern of a second amplification product derived from a hybridization complex formed between the genome-related nucleic acid and a second barcode nucleic acid.

Detection agent comprising second bead

A detection agent comprising second microbeads for use with first microbeads for collectively detecting nondestructive measurement information and genome-related information on a single cell, wherein the first microbeads are particles which are cleavably linked to first barcode nucleic acids corresponding to respective imaging information or organisms each containing the first barcode nucleic acids corresponding to the respective imaging information, the second microbeads are linked to a plurality of second barcode nucleic acids which are capable of hybridizing with genome-related nucleic acids corresponding to a genome of a cell or an expression product thereof or the first barcode nucleic acids, the detection agent is capable of detecting nondestructive measurement information on a single cell using, as an index, an expression pattern of a first amplification product derived from a hybridization complex formed by the first barcode nucleic acids and the second barcode nucleic acids, and the detection agent is capable of detecting nondestructive measurement information on a single cell using, as an index, an expression pattern of a second amplification product derived from a hybridization complex formed by the genome-related nucleic acids and the second barcode nucleic acids The formula (II) is used as an index to detect genome-related information of a single cell.

Method for obtaining non-destructive measurement information and genome-related information for individual cells of a test substance

In addition, as another embodiment of the present invention, there is provided a method for obtaining nondestructive measurement information and genome-related information of a single cell for a test substance using the method for detecting nondestructive measurement information and genome-related information of a single cell of the present invention as a whole. The method for obtaining nondestructive measurement information and genome-related information on a single cell of a test substance is characterized in that the single cell or a substance derived therefrom, the first bead and the second bead are allowed to coexist together with the test substance in the compartment. The timing of allowing the analyte to coexist (for example, the timing of adding) may be before, during, or after the formation of the compartment. Examples of the analyte include low-molecular compounds, peptides, proteins, nucleic acids, and drugs such as viruses. The substance to be measured in each compartment may be the same or different. Alternatively, the concentration of the analyte may be the same or different in each compartment.

Furthermore, analysis such as screening of the test substance can be performed using the nondestructive measurement information and genome-related information for a single cell of the test substance obtained by the above-described method. Based on the obtained genome-related information and/or nondestructive measurement information of the cell, the response of the cell to the test substance (for example, changes in molecular expression, local presence, morphology, and differentiation) is measured, and based on the measurement result of the response, analysis such as screening of the test substance is performed.

The above-described composition, the detection agent containing the first bead, the detection agent containing the second bead, and the method of obtaining the detection agent can be performed according to the description of the detection method of the present invention.