EP4689158A1

EP4689158A1 - Analyzing method, circular nucleic acid manufacturing method, and nucleic acid

Info

Publication number: EP4689158A1
Application number: EP24718287.6A
Authority: EP
Inventors: Masahiro Matsumoto; Isao Hidaka; Noe SATO; Kenzo Machida
Original assignee: Sony Group Corp
Current assignee: Sony Group Corp
Priority date: 2023-03-30
Filing date: 2024-03-28
Publication date: 2026-02-11
Also published as: CN121443751A; WO2024204517A1

Abstract

Provided is an analyzing method including generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid, concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid, and performing analysis using the circular nucleic acid.

Description

ANALYZING METHOD, CIRCULAR NUCLEIC ACID MANUFACTURING METHOD, AND NUCLEIC ACID

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Japanese Priority Patent Application JP 2023-056365 filed March 30, 2023, the entire contents of which are incorporated herein by reference.

The present disclosure relates to an analyzing method, a circular nucleic acid manufacturing method, and a nucleic acid. More specifically, the present disclosure relates to an analyzing method for cell analysis, a circular nucleic acid manufacturing method used for cell analysis, and a nucleic acid used for cell analysis.

Single cell analysis is one of very useful methods for analyzing a cell or a component within the cell. The single cell analysis analyzes, for example, a nucleic acid, particularly an mRNA (messenger ribonucleic acid), included in each cell. A few techniques have thus far been proposed to perform the single cell analysis.

PTL 1 below discloses a bioparticle analyzing method including a capturing step of capturing a bioparticle via a bioparticle capturing portion at a surface to which a molecule including the bioparticle capturing portion, a barcode sequence, and a cleavable linker is fixed via the linker, a cleavage step of setting the bioparticle free from the surface by cleaving the linker, and an isolating step of isolating the bioparticle into a minute space.

PCT Patent Publication No. WO2022/009642

Summary Technical Problems

The single cell analysis may analyze a nucleic acid possessed by each cell. For the analysis, it is conceivable to add, to a target sequence, for example, a cell identifier sequence (for example, a Cell barcode) for identifying the origin of the nucleic acid, a molecule identifier sequence (for example, a molecular barcode, a Unique Molecular Identifier/UMI, or the like) for identifying the number of original molecules of the nucleic acid, a nucleic acid amplification sequence (for example, an amplification sequence, a PCR (polymerase chain reaction) handle), and the like.
However, at a time of sequencing the target sequence, these added sequences may also need to be read, so that the reading of the sequences other than the target sequence partially consumes a total number of reads (length x depth).
In addition, because PCR at a time of cDNA (complementary deoxyribonucleic acid) amplification is an exponential amplification, target nucleic acids originally present in large numbers in cells tend to be increased, and such target nucleic acids tend to be detected. This can cause a bias in an analysis result. That is, in the exponential amplification of nucleic acids, nucleic acids present in small numbers in cells are difficult to reflect in the analysis result.

It is accordingly desirable to shorten a sequence provided to a target nucleic acid. In addition, it is desirable to reduce the above-described bias in addition to the shortening of the sequence provided to the target nucleic acid.

Solution to Problems

The present disclosure provides an analyzing method including generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid, concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid, and performing analysis using the circular nucleic acid.
The nucleic acid concatenating portion may include a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid.
In the generating of the complementary strands, target nucleic acid complementary strand generation may be performed with the nucleic acid concatenating portion as a primer.
In the generating of the complementary strands, a double strand of each target nucleic acid and the complementary strand of each target nucleic acid may be formed.
In the generating of the circular nucleic acid, the double strand may be concatenated via the nucleic acid concatenating portion.
The nucleic acid concatenating portion may include a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand.
In the generating of the circular nucleic acid, a 5’ terminus of one complementary strand and a 3’ terminus of another complementary strand may be concatenated to each other, and the concatenation may be performed in a state in which a complementary strand capturing portion of the nucleic acid concatenating portion bound to the one complementary strand and a 3’ terminus region of the other complementary strand are bound to each other.
In the generating of the circular nucleic acid, a single stranded circular nucleic acid in which the complementary strands are concatenated to each other may be obtained by forming a double stranded circular nucleic acid and then removing the target nucleic acid from the double stranded circular nucleic acid.
In the analyzing, a nucleic acid amplification reaction using the circular nucleic acid may be performed.
The nucleic acid amplification reaction may be RCA (rolling circle amplification) or PCR.
The nucleic acid concatenating portion may include a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid, a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand generated in the generating of the complementary strands, and a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
The target nucleic acid capturing portion may have a poly-T sequence, and the complementary strand capturing portion may have a base sequence complementary to a base sequence provided to a 3’ terminus at a time of reverse transcription by a reverse transcriptase.
The double stranded portion may have a restriction enzyme recognition sequence.
The double stranded portion may have a non-natural base sequence.
The double stranded portion may have a base sequence having an error correcting function.
The analyzing method may be an analyzing method for performing single cell analysis, and the nucleic acid concatenating portion including the double stranded portion different for each cell may be used.
The analyzing method may include destroying a cell, and the generating of the complementary nucleic acid may be performed on the target nucleic acid included in the cell.
The destroying the cell may be performed within a space partitioned for each cell.
The nucleic acid concatenating portion may be immobilized on a substrate.
In addition, the present disclosure also provides a circular nucleic acid manufacturing method including generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid, and concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid.
In addition, the present disclosure also provides a nucleic acid including a target nucleic acid capturing portion configured to capture a 3’ terminus region of a target nucleic acid, a complementary strand capturing portion configured to capture a 3’ terminus region of a complementary strand produced by complementary strand generation of the target nucleic acid, and a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
The target nucleic acid capturing portion may be a single strand, and the complementary strand capturing portion may be a single strand.
In addition, the nucleic acid may be used to generate a circular nucleic acid.

FIG. 1A is a schematic diagram illustrating an example of a configuration of a nucleic acid concatenating portion. FIG. 1B is a diagram illustrating an example of sequence groups having an error correcting function. FIG. 1C is a diagram illustrating an example of configurations of the nucleic acid concatenating portion. FIG. 1D is a diagram illustrating an example of a configuration of the nucleic acid concatenating portion. FIG. 1E is a schematic diagram illustrating a modification of the configuration of the nucleic acid concatenating portion. FIG. 1F is a schematic diagram illustrating a modification of the configuration of the nucleic acid concatenating portion. FIG. 2A is a schematic diagram of assistance in explaining an example of a circular nucleic acid generating process using the nucleic acid concatenating portion. FIG. 2B is a schematic diagram of assistance in explaining mRNA structures. FIG. 2C is a schematic diagram of assistance in explaining an example of sequencing. FIG. 2D is a conceptual diagram of a nucleic acid concatenating portion and the like used in a first modification of the circular nucleic acid generating process. FIG. 2E is a schematic diagram of assistance in explaining the first modification of the circular nucleic acid generating process using the nucleic acid concatenating portion. FIG. 2F is a conceptual diagram of a nucleic acid concatenating portion and the like used in a second modification of the circular nucleic acid generating process. FIG. 2G is a schematic diagram of assistance in explaining the second modification of the circular nucleic acid generating process using the nucleic acid concatenating portion. FIG. 2H is a conceptual diagram of a nucleic acid concatenating portion and the like used in a third modification of the circular nucleic acid generating process. FIG. 2I is a schematic diagram of assistance in explaining the third modification of the circular nucleic acid generating process using the nucleic acid concatenating portion. FIG. 3 depicts schematic diagrams of assistance in explaining a bias in exponential amplification. FIG. 4A is a diagram illustrating an example of a restriction site. FIG. 4B is a schematic diagram of assistance in explaining an example of generating tagged short reads. FIG. 5 depicts schematic diagrams illustrating an example of operations performed in a single cell analyzing method. FIG. 6A is a schematic diagram illustrating an example of a configuration of a complex. FIG. 6B is a schematic diagram illustrating an example of a configuration of the complex. FIG. 6C is a schematic diagram illustrating an example of manufacturing the complex. FIG. 6D is a schematic diagram illustrating an example of operations performed in a modification of the single cell analyzing method. FIG. 6E is a conceptual diagram of a nucleic acid concatenating portion used in the modification of the single cell analyzing method. FIG. 7 depicts schematic diagrams illustrating an example of operations performed in the single cell analyzing method. FIG. 8 depicts schematic diagrams illustrating an example of operations performed in the single cell analyzing method. FIG. 9 depicts schematic diagrams illustrating an example of operations performed in the single cell analyzing method. FIG. 10 depicts schematic diagrams illustrating an example of operations performed in the single cell analyzing method. FIG. 11 depicts schematic diagrams illustrating an example of nucleic acids generated by RCA processing. FIG. 12 is a schematic diagram of an example of wells used to perform a particle isolating step. FIG. 13A is a schematic diagram illustrating an example of a device used to perform the particle isolating step. FIG. 13B is a schematic diagram illustrating an example of the device used to perform the particle isolating step. FIG. 14A is a schematic diagram of assistance in explaining an example of a device for forming an emulsion. FIG. 14B is a schematic diagram of assistance in explaining an example of a chip for forming the emulsion. FIG. 14C is a conceptual diagram of an antibody-nucleic acid molecule complex used when single cell analysis and analysis of a secreted molecule on a cell surface are performed simultaneously. FIG. 14D is a schematic diagram illustrating an example in which a cell to be subjected to the single cell analysis and the analysis of the secreted molecule on the cell surface are isolated on a chip for forming an emulsion. FIG. 14E is a schematic diagram illustrating an example of operations performed in the single cell analysis and the analysis of the secreted molecule on the cell surface. FIG. 15 depicts schematic diagrams of assistance in explaining the example of the chip for forming the emulsion. FIG. 16 is a block diagram of assistance in explaining the example of the device for forming the emulsion. FIG. 17 illustrates an example of a flowchart of operations performed for the emulsion. FIG. 18A is a schematic diagram illustrating an enlarged view of a connection flow passage and the vicinities thereof. FIG. 18B is a schematic diagram illustrating an enlarged view of the connection flow passage and the vicinities thereof. FIG. 19 is a schematic diagram illustrating an example of a case where priming sequences are provided.

Preferred modes for carrying out the present disclosure will hereinafter be described. It is to be noted that embodiments to be described in the following represent representative embodiments of the present disclosure, and that the scope of the present disclosure is not limited only to these embodiments. Incidentally, the description of the present disclosure will be made in the following order.
1. First Embodiment (Analyzing Method)
2. Second Embodiment (Circular Nucleic Acid Manufacturing Method)
3. Third Embodiment (Nucleic Acid)

1. First Embodiment (Analyzing Method)

(1) Basic Concept

(Problems)
When the sequence of an identifier provided to a target sequence is shortened, identifier variation is decreased, and the number of identifiers for analyzing a large number of cells and molecules becomes insufficient. An identifier including a single stranded DNA is produced by a combination of four bases of A/T/G/C, and the variation thereof is simply 4ⁿ (n = Identifier Length). In a case where 10⁶ identifiers are necessary, n = 10. Further, in actual analysis, an error correcting mechanism is often incorporated, so that n is further increased. In addition, simply, each time the length is shortened by one, the variation becomes 1/4. Hence, a method of shortening the length of the identifier is not desirable.

It is also conceivable to reduce the kinds of identifiers described above. However, each identifier has a role and is therefore difficult to simply exclude.
For example, in a case of eliminating a cell identifier (cell barcode), it is conceivable to analyze pooled cells by NGS (next generation sequencing). However, in this case, it is difficult to classify analyzed molecules for each cell. In addition, while it is conceivable to perform NGS for each cell, a high cost is involved in a case where a large number of cells are analyzed.
In a case of eliminating a molecule identifier (molecular barcode, UMI), it is necessary not to replicate molecules, or it is necessary for replicated molecules to have a common sequence. Not all mRNAs have different sequences, and multiple mRNAs having the same sequence occur. Therefore, in a case where replication is performed after cDNA synthesis, it is not possible to make a distinction between an original number and a number resulting from replication unless a sequence such as an UMI is provided.
In a case of eliminating an amplification sequence (amplification sequence, PCR handle), it is difficult, for example, to increase molecules converted to cDNA. In order to perform amplification without the amplification sequence, in a case where target sequences may need to be known, molecules of unknown sequences are not possible to be detected.
In addition, with an amplifying method using PCR as exponential amplification, molecules with a higher existence frequency tend to be increased, and molecules with a lower frequency do not increase easily. Hence, molecules with a high frequency tend to be detected easily.

(Outline of Present Disclosure)
An analyzing method according to an embodiment of the present disclosure uses a circular nucleic acid in which complementary strands of two or more kinds of target nucleic acid are concatenated to each other via a nucleic acid concatenating portion.
In the analyzing method, the nucleic acid concatenating portion can play a role as a cell identifier and can further play a role as a molecule identifier. Further, the nucleic acid concatenating portion can play a role as an amplification sequence. Further, the length of a base sequence constituting the nucleic acid concatenating portion can be shortened. That is, in the analyzing method according to an embodiment of the present disclosure, the length of a sequence added to a target nucleic acid can be shortened.
Here, the target nucleic acid refers to a nucleic acid to be captured by a nucleic acid concatenating portion to be described later.
In addition, in the circular nucleic acid, two or more kinds of target nucleic acid or a sequence derived from the target nucleic acid (including a sequence complementary to the sequence) are concatenated to each other. For example, one or more kinds of target nucleic acid present in a slight amount within cells or a sequence derived from the target nucleic acid and one or more kinds of target nucleic acid present in a large amount within cells or a sequence derived from the target nucleic acid may be concatenated to each other. When such a target nucleic acid with a low existence frequency and a target nucleic acid with a high existence frequency are concatenated to each other, a possibility of detecting the target nucleic acid with a low existence frequency can be increased. Consequently, the bias described in the above description can be reduced.

In one embodiment, the analyzing method according to an embodiment of the present disclosure includes a complementary nucleic acid generating step of generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid, and a circular nucleic acid generating step of concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid. The analyzing method may further include an analyzing step of performing analysis using the circular nucleic acid. In the analyzing method using the circular nucleic acid thus generated, the length of a sequence provided to the target nucleic acid can be shortened, as described above. Further, it is also possible to reduce the bias.
In the following, the analyzing method will be described with reference to the drawings.

(2) Example 1 (Bulk analysis)

(2-1) Nucleic acid concatenating portion
The nucleic acid concatenating portion used in the analyzing method according to an embodiment of the present disclosure will first be described. A part involved in the concatenation of two or more complementary strands of the nucleic acid concatenating portion will be referred to also as a concatenator in the present specification. An example of a configuration of the nucleic acid concatenating portion is illustrated in FIG. 1A. A nucleic acid concatenating portion 10 illustrated in the figure includes a target nucleic acid capturing portion 11 configured to capture a 3’ terminus region of a target nucleic acid, a complementary strand capturing portion 12 configured to capture a 3’ terminus region of a complementary strand of a target nucleic acid, and a double stranded portion 13 that connects the target nucleic acid capturing portion 11 and the complementary strand capturing portion to each other.
In addition, the nucleic acid concatenating portion used in the analyzing method according to an embodiment of the present disclosure can also adopt a configuration illustrated in FIG. 1E or FIG. 1F. As in the configuration example of FIG. 1A, the nucleic acid concatenating portions 10 illustrated in these figures include a target nucleic acid capturing portion 11 configured to capture a 3’ terminus region of the target nucleic acid, and a complementary strand capturing portion 12 configured to capture a terminus region of a sequence derived from the target nucleic acid such as a 3’ terminus region of a complementary strand of the target nucleic acid. On the other hand, the nucleic acid concatenating portions 10 illustrated in these figures do not have a double stranded portion 13. Incidentally, the nucleic acid concatenating portion 10 illustrated in FIG. 1F includes a sequence additional portion 14 in addition to the configuration of FIG. 1E.
In the present technology, by using the thus formed nucleic acid concatenating portion, it is possible to shorten a sequence introduced into the target nucleic acids or a circular nucleic acid having a sequence derived from the target nucleic acid and decrease a detection bias.

The target nucleic acid capturing portion 11 is, for example, configured to capture the 3’ terminus region of the target nucleic acid. In a case where the target nucleic acid is mRNA, for example, the mRNA has a poly-A tail portion in the 3’ terminus region. Hence, the target nucleic acid capturing portion 11 may be configured to capture the poly-A tail portion. In particular, the target nucleic acid capturing portion 11 may be a base sequence configured to capture the poly-A tail portion.

In order for the target nucleic acid capturing portion 11 to capture the poly-A tail portion, the target nucleic acid capturing portion may have a poly-T sequence, for example. The length of the poly-T sequence may, for example, be a length of 10 to 50 bases and may preferably be a length of 15 to 30 bases. That is, the poly-T sequence may, for example, include Ts of 10 to 50 bases and may preferably include Ts of 15 to 30 bases. The target nucleic acid capturing portion 11 may include only the poly-T sequence.
In addition, the target nucleic acid capturing portion 11 may be a single stranded DNA or RNA. This facilitates binding to the target nucleic acid, and particularly facilitates complementary binding.
Incidentally, the length of the poly-T sequence may be even longer or even shorter and may be changed according to the length of the poly-A tail portion of the target nucleic acid, for example.
In addition, in a case of an RNA not having the poly-T sequence or the sequence of a target RNA being known, a random sequence (a random primer, a random hexamer, or the like) or a sequence that specifically binds to the target RNA may be set as the target nucleic acid capturing portion. The length of the random sequence may, for example, be a length of 6 to 20 bases and may preferably be a length of 6 to 10 bases. The length of the sequence that specifically binds to the target RNA may be a length of 10 to 40 bases or may preferably be a length of 15 to 35 bases.

Incidentally, an example of sequences complementary to the target RNA are illustrated in Table 1 and Table 2 below. Table 1 illustrates an example of base sequences that specifically bind to ERBB2 (ERBB2_Probe1 to ERBB2_Probe19; these correspond to sequence ID No. 109 to 127). Table 2 illustrates an example of base sequences that specifically bind to XPO1 (XPO1_Probe1 to XPO1_Probe13; these correspond to sequence ID No. 128 to 140).
At least one base sequence that specifically binds to the target RNA may be included in the target nucleic acid capturing portion. In a case where there are multiple different target RNAs, at least one base sequence respectively selected from a group of sequences that specifically bind to each of the target RNAs may be included in the target nucleic acid capturing portion. In a case where there is one target RNA, at least one base sequence selected from a group of sequences that specifically bind to the one target RNA may be included in the target nucleic acid capturing portion.
The design of sequences that specifically bind to the target RNA can be made by a method similar to that of a PCR primer and a FISH (Fluorescence in situ hybridization) probe. To briefly describe an example thereof, a sequence group of all combinations of a set length is generated, and for each of sequences included in the sequence group, stability of binding to the target sequence is calculated by a Nearest neighbor method. A sequence group of, for example, -5 kcal/mol or less, preferably -28 kcal/mol or less, at 37°C is extracted from the sequence group. Further, a sequence group with a GC content of 40% to 60% is further extracted from the extracted sequence group. Further, sequences satisfying one or more additional filters (conditions) such as an A content of less than 28%, exclusion of successions of four bases or more, a C content of 22% to 28%, and the like are optionally extracted. A sequence group that specifically binds to the target RNA is thus selected.

The complementary strand capturing portion 12 is, for example, configured to capture the target nucleic acid or a terminus region of a nucleic acid having a sequence derived from the target nucleic acid (for example, 3’ terminus regions of complementary strands of these nucleic acids). In particular, in the analyzing method according to an embodiment of the present disclosure, the complementary strand capturing portion possessed by one nucleic acid concatenating portion can capture a 3’ terminus region of a complementary strand of a target nucleic acid (complementary strand produced by cDNA synthesis of the other target nucleic acid) other than a target nucleic acid captured by the target nucleic acid capturing portion of the one nucleic acid concatenating portion. It is thereby possible to concatenate two or more target nucleic acids or a complementary strand of a sequence derived from the target nucleic acid to each other via the nucleic acid concatenating portion.
Here, being “concatenated via the nucleic acid concatenating portion” is not limited to a case of concatenation via the nucleic acid concatenating portion, and includes a case of concatenation via a sequence derived from the nucleic acid concatenating portion.

In the analyzing method according to an embodiment of the present disclosure, a complementary nucleic acid generating step is a step of generating two or more complementary strands to be concatenated to each other in a next circular nucleic acid generating step. More specifically, in a state in which the nucleic acid concatenating portion is bound to one end of each of one or more kinds of target nucleic acid, complementary strands of the one or more kinds of target nucleic acid are generated.
Incidentally, as will be described later, the complementary nucleic acid generating step may generate nucleic acids having a sequence derived from the target nucleic acid with the generated complementary strands of the target nucleic acid as a template, and may provide a sequence to be captured by the complementary strand capturing portion included in the nucleic acid concatenating portion.
For example, in a case where a target nucleic acid is mRNA, a complementary strand produced by the reverse transcription of the mRNA has a CCC sequence (C: cytosine) generated at a 3’ terminus thereof by a reverse transcriptase that performs the reverse transcription. Accordingly, the complementary strand capturing portion 12 may be configured to capture the CCC sequence. In particular, the complementary strand capturing portion 12 may be a base sequence configured to capture the CCC sequence.
Thus, the complementary strand capturing portion may have a base sequence complementary to the base sequence provided to the 3’ terminus at a time of the reverse transcription by the reverse transcriptase.
On the other hand, in a case of using the nucleic acid concatenating portion not having the double stranded portion, the complementary strand capturing portion included in the nucleic acid concatenating portion may be an optional sequence, and a nucleic acid having a sequence derived from the target nucleic acid may be generated by using a primer having, on a 5’ side, a sequence complementary to the sequence of the complementary strand capturing portion. In this case, the complementary strand capturing portion of the nucleic acid concatenating portion located at the 5’ terminus of a complementary strand (cDNA) generated by the reverse transcription of an mRNA can capture the sequence complementary to the sequence of the above-described complementary strand capturing portion which complementary sequence is derived from the primer present at the 5’ terminus of a nucleic acid having a sequence derived from another target nucleic acid. The two or more complementary strands are thus concatenated to each other via the nucleic acid concatenating portion.
In a case of using such a nucleic acid concatenating portion, the sequence of the complementary strand capturing portion is not limited to a GGG sequence, and can be formed as a sequence of an optional length by combining four bases of A, T, C, and G. In a case where the sequence of the complementary strand capturing portion is lengthened, it is possible to form a circular nucleic acid while reducing failures in capturing the generated complementary strands when the circular nucleic acid is formed by concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion. It can thus be expected that a probability of being able to detect even a complementary strand derived from the sequence of an mRNA occurring with a low frequency, for example, will be increased and that analysis accuracy will be consequently improved.

The complementary strand capturing portion 12 that captures the CCC sequence, for example, includes a GGG sequence. The GGG sequence may be DNA or may be RNA. That is, the GGG sequence may be GGG or rGrGrG (r: ribonucleotide, G: guanine).
In addition, the complementary strand capturing portion 12 may be a single stranded DNA or RNA. This facilitates binding to the complementary strand.

In addition, the complementary strand capturing portion may include a self-bonding suppressing sequence Hn or Nn in addition to the GGG sequence. Here, H is a base other than G, that is, H is A, T, or C. N is A, T, G, or C. n is the number of Hs or Ns and may, for example, be an integer of 1 or more. n may, for example, be an integer of any of 1 to 8. In a case where there are said to be approximately 20,000 kinds of mRNAs, the mRNAs can be covered by such a numerical range. In some embodiments, n may, for example, be 1, 2, 3, 4, or 5 and may further be 1 or 2. In a case where n is 2 or more, each H or N constituting the self-bonding suppressing sequence may be selected independently and randomly. The complementary strand capturing portion may, for example, have a base sequence of GGGH, GGGN, GGGHN, GGGNH, GGGHH, or GGGNN. The complementary strand capturing portion may be DNA or may be RNA.

The double stranded portion 13 is a part that connects the target nucleic acid capturing portion 11 and the complementary strand capturing portion 12 to each other. The double stranded portion 13 may be formed by a double stranded DNA, may be formed by a double stranded RNA, or may be formed by a hybrid of DNA and RNA. Preferably, the double stranded portion 13 is DNA. This prevents decomposition in RNA digestion processing to be described later and facilitates the formation of the circular nucleic acid. In addition, the double stranded portion 13 is easily used as a primer in nucleic acid amplification.

The target nucleic acid capturing portion 11 is concatenated to the 3’ terminus of one strand of the double stranded portion 13. The complementary strand capturing portion 12 is concatenated to the 3’ terminus of the other difference of the double stranded portion 13. Because such a structure is provided, a circular nucleic acid can be formed by concatenating generated complementary strands, as will be described later.

The double stranded portion 13 may include a sequence including a random combination of four bases of A, T, C, and G. The random sequence preferably includes a sequence group having an error correcting function. Sequence groups having the error correcting function include, for example, a Sequence-Levenshtein code and filled/truncated right end edit (FREE) barcodes. A method of generating these sequence groups and an error correcting mechanism using these sequence groups are described in Buschmann and Bystrykh BMC Bioinformatics 2013, 14: 272 and Proc Natl Acad Sci U S A. 2018 Jul 3; 115 (27): E6217-E6226. Those skilled in the art can generate and use a sequence group having the error correcting function as appropriate by referring to these pieces of literature. In addition, other than the above-described two kinds of sequence groups, sequence groups having the error correcting function known in this technical field are known, such as Levenshtein codes, Hamming codes, and Reed-Solomon codes, and any one of these may be used in the present disclosure. Incidentally, the sequence group having the error correcting function can be referred to also as Indel-correcting DNA barcodes. In addition, software that can be used for the generation of these sequence groups having the error correcting function and error correction using the sequence groups is also known to those skilled in the art. Those skilled in the art can prepare and use a sequence group having the error correcting function by using such software. Thus, a sequence group is extracted which enables an original sequence to be identified even when a readout error (insertion, deletion, or substitution) of a few bases occurs at a time of sequencing.

An example of mechanisms of sequences having the error correcting function and a generating method thereof will be described in the following.

Mechanisms of sequences having the error correcting function will be described in the following. However, the following description represents typical examples, and sequences having the error correcting function applicable in the present disclosure do not have to be limited to sequences based on these mechanisms.
With a Hamming distance, the distance is considered to be increased by one when one in a barcode sequence is replaced (replacement: substitution). For example, “TCT” in which one in “ACT” is replaced has a distance of 1 (substitution of T for A), there is a distance of 1 between “TCT” and “TAT” (substitution of A for C), and there is a distance of 1 between “TAT” and “TAC” (substitution of C for T). In a case of “ACT” and “TAC,” there is a distance of 3, which is obtained as a sum. In a case where “ACT,” “GTG,” “TAC,” and “CGA” are prepared as barcodes, for example, these have a mutual distance of 3. In a case where a result read by sequencing is “ACG,” “ACG” is different from the prepared barcodes, and therefore, an error is considered to have occurred. The distance is used to estimate which sequence has an error. The distances between the read “ACG” and the prepared four kinds of sequences are calculated. A most likely barcode is estimated to be “ACT,” which has a shortest distance, on an assumption that error frequencies are uniform. Thus, it is possible to detect a replacement of a code having a hamming distance with another code and make a correction. A length of at least 2*k + 1 may be needed to correct k errors.
A Levenshtein distance makes it possible to deal with not only substitution but also insertion and deletion. The distance is considered to be increased by 1 for a substitution, an insertion, or a deletion of a base. For example, it is considered that there is a distance of 1 between “GCG” and “GC” (deletion of G), that there is a distance of 1 between “GC” and “GA” (substitution of A for C), and that there is a distance of 1 between “GA” and “AGA” (insertion of A). A distance between “GCG” and “AGA” is 3 as a sum total. For example, a sequence group of “GCG,” “TTT,” “AGA,” and “CAC,” which have a mutual distance of 3, is prepared as barcodes. In a case where a result read by sequencing is “GC,” “GC” is different from the prepared barcodes, and therefore, an error is considered to have occurred. Distances between the obtained “GC” and the prepared barcodes are calculated. A sequence having a shortest distance is estimated as a most likely barcode. In the example described above, “GCG” having a distance of 1 is estimated to be the barcode. This method is applicable only in cases where a barcode length is known in advance.
A Sequence-Levenshtein distance makes it possible to deal with an optional length by regarding a distance between barcodes A and B of an optional length as the number of times of substitution, insertion, and deletion, and once performing, for A obtained by these operations, either reducing A to the same length as B or adding the same base until A has the same length as B.
FREE (filled/truncated right end edit barcodes) corrects substitution, insertion, and deletion on the basis of a Needleman-Wunsch algorithm. Two sequences for which alignment is desired to be performed are arranged vertically and horizontally as in a checkerboard. The two sequences are compared with each other. In a case where bases at each position match each other (match), for example, +2 is set. In a case where the bases do not match each other (mismatch), -1 is set. In a case where there are no bases (gap), -2 is set. An alignment is generated when a calculation is performed for each cell and thereafter arrows are traced back in decreasing order of scores. In a case where “ATTGC” and “ATGC” are compared with each other, for example, “ATTGC” is aligned as “AT-GC” (deletion or insertion of T).
As in the examples illustrated above, according to an embodiment of the present disclosure, a sequence having the error correcting function may be a base sequence configured to be able to detect an error in a case where the error has occurred in a base sequence identified by sequencing. The sequence having the error correcting function may, for example, be configured to be able to detect a difference between the sequence identified by sequencing and the sequence including a barcode prepared in advance. The barcode prepared in advance may, for example, be a sequence identified on the basis of a distance related to a variation (for example, substitution, insertion, deletion, or the like). The distance may be a distance based on a predetermined rule, such as the Hamming distance, the Levenshtein distance, or the Sequence-Levenshtein distance, as described in the above description. In addition, the barcode prepared in advance may, for example, be configured to be able to identify a variation by alignment as in FREE described in the above description. Particularly preferably, the sequence having the error correcting function may be configured to, in a case where an error has occurred in the sequence, be able to detect the error and be able to estimate a most likely barcode. The estimation may be performed on the basis of a predetermined rule according to the kind of the sequence having the error correcting function, as described in the above description. For example, as described in the above description, the estimation may be performed on the basis of a distance between the sequence identified by sequencing and the barcode prepared in advance, or the estimation may be performed on the basis of alignment between the sequence identified by sequencing and the barcode prepared in advance.

Methods of generating sequences having the error correcting function will be described in the following. However, the following description represents typical examples, and sequences having the error correcting function applicable in the present disclosure do not have to be limited to sequences generated by these generating methods.
Conway’s lexicographic code algorithm, for example, is performed in order to generate sequences having the error correcting function. This algorithm generates all sequences for a set length, and thereafter selects sequences having a mutual distance d. First, one sequence is unconditionally selected from a sequence group. Next, distances to the selected sequence are calculated for the remaining sequence group. A sequence having a distance less than d is excluded. A sequence having a distance equal to or more than d is selected. When similar processing is performed on the remaining sequence group, sequences whose distances to the selected sequence are equal to or more than d are selected. Similar processing is performed on all of sequence groups until there are no alternatives.
Thus, sequences having the error correcting function may be sequences generated by performing a predetermined algorithm. The predetermined algorithm may be an algorithm that selects sequences satisfying a predetermined criterion related to distances. Examples of the distances are as described in the above description. The predetermined criterion may be selected according to the kind of the sequences having the error correcting function which sequences are to be used.
In addition, sequences having the error correcting function may, for example, be generated by a dictionary type code generating method. In the generating method, sequences of a set length are generated in alphabetical order, and when a newly generated sequence does not overlap decoded sequences of a candidate sequence, the newly generated sequence is registered as a valid sequence. For example, when consideration is given to a case of correcting m errors of “CTCA,” barcodes (ex. CTGA, CCA, CTGC, ...) when m or less insertions/losses/substitutions have occurred are stored in a decode sphere DecodeSphere (“CTCA”) of “CTCA.” When another barcode candidate having a length of four bases, for example, a barcode candidate “AACC” comes, and a DecodeSphere (“AACC”) does not overlap the DecodeSphere (“CTCA”), AACC is registered as a valid barcode. There is limitation to the number m of correctable errors and a barcode sequence length n, and at least a length of 2*m + 1 may be needed.
Thus, sequences having the error correcting function may be sequences generated by the dictionary type code generating method. In the generating method, as described above, a barcode is registered on the basis of the presence or absence of overlapping of barcodes stored in a decode sphere, and a sequence having the error correcting function may be generated from the registered barcode.

Each sequence included in a sequence group having the error correcting function may, for example, be a sequence that is not complementary to existing RNA. For example, each sequence included in the sequence group having the error correcting function may have a guanine and cytosine content of 40% to 60%, for example, from a viewpoint of an improvement in sequencing efficiency. In addition, from the viewpoint described above, each sequence included in the sequence group having the error correcting function may be a sequence that does not have three or more consecutive homopolymer sequences. Further, from the viewpoint described above, each sequence included in the sequence group having the error correcting function may be a sequence that does not have a self-complementary sequence of two or more bases. Sequences at least satisfying such conditions may be used as sequences included in the sequence group having the error correcting function. The conditions to be satisfied by each sequence included in the sequence group having the error correcting function may be changed as appropriate according to the kind of the sequence.
In the present specification, sequences having the error correcting function may be represented by “(N)i” (where N is an optional base (A, T, G, or C), and i is the number of bases) and may have the error correcting function. i may, for example, be 3 or more, 4 or more, or 5 or more, may further be 10 or more, and may further be 15 or more. In addition, i may, for example, be 200 or less, 150 or less, or 100 or less, and may further be 50 or less. The length of the sequences having the error correcting function may be changed as appropriate according to the number of sequences (number of barcodes) to be prepared. In order to extract one million kinds or more of sequence groups having a function of correcting an error of one base, for example, the double stranded portion (sequence having the error correcting function in particular) may need to have a length of 16 bases or more. Sequence groups illustrated in FIG. 1B, for example, are cited as an example of sequence groups having the error correcting function. SEQ ID No. 1 to 50 illustrated in the figure indicate an example of sequences having a 16mer error correcting function. SEQ ID No. 51 to 100 indicate an example of sequences having a 17mer error correcting function. Incidentally, it is to be understood that where there are one million kinds or more of sequence groups having the error correcting function, as described above, in a case of 16mers, for example, the figure illustrates an example of a very small part of the sequence groups. The sequence groups listed in the figure have a left side end as a 3’ terminus and have a right side end as a 5’ terminus.
In addition, two or more of these extracted sequence groups having the error correcting function can be combined with each other to thereby increase the kinds. Sequences having the error correcting function can be generated as appropriate by those skilled in the art, and the sequences include sequences generated by the above-described generating methods, for example. However, the sequences having the error correcting function may not need to be limited to these. That is, the double stranded portion may have two kinds or more of sequences having the error correcting function.

As will be described in an example of the analyzing method to be described later, random sequences described earlier may be different from each other for each region in which the nucleic acid concatenating portion is disposed. That is, in the analyzing method, where multiple regions in which nucleic acid concatenating portions are arranged are used, the random sequences possessed by multiple nucleic acid concatenating portions arranged in one region each have the same base sequence, but the base sequences of random sequences may be different from each other between regions. Thus, the random sequence described earlier can be used as an identifier or can, for example, be used as a cell identifier and can further be used also as a positional information identifier.

Preferably, of the two base sequence strands constituting the double stranded portion 13, the strand coupled to the target nucleic acid capturing portion 11 (strand having a poly-T in particular) has a 5’ terminus thereof modified by phosphorylation. This makes it possible to perform ligation processing to be described later more reliably.
Preferably, of the two base sequence strands constituting the double stranded portion 13, the strand not coupled to the target nucleic acid capturing portion 11 (strand not having a poly-T in particular) has a 3’ terminus to which the complementary strand capturing portion is coupled.

The double stranded portion may preferably have a non-natural base sequence. In particular, the random sequence may include a non-natural base sequence. A non-natural base sequence refers to a base sequence that does not occur naturally. Those skilled in the art can design such a base sequence as appropriate. By using the non-natural base sequence, it is possible to suppress, for example, unnecessary double strand formation and a sequence detection error.

The double stranded portion 13 may further include a priming sequence. The priming sequence may, for example, be a base sequence that functions as a primer in nucleic acid amplification processing to be described later. The sequence can be selected as appropriate by those skilled in the art according to, for example, the kind of the nucleic acid amplification processing and/or the kind of enzyme used in the nucleic acid amplification processing or the like.
As described in the above description, in the analyzing method according to an embodiment of the present disclosure, where multiple regions in which nucleic acid concatenating portions are arranged are used, the priming sequences possessed by multiple nucleic acid concatenating portions arranged in one region each have the same base sequence. Further, the priming sequences may be the same also between regions. Consequently, amplification reaction occurs simultaneously from multiple circular nucleic acids having nucleic acid concatenating portions having the same priming sequence at one time of amplification processing.

In a case where the nucleic acid concatenating portion includes a sequence additional portion 14 as in the example illustrated in FIG. 1F, the sequence additional portion 14 is disposed between the target nucleic acid capturing portion 11 and the complementary strand capturing portion 12. The sequence additional portion 14 may include a DNA, may include an RNA, or may include a hybrid of a DNA and an RNA.
The sequence additional portion 14 may include a sequence constituted by a random combination of four bases of A, T, C, and G. The sequence additional portion 14 may, for example, be provided with the random sequence as a cell identifier sequence (Cell barcode) for identifying the source of a nucleic acid. In addition, the sequence additional portion 14 may be provided with a sequence group having functions that can be provided to the double stranded portion described above.

The nucleic acid concatenating portion 10 (double stranded portion 13 or sequence additional portion 14 in particular) may include a UMI or may not include a UMI. When no UMI is included, the base sequence of the nucleic acid concatenating portion can be shortened.

An example of configurations of the nucleic acid concatenating portion 10 is illustrated in FIG. 1C.
Nucleic acid concatenating portions (corresponding to sequence ID NO. 101 to 104) illustrated in a1 and a2 of the figure have a poly-T sequence (on a left side of the figure) as the target nucleic acid capturing portion and have a GGG sequence (on a right side of the figure) as the complementary strand capturing portion and have random sequences illustrated in the figure as the double stranded portion.
Nucleic acid concatenating portions (corresponding to sequence ID NO. 105 to 108) illustrated in b1 and b2 of the figure have a poly-T sequence (on the left side of the figure) as the target nucleic acid capturing portion and have a GGG sequence (on the right side of the figure) as the complementary strand capturing portion, and have, as the double stranded portion, priming sequences (underlined parts) in addition to random sequences. The priming sequences are a priming site in the nucleic acid amplification processing for the circular nucleic acid to be described later. For example, in a case where the nucleic acid amplification processing is RCA, priming sequences for RCA may be adopted, and in a case where the nucleic acid amplification processing is PCR, priming sequences for PCR may be adopted. The base sequences of these priming sequences can be selected as appropriate by those skilled in the art, as described in the above description.
That is, in one embodiment, as illustrated in FIG. 1D, the nucleic acid concatenating portion 10 may have a first strand in which the target nucleic acid capturing portion (capture sequence) and a random sequence (random) are concatenated to each other and a second strand in which a GGG sequence (or the above-described GGG (Hn and/or Nn) sequence) and a random sequence (random’) complementary to the random sequence are concatenated to each other, and the nucleic acid concatenating portion 10 may be configured as a nucleic acid in which the first strand and the second strand are complementarily bound to each other by the two complementary random sequences.
The target nucleic acid capturing portion may include a poly-T sequence, for example, as described in the above description, may include a random sequence (a random primer, a random hexamer, or the like), or may include a sequence (target primer) that specifically binds to a sequence target RNA that specifically binds to a target RNA.
The two random sequences complementarily bound to each other correspond to the double stranded portion described in the above description. The GGG sequence corresponds to the complementary strand capturing portion described in the above description. With regard to the first strand, there is a 5’ terminus on the random sequence side, and there is a 3’ terminus on the poly-T sequence side. With regard to the second strand, there is a 5’ terminus on the random sequence side, and there is a 3’ terminus on the GGG sequence side.

(2-2) Circular Nucleic Acid Generating Process
In the following, with reference to FIG. 2A and FIG. 2B, description will be made of a circular nucleic acid generating process using the nucleic acid concatenating portion, and further description will be made of nucleic acid amplification using the circular nucleic acid. More specifically, in the circular nucleic acid generating process illustrated in the figure, reverse transcription of two kinds of mRNAs is performed to generate two kinds of cDNAs complementary to the respective mRNAs, and then these two kinds of cDNAs are concatenated to each other to generate a circular nucleic acid. Steps performed in the circular nucleic acid generating process are as follows. Incidentally, while two kinds of different mRNAs are concatenated to each other in the present example, two or more identical mRNAs may be concatenated to each other in the present disclosure.

As illustrated in an upper part of FIG. 2A, a case is assumed in which there are two kinds of mRNAs (mRNA1 and mRNA2). mRNA1 and mRNA2 are target nucleic acids.

As illustrated in FIG. 2B, mRNA1 includes a translation region CDS1 and further includes a poly-A sequence polyA1 at a 3’ terminus thereof. Similarly, mRNA2 includes a translation region CDS1 and further includes a poly-A sequence polyA2 at a 3’ terminus thereof.

In step S11 illustrated in FIG. 2A, the 3’ terminus of mRNA1 is captured by the nucleic acid concatenating portion 10. Where the 3’ terminus of mRNA1 has a poly-A tail portion, the poly-T sequence 11 of the nucleic acid concatenating portion captures the poly-A tail portion, or in particular, complementarily binds to the poly-A tail portion. The 3’ terminus of the mRNA is thereby captured by the nucleic acid concatenating portion (target nucleic acid capturing portion in particular).
As with mRNA1, the 3’ terminus of mRNA2 is also captured by a nucleic acid concatenating portion (target nucleic acid capturing portion in particular).

In step S12 in the figure, cDNA of mRNA1 is synthesized. The synthesis of the cDNA may be performed by a reverse transcriptase, for example. The cDNA synthesis is performed with the nucleic acid concatenating portion as a primer. Consequently, as illustrated in the figure, a hybrid H1 of mRNA1 and cDNA1 is formed.
As for mRNA2, similarly, cDNA2 is synthesized by a reverse transcriptase, and thereby a hybrid H2 of mRNA2 and cDNA2 is formed.
In step S2, in the case where the cDNA synthesis is performed by the reverse transcriptases, a CCC sequence is formed at the 3’ terminuses of the synthesized cDNAs, as illustrated in the figure. As will be described later, this CCC sequence is used in the following circular nucleic acid formation.
Thus, in the method according to an embodiment of the present disclosure, a complementary nucleic acid generating step of generating complementary strands of one or more kinds of target nucleic acid may be performed in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid.
In the complementary strand generating step, the complementary strands of the target nucleic acid may be generated with the nucleic acid concatenating portions as a primer. In the complementary strand generating step, a double strand of each target nucleic acid and the complementary strand of each target nucleic acid may be formed, and thus a hybrid of mRNA and cDNA, for example, may be formed, as described above.

In step S13 in the figure, the hybrid H1 and the hybrid H2 generated in step S2 are concatenated to each other. The concatenation is a concatenation based on complementary binding between the rGrGrG sequence of the hybrid H1 and the CCC sequence of the hybrid H2.

In step S14 in the figure, a single stranded circular nucleic acid is formed by using the hybrid H1 and the hybrid H2. In order to form the single stranded circular nucleic acid, specifically, the following steps are performed.

First, the hybrid H1 has a CCC sequence at an end on an opposite side of an end at which the rGrGrG sequence used for the concatenation in step S13 is present. This CCC sequence is a single stranded part. The hybrid H2 has a rGrGrG sequence at an end on an opposite side from an end at which the CCC sequence used for the concatenation in step S13 is present. This rGrGrG sequence is also a single stranded part. Hence, the CCC sequence of the hybrid H1 and the rGrGrG sequence of the hybrid H2 are complementarily bound to each other, thereby forming a double stranded circular nucleic acid.
Thus, in a circular nucleic acid generating step included in the method according to an embodiment of the present disclosure, the double strands may be concatenated to each other via the nucleic acid concatenating portion.
Further, in the circular nucleic acid generating step, a 5’ terminus of one complementary strand and a 3’ terminus of another complementary strand are concatenated to each other, and the concatenation may be performed in a state in which a complementary strand capturing portion of the nucleic acid concatenating portion bound to the one complementary strand and a 3’ terminus region of the other complementary strand are bound to each other.

There is a nick between the 5’ terminus of the strand connected to cDNA1 in the double strand of the nucleic acid concatenating portion bound to mRNA1 and the 3’ terminus of cDNA2 generated by the reverse transcription of mRNA2 in the double stranded circular nucleic acid.
Similarly, there is also a nick between the 5’ terminus of the strand connected to cDNA2 in the double strand of the nucleic acid concatenating portion bound to mRNA2 and the 3’ terminus of cDNA1 generated by the reverse transcription of mRNA1 in the circular nucleic acid.
Accordingly, in step S14, ligation that eliminates these nicks are performed in a state in which the double stranded circular nucleic acid is formed. Consequently, cDNA1 and cDNA2 are set in a bound state, and the state of the circular nucleic acid is maintained even when mRNA degradation to be described later is performed.

In step S14, after the ligation, mRNA degradation processing is performed. The degradation processing may be performed by using, for example, RNase, particularly RNase H. Consequently, as illustrated in the figure, a single stranded circular nucleic acid RN is formed.
Thus, in the circular nucleic acid generating step, the double stranded circular nucleic acid may be formed, and then the target nucleic acid (for example, mRNA) may be removed from the double stranded circular nucleic acid to provide the single stranded circular nucleic acid in which the complementary strands are concatenated to each other.

In step S15, a primer is added to the single stranded circular nucleic acid RN. In the figure, a primer is used which is configured to be bound to a part that has constituted the double stranded portion in the nucleic acid concatenating portion and the CCC sequence. That is, the primer is configured to cover a ligation point.

In step S16, RCA using a DNA polymerase is performed with the primer as a starting point. The DNA polymerase, for example, thus synthesizes single stranded linear nucleic acids complementary to the single stranded circular nucleic acid.

The base sequences of the thus formed single stranded linear nucleic acids are decoded. The decoding may be performed by a known technology in this technical field.
A Long Read Sequence technology, for example, may be applied as such a sequence analyzing technology. For example, the above-described single stranded cDNA is sequenced by nanopore sequencing (Oxford Nanopore Technologies plc).
In addition, the base sequences may be sequenced by HiFi sequencing (Pacific Biosciences, Inc.). In this case, as illustrated in FIG. 2C, a double stranded structure is generated after a step is performed which provides a single stranded cDNA extended by RCA with a poly-A tail portion or a poly-C tail portion. Sequencing is performed on a circular nucleic acid obtained by further providing an SMRTbell (registered trademark) adapter to the nucleic acid of the double stranded structure. Here, the sequence of the nucleic acid concatenating portion is known. Therefore, by demarcating the sequenced sequence by the known nucleic acid concatenating portion after the sequencing, it is possible to identify original mRNA sequences and count the number of original mRNAs. By decoding the mRNA sequences that occur repeatedly, it is possible to count molecules while regarding molecules produced by replication as the same even in a case where multi-RCA (multi-RCA) is performed. That is, identification as to whether the number is the number of original mRNAs or the number produced by replication can be made without the use of UMIs. In addition, in a case where rGrGrGNN or GGGNN is used, the identification can be made according to a location of random NN occurrence.
In addition, by performing long read sequencing of mRNA complementary sequences that occur repeatedly, it is possible to correct a readout error even when the error occurs at a time of the sequencing. That is, the error correction is made possible by concatenating two or more mRNAs, as described above. This can bring about an improvement in decoding accuracy.

In a case where rGrGrG is used as the complementary strand capturing portion of the nucleic acid concatenating portion, this rGrGrG is degraded by RNaseH processing. In addition, also in a case where the complementary strand of the double stranded portion in the nucleic acid concatenating portion (that is, the strand connected to the complementary strand capturing portion) is synthesized by a ribonucleotide, the complementary strand is degraded by the RNaseH processing. Therefore, in the above-described step S15, (that is, before RCA in step S16), a primer is added. The primer may, for example, be a primer having the same sequence as the complementary strand of the double stranded portion.
In addition, in a case where GGG is used as the complementary strand capturing portion and the complementary strand of the double stranded portion in the nucleic acid concatenating portion is synthesized by a nucleotide, these are not degraded by the RNaseH processing. In this case, the GGG and the complementary strand may be used as a primer.

In the example illustrated in FIG. 2A described above, the circular nucleic acid is formed by using two mRNAs. However, the circular nucleic acid may be formed by using three or more mRNAs. That is, a circular nucleic acid including cDNAs complementary to the three or more mRNAs, respectively, may be similarly formed. Then, the nucleic acid amplification processing such as RCA may be performed on the circular nucleic acid, as described in the above description.

When the circular nucleic acid is generated as illustrated in FIG. 2A described above, two or more cDNAs are concatenated to each other in the circular nucleic acid, and a cDNA corresponding to a low-expressed mRNA and a cDNA corresponding to a highly expressed mRNA, for example, can be concatenated to each other. It is thereby possible to improve efficiency of detection of the low-expressed mRNA.
In addition, in a case of a PCR method, the nucleic acid is exponentially amplified. Thus, the amplification amplifies an mRNA present in large numbers before the amplification much more than an mRNA present in small numbers before the amplification. Consequently, the mRNA present in small numbers before the amplification is not easily detected. That is, the PCR method brings about a bias due to the amplification (which bias will be referred to also as an amplification bias).
On the other hand, nucleic acid amplification based on an RCA method is not an exponential amplification and therefore reduces the bias. That is, in one embodiment, nucleic acid amplification using the circular nucleic acid may be performed by the RCA method.

Incidentally, the PCR method may be used in some embodiments of the analyzing method according to an embodiment of the present disclosure. This is because, even in a case of using the PCR method, the bias can be reduced by performing PCR on the circular nucleic acid. This will be described in the following.
Generally, after cDNA synthesis, a tag is attached to the nucleic acid amplified in the PCR method. In the PCR method, where a sequence is generated exponentially, a Unique Molecular Identifier (UMI) is generally provided at a time of reverse transcription in order to identify the origin of the generated sequence. Here, the number of kinds of UMIs may need to be equal to or more than the number of kinds of molecules to be distinguished (for example, the number of kinds of molecules originally possessed by each cell). Therefore, the UMIs generally include the number of bases (nucleotides) which is equal to or more than 6 (which may, for example, be 6 to 10 or may be equal to or more than 10). The sequences of the UMIs are decoded at a time of sequencing. However, these sequences are sequences not included in the target nucleic acid and are sequences that originally may not need to be subjected to sequence decoding. It is therefore preferable to be able to exclude these sequences.
In addition, the PCR method exponentially amplifies the nucleic acid and therefore causes a bias in that molecules expressed in large numbers tend to increase and those molecules tend to be consequently detected. As illustrated in A of FIG. 3, for example, in a case where the PCR method is performed for a sample in which there is one target nucleic acid M1, while the number of target nucleic acids M2 is m (7 in the figure), a reverse transcription product of the target nucleic acid M1 is amplified to 2ⁿ by the amplification, while the target nucleic acids M2 are amplified to (2ⁿ) × m.
In a case where the PCR method is performed for a nucleic acid in which two or more target nucleic acids are concatenated to each other according to an embodiment of the present disclosure, a molecule expressed in small numbers, for example, is concatenated to a molecule expressed in large numbers, and the PCR method is performed for the concatenated nucleic acid. An improvement in efficiency of detection of the low-expressed molecule is thereby expected. For example, a case is assumed in which concatenation processing according to an embodiment of the present disclosure is performed for the sample described with reference to A of FIG. 3, thereby, for example, producing a concatenation product in which one reverse transcription product of the target nucleic acid M1 and three reverse transcription products of the target nucleic acids M2 are concatenated to one another, and producing a concatenation product in which four reverse transcription products of the target nucleic acids M2 are concatenated to one another, as illustrated in B of FIG. 3. In this case, each concatenation product is amplified to 2ⁿ. Therefore, as compared with the case of A of FIG. 3, a difference between the number of amplification products of the target nucleic acid M1 and the number of amplification products of the target nucleic acids M2 is reduced.
Thus, the analyzing method according to an embodiment of the present disclosure reduces the amplification bias also in the case of using the PCR method rather than the RCA method.
As described above, the method according to an embodiment of the present disclosure may include an analyzing step of performing analysis using the circular nucleic acid. In the analyzing step, nucleic acid amplification reaction using the circular nucleic acid may be performed. The nucleic acid amplification reaction may be RCA or PCR, for example.

(2-3) First Modification of Circular Nucleic Acid Generating Process
The circular nucleic acid that can be used in the analyzing method according to an embodiment of the present disclosure is not particularly limited as long as the circular nucleic acid is generated by concatenating the two or more complementary strands generated by the complementary nucleic acid generating step described above to each other via the nucleic acid concatenating portion. Optional methods can be combined with each other to generate the circular nucleic acid.

A modification of the circular nucleic acid generating process will be described in the following with reference to drawings. It is to be noted that the circular nucleic acid generating process described in the present specification is an example thereof, and is not limited to modes described in the present specification.

FIG. 2D is a conceptual diagram of a nucleic acid concatenating portion and the like used in a first modification of the circular nucleic acid generating process.
In FIG. 2D, <2D-α> is a nucleic acid concatenating portion used in the first modification of the circular nucleic acid generating process. <2D-β> is a primer used when a nucleic acid having a sequence derived from a target nucleic acid is generated in the circular nucleic acid generating process according to the present modification. <2D-γ> is an RNA oligomer for protecting the sequence of a complementary strand capturing portion included in the nucleic acid concatenating portion when the nucleic acid having the sequence derived from the target nucleic acid is generated.

The nucleic acid concatenating portion of <2D-α> includes a target nucleic acid capturing portion (poly-T in the figure) capable of capturing a poly-A sequence present at the 3’ terminus of an mRNA and a complementary strand capturing portion (concatenator in the figure). Incidentally, though not depicted in the example illustrated in FIG. 2D, the nucleic acid concatenating portion may include a sequence additional portion between the target nucleic acid capturing portion and the complementary strand capturing portion. The sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion to be used in the present modification may be formed as an optional sequence. In addition, in a case where the nucleic acid concatenating portion includes the sequence additional portion, an optional sequence corresponding to functions to be provided to the sequence additional portion can be provided as the sequence of the sequence additional portion.

The primer illustrated in <2D-β> includes a poly-T sequence complementary to a poly-A sequence added to a cDNA by a terminal transferase in step S213 to be described later, and a sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2D-α>. Thus, a starting point is set at a time of generating the nucleic acid having the sequence derived from the target nucleic acid while the cDNA generated in the circular nucleic acid generating process according to the present modification is used as a template, and due to the sequence complementary to the sequence of the complementary strand capturing portion, the two or more generated nucleic acids can be suitably concatenated to each other via the nucleic acid concatenating portion.

The RNA oligomer of <2D-γ> includes an RNA. In addition, the sequence of the RNA oligomer is a sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2D-α>. Thus, the RNA oligomer can be suitably hybridized to the 5’ terminus side of the cDNA generated in the circular nucleic acid generating process according to the present modification.

In the first modification of the circular nucleic acid generating process, a polyA tailing method is used in the complementary nucleic acid generating step, and the nucleic acid having the sequence derived from the target nucleic acid is generated with the generated complementary strand of the target nucleic acid as a template by using the primer provided with a sequence to be captured by the complementary strand capturing portion included in the nucleic acid concatenating portion.
Description will be made in the following with reference to FIG. 2E.

In step S211 in FIG. 2E, the nucleic acid concatenating portion illustrated in <2D-α> is used, and the target nucleic acid capturing portion included in the nucleic acid concatenating portion captures the 3’ terminus of an mRNA. Step S211 is performed in a similar manner to the circular nucleic acid generating process of (2-2) described above except that the nucleic acid concatenating portion being used is different. Conditions similar to those applicable to the circular nucleic acid generating process can be suitably applied also in the present modification.

With regard also to step S212 in which a cDNA is generated with the sequence of the mRNA as a template in FIG. 2E, conditions similar to those applicable to the circular nucleic acid generating process of (2-2) described above can be suitably applied also in the present modification.

In step S213 of FIG. 2E, the mRNA as the template of the cDNA is degraded by processing an RNA-DNA complex generated in step S212 by RNaseH.
Thereafter, a poly-A sequence is added to the 3’ terminus of the cDNA by performing processing using a terminal transferase. Thus, even in a case where a reverse transcription using a reverse transcriptase is not performed to the 5’ terminus of the mRNA, and no CCC sequence is provided, for example, the primer illustrated in <2D-β> can be suitably hybridized to the cDNA by the poly-T sequence possessed by the primer in the next step S214.

In step S214 of FIG. 2E, the primer of <2D-β> is hybridized to the 3’ terminus side of the cDNA generated in step S213, and the RNA oligomer of <2D-γ> is hybridized to the 5’ terminus side of the cDNA.
The order of the hybridizations at this time is not limited. For example, the RNA oligomer may be hybridized first, or the hybridizations may be performed at the same time with the concentration of the RNA oligomer made higher than the concentration of the primer.

Thereafter, in step S215 of FIG. 2E, a nucleic acid having a sequence derived from the target nucleic acid is generated by a DNA polymerase not having strand replacement activity with the primer of <2D-β> as a starting point and with the cDNA as a template. Incidentally, because the RNA oligomer of <2D-γ> is hybridized to the 5’ terminus side of the cDNA, the generation reaction of the nucleic acid by the DNA polymerase not having the strand replacement activity is ended at a position at which the RNA oligomer is hybridized.

In step S216 of FIG. 2E, an RNA-DNA complex generated in step S215 is processed by RNaseH. Consequently, the RNA oligomer is degraded, and a DNA-DNA complex including the complementary strand of the target nucleic acid is obtained.

In step S217 of FIG. 2E, two or more DNA-DNA complexes including the obtained complementary strand of the target nucleic acid are concatenated to each other via the complementary strand capturing portion of the nucleic acid concatenating portion and the sequence complementary to the sequence of the complementary strand capturing portion which complementary sequence is derived from the primer of <2D-β>. As conditions of this step, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2) described above can be suitably applied also in the present modification.

A concatenation reaction illustrated in step S217 forms a single-stranded circular nucleic acid as in the circular nucleic acid generating process of (2-2) described above, that is, as illustrated in step S14 illustrated in FIG. 2A. Incidentally, with regard to conditions for forming this single-stranded circular nucleic acid, optional conditions can be suitably applied.

In the first modification of the circular nucleic acid generating process, the polyA tailing method is used in the complementary nucleic acid generating step, and the primer provided with the sequence to be captured by the complementary strand capturing portion included in the nucleic acid concatenating portion is used. Thus, even in a case where the reverse transcription using the reverse transcriptase is not performed to the 5’ terminus of the mRNA, and the CCC sequence is not provided, for example, the sequence complementary to the sequence of the complementary strand capturing portion of the nucleic acid concatenating portion can be provided. It is consequently possible to form the circular nucleic acid while reducing failures in capturing the generated complementary strands when the circular nucleic acid is formed by concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion. It can thus be expected that a probability of being able to detect even a complementary strand derived from the sequence of an mRNA occurring with a low frequency will be increased, and that analysis accuracy will be consequently improved.

Incidentally, the combination of the DNA-DNA complexes concatenated to each other by the concatenation reaction illustrated in step S217 is not able to be selected. Thus, the single-stranded circular nucleic acid formed in step S217 is one in which the sequences derived from cDNAs generated with the sequences of mRNAs as a template are randomly concatenated to each other. In a case where there are many kinds of concatenated cDNAs, and the order of concatenated cDNAs is substantially unique, the single-stranded circular nucleic acid can be identified by the order of the concatenated cDNAs.

Thereafter, though not depicted in FIG. 2E, as in the circular nucleic acid generating process of (2-2) described above, the primer of <2D-β> or the like, for example, is added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized by RCA using a DNA polymerase with the primer as a starting point. Incidentally, <2D-β> can be suitably used as the primer to be used at this time. However, the primer to be used is not limited to this. In addition, with regard also to these conditions, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2) described above can be suitably applied also in the present modification. As for the decoding of the base sequence of the obtained single-stranded linear nucleic acid, a method similar to that applicable in the circular nucleic acid generating process of (2-2) described above, for example, can be suitably applied also in the present modification.

(2-4) Second Modification of Circular Nucleic Acid Generating Process
FIG. 2F is a conceptual diagram of a nucleic acid concatenating portion and the like used in a second modification of the circular nucleic acid generating process.
In FIG. 2F, <2F-α> is a nucleic acid concatenating portion used in the first modification of the circular nucleic acid generating process. <2F-β> is a primer used when a nucleic acid having a sequence derived from a target nucleic acid is generated in the circular nucleic acid generating process in the present modification. <2F-γ> is a primer used in PCR that uses a DNA-RNA complex formed in the present modification as a template.

The nucleic acid concatenating portion of <2F-α> includes a target nucleic acid capturing portion (poly-T in the figure) capable of capturing a poly-A sequence present at the 3’ terminus of an mRNA and a complementary strand capturing portion (concatenator in the figure). A sequence additional portion (cell barcode in the figure) is provided between the target nucleic acid capturing portion and the complementary strand capturing portion. Incidentally, a mode in which the nucleic acid concatenating portion used in the present modification is not provided with the sequence additional portion can also be used. The sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion used in the present modification may be formed as an optional sequence. In addition, in a case where the nucleic acid concatenating portion includes the sequence additional portion, an optional sequence corresponding to functions to be provided to the sequence additional portion can be provided as the sequence of the sequence additional portion.

The primer illustrated in <2F-β> includes a poly-T sequence complementary to a poly-A sequence added to a cDNA by a terminal transferase in step S313 to be described later, and a sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2F-α>. Incidentally, in the primer, the sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion described above is formed by an RNA. The primer illustrated in <2F-β> serves as a starting point when a nucleic acid having a sequence derived from a target nucleic acid is generated by using the cDNA generated by the circular nucleic acid generating process according to the present modification as a template, and the primer serves as a template in PCR to be described later and thereby provides the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion to the 3’ terminus side of a nucleic acid synthesized in the PCR.

The primer illustrated in <2F-γ> is formed by an RNA. In addition, the sequence of the primer is a sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2F-α>. Thus, a starting point of a nucleic acid extension reaction is set in PCR that uses a DNA-RNA complex formed in the present modification as a template.

Also in the second modification of the circular nucleic acid generating process, the polyA tailing method is used in the complementary nucleic acid generating step, and the nucleic acid having the sequence derived from the target nucleic acid is generated with the generated complementary strand of the target nucleic acid as a template by using the primer provided with a sequence to be captured by the complementary strand capturing portion included in the nucleic acid concatenating portion.
Description will be made in the following with reference to FIG. 2G.

Step S311, step S312, and step S313 in FIG. 2G are similar to step S211, step S212, and step S213 in the first modification of the circular nucleic acid generating process described above. Conditions similar to those applicable to the circular nucleic acid generating process can be suitably applied also in the present modification.

In step S314 of FIG. 2G, a nucleic acid having a sequence derived from the target nucleic acid is generated by a DNA polymerase with the primer of <2F-β> as a starting point and with the cDNA as a template. A sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2F-α> can be provided to the 5’ terminus side of the generated nucleic acid by using the primer of <2F-β>.

In step S315 of FIG. 2G, PCR that uses the primer illustrated in <2F-γ> formed by an RNA is performed while the generated nucleic acid having the sequence derived from the target nucleic acid is used as a template. Incidentally, as described above, the above-described nucleic acid serving as a template has, at the 5’ terminus thereof, the sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion which complementary sequence is derived from the primer of <2F-β> and is formed by an RNA. Thus, in the PCR, a DNA polymerase having reverse transcription activity such as a Tth DNA polymerase can be suitably used.

In step S316 of FIG. 2G, an RNA-DNA complex generated in step S315 is processed by RNaseH. Consequently, the RNA oligomer is degraded, and a DNA-DNA complex including the complementary strand of the target nucleic acid is obtained.

In step S317 of FIG. 2G, two or more DNA-DNA complexes including the obtained complementary strand of the target nucleic acid are concatenated to each other via the complementary strand capturing portion of the nucleic acid concatenating portion and the sequence complementary to the sequence of the complementary strand capturing portion which complementary sequence is derived from the primer of <2F-β>. As conditions of this step, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2) or (2-3) described above can be suitably applied also in the present modification.

A concatenation reaction illustrated in step S317 forms a single-stranded circular nucleic acid as in the circular nucleic acid generating process of (2-2) or (2-3) described above, that is, as illustrated in step S14 illustrated in FIG. 2. Incidentally, with regard also to conditions for forming this single-stranded circular nucleic acid, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2) or (2-3) described above can be suitably applied also in the present modification.

Thereafter, though not depicted in FIG. 2G, as in the circular nucleic acid generating process of (2-2) or (2-3) described above, the primer is added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized by RCA using a DNA polymerase with the primer as a starting point. With regard also to these conditions, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2) or (2-3) described above can be suitably applied also in the present modification.

(2-5) Third Modification of Circular Nucleic Acid Generating Process
FIG. 2H is a conceptual diagram of a nucleic acid concatenating portion and the like used in a third modification of the circular nucleic acid generating process.
In FIG. 2H, <2H-α> is a nucleic acid concatenating portion used in the first modification of the circular nucleic acid generating process. <2H-β> is a primer used when a nucleic acid having a sequence derived from a target nucleic acid is generated in the circular nucleic acid generating process in the present modification.

As with the nucleic acid concatenating portion of <2F-α>, the nucleic acid concatenating portion of <2H-α> includes a target nucleic acid capturing portion (poly-T in the figure) capable of capturing a poly-A sequence present at the 3’ terminus of an mRNA and a complementary strand capturing portion (concatenator in the figure). A sequence additional portion (cell barcode in the figure) is provided between the target nucleic acid capturing portion and the complementary strand capturing portion. However, the nucleic acid concatenating portion of <2H-α> is different from the nucleic acid concatenating portion of <2F-α> in that the complementary strand capturing portion of the nucleic acid concatenating portion of <2H-α> is formed by an RNA.
Incidentally, a mode in which the nucleic acid concatenating portion used in the present modification is not provided with the sequence additional portion can also be used. The sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion used in the present modification may be formed as an optional sequence. In addition, in a case where the nucleic acid concatenating portion includes the sequence additional portion, an optional sequence corresponding to functions to be provided to the sequence additional portion can be provided as the sequence of the sequence additional portion.

As with the primer illustrated in <2F-β>, the primer illustrated in <2H-β> includes a poly-T sequence complementary to a poly-A sequence added to a cDNA by a terminal transferase in step S413 to be described later, and a sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2H-α>. However, this primer is different from the primer illustrated in <2F-β> in that the sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion described above is formed by a DNA in this primer.
The primer illustrated in <2H-β> serves as a starting point when a nucleic acid having a sequence derived from the target nucleic acid is generated by using the cDNA generated by the circular nucleic acid generating process according to the present modification as a template, and the sequence complementary to the sequence of the complementary strand capturing portion enables two or more generated nucleic acids to be suitably concatenated to each other via the nucleic acid concatenating portion.

Also in the third modification of the circular nucleic acid generating process, the polyA tailing method is used in the complementary nucleic acid generating step, and the nucleic acid having the sequence derived from the target nucleic acid is generated with the generated complementary strand of the target nucleic acid as a template by using the primer provided with a sequence to be captured by the complementary strand capturing portion included in the nucleic acid concatenating portion.
Description will be made in the following with reference to FIG. 2I.

Step S411, step S412, and step S413 in FIG. 2I are similar to step S211, step S212, and step S213 in the first modification of the circular nucleic acid generating process described above. Conditions similar to those applicable to the circular nucleic acid generating process can be suitably applied also in the present modification.

In step S414 of FIG. 2I, a nucleic acid having a sequence derived from the target nucleic acid is generated by a DNA polymerase not having reverse transcription activity with the primer of <2H-β> as a starting point and with the cDNA as a template. A sequence complementary to the sequence of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2H-α> can be provided to the 5’ terminus side of the generated nucleic acid by using the primer of <2H-β>. In addition, as described above, the complementary strand capturing portion included in the nucleic acid concatenating portion of <2H-α> is formed by an RNA, and the DNA polymerase used in step S414 does not have reverse transcription activity. Thus, an extension reaction of the nucleic acid is stopped in front of the complementary strand capturing portion included in the nucleic acid concatenating portion of <2H-α>. A DNA-DNA complex including the complementary strand of the target nucleic acid is thereby obtained.

In step S415 of FIG. 2I, two or more DNA-DNA complexes including the obtained complementary strand of the target nucleic acid are concatenated to each other via the complementary strand capturing portion of the nucleic acid concatenating portion and the sequence complementary to the sequence of the complementary strand capturing portion which complementary sequence is derived from the primer of <2H-β>. As conditions of this step, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2), (2-3), or (2-4) described above can be suitably applied also in the present modification.

A concatenation reaction illustrated in step S415 forms a single-stranded circular nucleic acid as in the circular nucleic acid generating process of (2-2), (2-3), or (2-4) described above, that is, as illustrated in step S14 illustrated in FIG. 2. Incidentally, with regard also to conditions for forming this single-stranded circular nucleic acid, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2), (2-3), or (2-4) described above can be suitably applied also in the present modification.

Thereafter, though not depicted in FIG. 2G, as in the circular nucleic acid generating process of (2-2), (2-3), or (2-4) described above, the primer is added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized by RCA using a DNA polymerase with the primer as a starting point. With regard also to these conditions, conditions similar to those applicable to the generating process of the circular nucleic acid generating process of (2-2), (2-3), or (2-4) described above can be suitably applied also in the present modification.

(3) Example 2 (Use of Restriction Enzyme Recognition Site)

According to an embodiment of the present disclosure, a restriction enzyme recognition sequence may be incorporated in the nucleic acid concatenating portion. The restriction enzyme recognition sequence may, for example, be incorporated in the double stranded portion. It is thereby possible to cut an amplification product (amplification product resulting from RCA or PCR, for example) of the concatenation product (circular nucleic acid in particular) concatenated according to an embodiment of the present disclosure at the position of the restriction enzyme recognition sequence by restriction enzyme processing. Consequently, the amplification product is analyzed easily. For example, sequences produced by the cutting can be tagged for sequencing (tagged for short read sequencing in particular). In a case where Hind III is used as a restriction enzyme, for example, a restriction site illustrated in FIG. 4A is introduced into the double stranded portion. As indicated by a broken line in the figure, the double stranded DNA is cut, and tags for sequencing are added here.

An example of generating tagged short reads is illustrated in FIG. 4B.
As illustrated in the figure, in step S31, a complementary strand CN of a circular nucleic acid GN generated according to an embodiment of the present disclosure is synthesized by a DNA polymerase. A double stranded DNA is thereby generated.
In step S32, the double stranded DNA is cut in restriction sites RN present in the double stranded portions by using a restriction enzyme. Double stranded DNA fragments having one cDNA are generated by the cutting.
In step S33, tags T1 to T4 for sequencing are added to both ends of each double stranded DNA fragment. These tags may, for example, be configured to specifically recognize and bind to the sequences and/or structures of the sites cut by the restriction enzyme. The kinds of these tags may be selected as appropriate by those skilled in the art according to a sequencing method. For example, as for short read sequencing provided by Illumina, Inc. tags of Read1, Read2, Sample index (i5), Sample index (i7), P5, and P7 are added. Thereafter, sequencing is performed by a sequencing device provided by Illumina, Inc. The sequence of each double stranded DNA fragment (and the number of double stranded fragments) is thereby identified.

(4) Example 3 (Single Cell Analysis)

The analyzing method according to an embodiment of the present disclosure may be configured as a single cell analyzing method, for example. That is, the present disclosure also provides a single cell analyzing method. Preferably, nucleic acid concatenating portions having double stranded portions different for respective cells may be used in the single cell analysis.
Operations performed in the single cell analyzing method will be described in the following with reference to FIG. 5.

In order to perform the single cell analyzing method, as illustrated in A of the figure, nucleic acid concatenating portions 10 are arranged on a surface (two-dimensional flat surface in particular) of a substrate 40. In the figure, as described in the above description, the nucleic acid concatenating portions include at least a target nucleic acid capturing portion configured to capture the 3’ terminus region of a target nucleic acid, a complementary strand capturing portion configured to capture the 3’ terminus region of a complementary strand produced by complementary strand generation of the target nucleic acid, and a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
Here, it is desired that nucleic acid concatenating portions having different sequences bind to respective cells and that nucleic acid concatenating portions having the same sequence bind to one cell. Accordingly, the surface of the substrate 40 may be divided. Then, nucleic acid concatenating portions having the same sequence may be fixed to one region, and nucleic acid concatenating portions having different sequences may be fixed to respective regions. Specifically, differences in the sequences may be differences in the double stranded portions.
As illustrated in the figure, for example, the surface of the substrate may be divided into a region A1 and a region A2, multiple nucleic acid concatenating portions having the same base sequence may be immobilized in the region A1, and multiple nucleic acid concatenating portions having the same base sequence may be immobilized also in the region A2. Then, the nucleic acid concatenating portions immobilized in the region A1 and the nucleic acid concatenating portions immobilized in the region A2 are different in base sequences, and particularly different in base sequences in double stranded portions.
It is to be noted that, in the present disclosure, the surface to which the nucleic acid concatenating portions are immobilized may, for example, be the flat surface of a substrate, a well, a plate, or the like, as described in the above description, but is not limited to these. Alternatively, the surface to which the nucleic acid concatenating portions are immobilized may, for example, be the surfaces of beads or the like. In a case of the beads, the beads correspond to the respective regions described in the above description. For example, one cell may be captured by one bead. In this case, multiple nucleic acid concatenating portions having the same base sequence may be immobilized on one bead. Then, nucleic acid concatenating portions of the respective beads may, for example, be different from each other in random sequences. The nucleic acid concatenating portions of the respective beads may each include the same target nucleic acid capturing portion and/or the same complementary strand capturing portion and may further include the same priming sequence.

(Complex Including Nucleic Acid Concatenating Portion)
A cell capturing portion for capturing a cell and/or an immobilizing portion for immobilization on the substrate may be bound to the nucleic acid concatenating portion. The cell capturing portion may be an antibody or a lipid. Further, various kinds of barcode sequences may be bound to the nucleic acid concatenating portion. The nucleic acid concatenating portion may be present as one element of a complex including the cell capturing portion and/or the immobilizing portion. Examples of the complex will be described in the following with reference to FIGS. 6A to 6C.

FIG. 6A illustrates an example of schematic configuration of the nucleic acid concatenating portion to which an antibody is bound as the cell capturing portion.

A nucleic acid concatenating portion 50 (part enclosed by a broken line) may be present as a part of a complex 100 immobilized on a substrate 61. The complex 100 includes a cell capturing portion 63 configured to capture a cell, the nucleic acid concatenating portion 50 that captures a target nucleic acid within the cell, and an immobilizing portion for immobilizing the nucleic acid concatenating portion 50 on the substrate. In the figure, a structural body indicated by reference signs 54a, 54b, 55a, 55b, 56, 57, 58, 59, 60-1, and 62 corresponds to the immobilizing portion. However, the configuration of the immobilizing portion is not limited to this. The immobilizing portion may not need to have all of the elements indicated by these reference numerals and may have another configuration as long as the immobilizing portion can fix the nucleic acid concatenating portion to a predetermined surface.
The constituent elements of the complex 100 will be described in the following.

First, the figure illustrates four structural bodies 60-1 (nucleic acids in the figure) present in the region A1 and illustrates the constituent elements such as the nucleic acid concatenating portion bound only to one structural body 60-1 among these structural bodies 60-1. It is to be understood that this is in a simplified form for the ease of understanding of the drawing. In actuality, a large number of structural bodies 60-1 are present in the region A1, and constituent elements such as the nucleic acid concatenating portions are similarly bound to the respective structural bodies.
In addition, as in the region A1, a large number of structural bodies 60-2 are present also in the region A2, and constituent elements such as the nucleic acid concatenating portions are bound to the respective structural bodies.
Further, the substrate 61 is not limited to the two regions illustrated in the figure and may have the number of regions which corresponds to the number of cells desired to be captured.

The cell capturing portion 63 may be a compound configured to capture a desired cell.
In one embodiment, the cell capturing portion 63 may, for example, be an antibody, as illustrated in the figure. The antibody may, for example, be an antibody configured to capture a molecule present on a cell surface (particularly a surface marker or the like). The antibody can be selected as appropriate by those skilled in the art according to the kind of cell to be captured.
In another embodiment, the cell capturing portion may, for example, be a lipid, as illustrated in FIG. 6B. A complex in the figure is similar to that of FIG. 6A except that the complex in the figure has the lipid as the cell capturing portion 63 in place of the antibody. The lipid may be a lipid configured to capture a cell. The kind of the lipid can be selected as appropriate by those skilled in the art.

The nucleic acid concatenating portion 50 includes a target nucleic acid capturing portion 51 configured to capture the 3’ terminus region of a target nucleic acid, a complementary strand capturing portion 52 (GGG in the figure) configured to capture the 3’ terminus region of a complementary strand of a target nucleic acid, and a double stranded portion 53 (53a, 53b, 53c, and 53d) that connects the target nucleic acid capturing portion 51 and the complementary strand capturing portion to each other. These are the same as the target nucleic acid capturing portion 11, the complementary strand capturing portion 12, and the double stranded portion 13 described above, and the description thereof applies also in the present example.

The target nucleic acid capturing portion 51 may be a poly-T sequence, as described in the above description.
The complementary strand capturing portion 52 may be a GGG sequence, as described in the above description.
With regard to the target nucleic acid capturing portion 51 and the complementary strand capturing portion 52, all of the nucleic acid concatenating portions present on the substrate 61 may have the same target nucleic acid capturing portion 51 and the same complementary strand capturing portion 52.

The double stranded portion 53 includes random sequences 53a and 53b and priming sequences 53c and 53d. The random sequences 53a and 53b are in complementary relation to each other. In addition, the priming sequences 53c and 53d are also in complementary relation to each other.
The random sequences 53a and 53b have a base sequence different for the respective regions. That is, the random sequences included in the nucleic acid concatenating portions immobilized in the region A1 each have the same base sequence. The random sequences included in the nucleic acid concatenating portions immobilized in the region A2 also each have the same base sequence. Then, the random sequences present in the region A1 have the base sequence different from that of the random sequences present in the region A2.
With regard to the priming sequences 53c and 53d, all of the nucleic acid concatenating portions present on the substrate 61, for example, may have the priming sequences 53c and 53d of the same base sequence. Consequently, the generation of circular nucleic acids can be performed en bloc by using the priming sequences.

The complex 100 may have a cleavage site 54 (54a and 54b). The cleavage site 54 may, for example, be a restriction enzyme recognition site. In particular, the cleavage site 54 may be a site in which multiple restriction enzyme recognition sites are arranged successively. The cleavage site 54 may, for example, be a site in which 1 to 10 restriction enzyme recognition sites are arranged successively. Particularly, the cleavage site 54 may be a site in which two to eight restriction enzyme recognition sites are arranged successively. More particularly, the cleavage site 54 may be a site in which four to six restriction enzyme recognition sites, for example, five restriction enzyme recognition sites are arranged successively. The number of bases of each restriction enzyme recognition site may, for example, be 4 to 10, particularly may be 4 to 8. The restriction enzyme may be an endonuclease. A possibility of cleavage can be increased by thus providing multiple restriction enzyme recognition sites arranged in series with each other.
In a case where the cleavage site 54 is a restriction enzyme recognition site, the cleavage site 54 may be configured as a double stranded nucleic acid. One strand of the double stranded nucleic acid may be coupled to the nucleic acid concatenating portion 50 (target nucleic acid capturing portion 51 in particular). Another strand of the double stranded nucleic acid may be coupled to a barcode sequence, as will be described later.
The base length of the cleavage site 54 may, for example, be 10 to 50 bases, particularly 20 to 40 bases.

For the cleavage of the restriction enzyme recognition site, an appropriate restriction enzyme (http://catalog.takara-bio.co.jp/product/basic_info.php?unitid = U100003632) is used according to each sequence. A restriction enzyme activity of 1U is an amount of enzyme that fully degrades 1 μg of λDNA in one hour at 37°C in principle in 50 μl of each enzyme reaction solution. The amount of enzyme is adjusted according to an amount of restriction enzyme recognition sequences.

The complex 100 may include a positional information barcode sequence portion 55 (55a and 55b). The positional information barcode sequence portion 55 can be referred to also as an array barcode sequence portion, for example.
The positional information barcode sequence portions 55a and 55b have a base sequence different for the respective regions. That is, the positional information barcode sequence portions immobilized in the region A1 each have the same base sequence. The positional information barcode sequence portions immobilized in the region A2 also each have the same base sequence. Then, the positional information barcode sequence portions present in the region A1 have the base sequence different from that of the positional information barcode sequence portions present in the region A2.
On the basis of positional information corresponding to the base sequence possessed by the positional information barcode sequence portion, a cell captured in each region, and, for example, an image of each cell can be associated with each other. The image may be an image obtained by an image obtaining device such as a microscope device, for example, after the cell is captured in each region but before the cell is set free from each region by cleavage in the cleavage site.
The base length of the positional information barcode sequence portion 55 may be adjusted such that the number of variations of the positional information barcode sequence portion 55 is equal to or more than the number of regions on the substrate (or equal to or more than the number of cells to be captured). The base length may, for example, be equal to or more than 10 bases, particularly equal to or more than 12, more particularly equal to or more than 14, even more particularly equal to or more than 16. The base length may, for example, be equal to or less than 100 bases, particularly equal to or less than 70 bases, more particularly equal to or less than 50 bases, and may, for example, be equal to or less than 30 bases.

The complex 100 may include an immobilizing barcode sequence portion 58.
The immobilizing barcode sequence portion 58 has a base sequence different for the respective regions. That is, the immobilizing barcode sequence portions immobilized in the region A1 each have the same base sequence. The immobilizing barcode sequence portions immobilized in the region A2 also each have the same base sequence. Then, the immobilizing barcode sequence portions present in the region A1 have the base sequence different from that of the immobilizing barcode sequence portions present in the region A2.
The immobilizing barcode sequence portion 58 has a base sequence complementary to the nucleic acids 60-1 immobilized to the substrate in advance. The nucleic acids 60-1 are present only within a specific region. Thus, the immobilizing barcode sequence specifically binds to a nucleic acid 60-1. Then, the complex 100 having the immobilizing barcode sequence is immobilized within the specific region.
The base length of the immobilizing barcode sequence portion 58 may be adjusted such that the number of variations of the immobilizing barcode sequence portion 58 is equal to or more than the number of regions on the substrate (or equal to or more than the number of cells to be captured). The base length may, for example, be equal to or more than 10 bases, particularly equal to or more than 12, more particularly equal to or more than 14, even more particularly equal to or more than 16. The base length may, for example, be equal to or less than 100 bases, particularly equal to or less than 70 bases, more particularly equal to or less than 50 bases, and may, for example, be equal to or less than 30 bases.

The complex 100 may include priming portions 57 and 59. The priming portion 57 has a base sequence used to synthesize a nucleic acid strand including the above-described barcode sequence portions and the above-described restriction enzyme recognition site. The base length of each priming portion is, for example, 10 to 30 bases, particularly 15 to 25 bases, more particularly 15 to 20 bases.

As described above, the complex 100 may include the nucleic acid concatenating portion, the cell capturing portion for capturing a cell, and the immobilizing portion for immobilizing the nucleic acid concatenating portion at a specific position. The immobilizing portion may include the restriction enzyme recognition site and/or the barcode sequence portions described in the above description. In addition, with regard to the complex 100, the immobilizing barcode sequence portion 58 functions as a structural body for immobilizing the complex at a specific position. However, the positional information barcode portion may be configured as a structural body for immobilizing the complex at a specific position.

As described in the above description, the complex 100 may be bound to a nucleic acid 60-1 immobilized on the substrate. The nucleic acid 60-1 may have a base sequence different for the respective regions. The complex 100 is immobilized on the substrate through binding between the nucleic acid 60-1 and the immobilizing barcode portion 58. That is, the nucleic acid concatenating portion may be immobilized on the substrate.

A nucleic acid 60 (60-1 and 60-2) may be immobilized to the substrate 61 via a linker 62. As the linker 62, a material known in this technical field may be used and can be selected as appropriate by those skilled in the art.

In one embodiment, the linker 62 may be a linker cleavable by stimulation, and is, for example, a linker cleavable by optical stimulation or chemical stimulation. The optical stimulation is particularly suitable for selectively applying a stimulus to a specific position in a cleavage step to be described later.
In a case where such a cleavable linker is used, the cleavage site 54 including the restriction enzyme recognition site described in the above description does not have to be provided but may be provided.
In a case where such a cleavable linker is used, the strand coupled to the nucleic acid concatenating portion (strand coupled to the target nucleic acid capturing portion in particular) may be immobilized on the substrate 61 directly (that is, without the intervention of the nucleic acid 60) via the linker.

The cleavable linker can include one selected from an arylcarbonylmethyl group, a nitroaryl group, a coumarin-4-ylmethyl group, an arylmethyl group, a metal containing group, and other groups as a linker cleavable by optical stimulation, for example. Groups described in Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191, for example, may be used as these groups.
The arylcarbonylmethyl group may, for example, be a phenacyl group, an o-alkylphenacyl group, or a p-hydroxyphenacyl group. The nitroaryl group may, for example, be an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, or o-nitroanilides. The arylmethyl group may, for example have a hydroxy group introduced therein or may not have a hydroxy group introduced therein.

In a case where the cleavable linker is a linker cleavable by optical stimulation, the linker may preferably be a linker cleaved by light having a wavelength of 360 nm or more. The linker may preferably be a linker cut by an energy of 0.5 μJ/μm² or less (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 Jan;12(1):23-6. doi: 10.1038/nmeth.3219.). The adoption of the linker cut by light having the above-described wavelength or the above-described energy can reduce cell damage (particularly the cutting of DNA or RNA or the like) that can occur when an optical stimulus is applied.

Particularly preferably, the cleavable linker may be a linker cleaved by light in a short wavelength region, specifically light in a wavelength region of 360 to 410 nm, or may be a linker cleaved by light in a near infrared region or an infrared region, specifically light in a wavelength region of 800 nm and more. In a case where the cleavable linker is a linker that is efficiently cut by light having a wavelength in a visible light region, a surface for analysis can be difficult to handle. Therefore, the linker is preferably a linker cleaved by light in the short wavelength region described above or light in the near infrared region or the infrared region described above.

The cleavable linker can include a disulfide bond or the like as a linker cleavable by chemical stimulation, for example. For the cleavage of the disulfide bond, a reducing agent such as Tris (2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), or 2-Mercaptoethanol is used. In a case where TCEP is used, for example, the TCEP is made to react for approximately 15 min. in 50 mM, for example.

The complex 100 may include multiple cleavable linkers. Preferably, the multiple linkers may be concatenated in series with each other. In a case where a cleavage probability of one linker is 0.8, for example, the cleavage probability is improved to 0.992 (= 1 - 0.2³) by concatenating three such linkers in series with each other.

(Method of Manufacturing Complex Including Nucleic Acid Concatenating Portion)
A method of manufacturing the complex will be described in the following with reference to FIG. 6C.

For the complex 100, a first pool of a first strand (single strand in particular) including the target nucleic acid capturing portion of the nucleic acid concatenating portion and a second pool of a second strand (single strand in particular) including the barcode sequence portions are prepared. A double strand is formed by binding the strands of the respective pools to each other. Here, the first strand includes one of the two strands constituting the double stranded portion of the nucleic acid concatenating portion.
A third strand (single strand in particular) including the complementary strand capturing portion of the nucleic acid concatenating portion is prepared for the double strand. The third strand includes the other of the two strands constituting the double stranded portion of the nucleic acid concatenating portion. Then, the third strand is bound to the double strand obtained by binding the first strand and the second strand to each other. The complex 100 is thereby obtained.
An oligo pool 1 (Oligo-pool 1) side of FIG. 6C illustrates a process for obtaining the first strand. An oligo pool 2 (Oligo-pool 2) side illustrates a process for obtaining the second strand.

(Oligo Pool 1)
As illustrated in the oligo pool 1, first, a single stranded nucleic acid N1 (DNA or RNA) is prepared which includes two priming sites (Priming) as well as a sequence (Concatenator*) forming the double stranded portion, a sequence (PolyT) forming the target nucleic acid capturing portion, a sequence (Cleavage site) forming the cleavage site, and a sequence (Array1 barcode*) forming the positional information barcode sequence portion between these priming sites.
Here, the sequences provided with “*,” that is, the sequence (Concatenator*) forming the double stranded portion and the sequence (Array1 barcode*) forming the positional information barcode sequence portion, have a base sequence different for the respective regions. Therefore, multiple kinds of single stranded nucleic acids in which these sequences differ but the sequences of the other parts are the same are prepared as the oligo pool 1.

Next, PCR using the two priming sites is performed. Specifically, as illustrated in the figure, for example, a primer that has a T7 promoter and binds to one priming site and a reverse primer that binds to the other priming site may be prepared, and PCR may be performed by using these two kinds of primers. A double stranded nucleic acid is obtained by the PCR.

The double stranded nucleic acid is subjected to in vitro transcription (IVT). An RNA transcript is thereby obtained.

The RNA transcript is subjected to reverse transcription (RT) by using a reverse primer that binds to the priming site, as illustrated in the figure. A cDNA corresponding to the RNA transcript is thereby generated.

After the cDNA synthesis, RNA digestion processing (RNA digestion) is performed. The processing removes the RNA and leaves only the cDNA. Thus, a single stranded nucleic acid is obtained which includes the sequence forming the double stranded portion, the sequence forming the target nucleic acid capturing portion, the sequence forming the cleavage site, and the sequence forming the positional information barcode sequence portion.

(Oligo Pool 2)
As illustrated in the oligo pool 2, first, a single stranded nucleic acid N2 (DNA or RNA) is prepared which includes two priming sites (Priming) as well as a sequence (Cleavage site) forming the cleavage site, a sequence (Array1# barcode*) forming the positional information barcode sequence portion, and a sequence (Array2# barcode*) forming the immobilizing barcode sequence portion between these priming sites. Incidentally, “#” denotes being complementary. For example, Array1 barcode* is complementary to Array1# barcode*, and Array2 barcode* is complementary to Array2# barcode*.
Here, the sequences provided with “*,” that is, the sequence (Array1/Array1# barcode*) forming the positional information barcode sequence portion and the sequence (Array2# barcode*) forming the immobilizing barcode sequence portion have a base sequence different for the respective regions. Therefore, multiple kinds of single stranded nucleic acids in which these sequences differ but the sequences of the other parts are the same are prepared as the oligo pool 2.

After the cDNA synthesis, RNA digestion processing (RNA digestion) is performed. The processing removes the RNA and leaves only the cDNA. Thus, a single stranded nucleic acid is obtained which includes the sequence forming the cleavage site, the sequence forming the positional information barcode sequence portion, and the sequence forming the immobilizing barcode sequence portion.

As illustrated in a center of the figure, the single stranded nucleic acid synthesized in the oligo pool 1 described above and the single stranded nucleic acid synthesized in the oligo pool 2 bind to each other on the basis of complementarity in the positional information barcode sequence portion and the cleavage site. A conjugate is thereby obtained. A single strand including the complementary strand capturing portion and the other strand of the two strands forming the double stranded portion further binds to the conjugate. The complex 100 is thereby obtained. Thus, the complex 100 may include the three nucleic acid strands. In addition, the binding may be performed on the basis of complementarity between the two strands forming the double stranded portion.
Incidentally, while the cell capturing portion (antibody) may bind to the single strand in advance as illustrated in the figure, the cell capturing portion may be bound to the complex after the formation of the complex of the three nucleic acid strands.

In addition, the complex 100 includes the immobilizing barcode portion, as described in the above description. The specific complex is fixed at a specific position on the basis of complementarity between the immobilizing barcode portion and a nucleic acid 60-1 provided on the substrate in advance.

As described in the above description, for the complex 100, the first pool of the first strand (single strand in particular) including the target nucleic acid capturing portion of the nucleic acid concatenating portion and the second pool of the second strand (single strand in particular) including the barcode sequence portion are prepared. A double strand is formed by binding the strands of the respective pools to each other. Here, the first strand includes one of the two strands constituting the double stranded portion of the nucleic acid concatenating portion.

The third strand (single strand in particular) including the complementary strand capturing portion of the nucleic acid concatenating portion is prepared for the double strand. The third strand includes the other of the two strands constituting the double stranded portion of the nucleic acid concatenating portion. Then, the third strand is bound to the double strand obtained by binding the first strand and the second strand to each other. The complex 100 is thereby obtained.

In one embodiment, the cell capturing portion may be bound to the third strand in advance. In this case, the third strand is bound to the double strand obtained by binding the first strand and the second strand to each other. The complex having the cell capturing portion is thereby obtained.
In another embodiment, the cell capturing portion may not be bound to the third strand in advance. In this case, the cell capturing portion may be further bound to the complex obtained by binding the third strand to the double strand obtained by binding the first strand and the second strand to each other.

While the complex and the nucleic acid concatenating portion may be prepared by the manufacturing method as described above, the method of manufacturing the complex and the nucleic acid concatenating portion is not limited to this. The method of manufacturing the complex and the nucleic acid concatenating portion may be changed as appropriate according to the configurations of the complex and the nucleic acid concatenating portion.

In the following, the description returns to FIG. 5.
In step S41, cells are supplied onto the substrate having the surface on which the complex is immobilized. The cell capturing portion in the complex captures a cell. Consequently, as illustrated in B of the figure, one cell is captured in one region. Incidentally, while only one complex is displayed to have the strand including the complementary strand capturing portion in B of the figure, this is an omission for the simplification of the drawing. In actuality, each of the complexes may have the strand including the complementary strand capturing portion. The strands including the complementary strand capturing portions may be similarly omitted also in the other figures referred to in the present specification.

In step S42, the cleavage site 54 in the complex, for example, is cleaved. The cleavage may be performed by a restriction enzyme that recognizes the cleavage site.
In addition, in a case where the complex includes the cleavable linker described in the above description, processing for cleaving the linker (optical stimulation or chemical stimulation) may be performed.
After the restriction enzyme processing, the residual is degraded by an exonuclease. The target nucleic acid capturing portion 51 thus becomes a 3’ terminus region. The exonuclease may preferably be Exonuclease III (E. coli) and can perform degradation from the 3’ terminus side of a double stranded DNA having a 5’ overhang, a blunt end, or a 3’ overhang of less than four bases (less than 4 bases).
The cleavage sets each cell free from the substrate while each cell has the nucleic acid concatenating portion bound thereto via the cell capturing portion. The cell set free is isolated in a minute space on a one-by-one basis. The minute space may be a space within an emulsion particle or may be a space within a well, as will be described later.
Then, each cell is destroyed in the isolated state. The destruction may be cytolysis processing, for example.
With the destruction, a target nucleic acid within the cell is captured by the target nucleic acid capturing portion constituting the nucleic acid concatenating portion. Consequently, as illustrated in C of FIG. 5, a conjugate of the target nucleic acid and the nucleic acid concatenating portion is generated. That is, a hybrid of the target nucleic acid and the nucleic acid concatenating portion is generated.
Because the cell destruction is performed in the isolated state, the target nucleic acid within the cell does not go out of the minute space. Therefore, nucleic acid concatenating portions having the same base sequence can be bound to all of target nucleic acids originating from one cell. In addition, as described above, the nucleic acid concatenating portions of respective cells are different from each other in the double stranded portions. Therefore, the cells and the base sequences of the nucleic acid concatenating portions (double stranded portions in particular) are associated with each other in one-to-one relation. Single cell analysis is thereby made possible. A method for the isolation will be separately described later.
Thus, the analyzing method according to an embodiment of the present disclosure may include a cell destroying step of destroying the cells, and the complementary nucleic acid generating step may be performed on the target nucleic acids included in the cells.

Step S43 is cDNA synthesis that is the same as that in step S12 in FIG. 2A described in the above description. The description of step S12 applies also to step S43.
When step S43 is performed, a cDNA having a CCC sequence at a terminus of a generated strand is generated, as illustrated in D of FIG. 5.

Step S44 is nucleic acid concatenation that is the same as that in step S13 in FIG. 2A described in the above description. The description of step S13 applies also to step S44.
When step S44 is performed, two or more cDNAs are concatenated to each other via the complementary strand capturing portion(s), as illustrated in E of FIG. 5.

Step S45 is circular nucleic acid formation, ligation, and primer addition that are the same as those in steps S14 and 15 of FIG. 2A described in the above description. The description of steps S14 and S15 applies also to step S45.
When step S45 is performed, a circular nucleic acid to which a primer is added is formed, as illustrated in F of FIG. 5.

Step S46 is nucleic acid amplification that is the same as that in step S16 in FIG. 2A described in the above description. The description of step S16 applies also to step S46.
When step S46 is performed, the nucleic acid is amplified as illustrated in G of FIG. 5. The nucleic acid thus amplified is used in single cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed by using a result of the sequencing.

(Isolation into Minute Space)
An example of a method for the isolation will be described in the following.

The minute space may, for example, be a space within an emulsion particle or a space within a well. The isolation isolates one cell to which one or more nucleic acid concatenating portions having the same double stranded portion base sequence are bound within one emulsion particle or one well. Then, the cell destroying step may be performed within the minute space (that is, a space partitioned for each cell).

In one embodiment, an isolating step of performing the isolation can include a determining step of determining whether to isolate a cell into a minute space, and a particle isolating step of isolating, into the minute space, the cell whose isolation is determined in the determining step. It is thereby possible to isolate only target cells. Therefore, cells other than the target cells, for example, can be excluded from analysis targets, so that analysis efficiency can be improved.

The determination may, for example, be performed on the basis of light produced from the cell (for example, scattered light and/or autofluorescence or the like), light produced from a substance bound to the cell, or a form image. The substance bound to the cell may, for example, be an antibody (fluorochrome-labeled antibody in particular) bound to the cell. The scattered light produced from the cell may, for example, be forward-scattered light and/or side-scattered light. Doublet detection can be made from signal height and/or area values obtained by detection of the scattered light. Single cell determination based on form image information is also possible. Whether the cell is a dead cell can be determined from the scattered light and/or the form image or fluorescence after staining by a dead cell staining reagent. The dead cell can be thereby removed. In the present disclosure, the determining step may be performed immediately before the isolating step. It is thereby possible to reliably isolate only a single cell to which the nucleic acid concatenating portion is bound.

In another embodiment, the particle isolating step may be performed without the determining step being performed. The number of steps in the method according to an embodiment of the present disclosure can be reduced by omitting the determining step.
In yet another embodiment, the determining step may be performed in the linker cleavage. For example, cells fixed on the substrate may be observed, and linker cleavage processing may be performed only for cells to be isolated into minute spaces. In this case, a device such as a cell sorter may not need to be used.

The determining step and the particle isolating step will be described in the following.

(Determining Step)

The determining step determines whether to isolate a cell set free from the substrate into a minute space. The determination may be made on the basis of light produced from the cell or light produced from a substance bound to the cell, as described above.

The determining step can include, for example, an irradiating step of irradiating the cell with light and a detecting step of detecting light produced by the irradiation.

The irradiating step may, for example, be performed by a light irradiating unit that irradiates the cell with light. The light irradiating unit may include, for example, a light source that emits the light. In addition, the light irradiating unit can include an objective lens that condenses the light onto the cell. The light source may be selected as appropriate by those skilled in the art according to a purpose of analysis. The light source may, for example, be a laser diode, an SHG (second harmonic generation) laser, a solid-state laser, a gas laser, a high luminance LED, or a halogen lamp, or may be a combination of two or more of these. The light irradiating unit may include another optical element as needed in addition to the light source and the objective lens.

The detecting step may, for example, be performed by a detecting unit that detects light produced from the cell or the substance bound to the cell, for example. For the detecting unit, for example, the light produced from the cell or the substance bound to the cell by the light irradiation of the light irradiating unit may, for example, be scattered light and/or fluorescence. The detecting unit can, for example, include a condensing lens that condenses light produced from bioparticles and a detector. A PMT (photomultiplier tube), a photodiode, a CCD, a CMOS, and the like can be used as the detector. However, the detector is not limited to these. The detecting unit may include another optical element as needed in addition to the condensing lens and the detector. The detecting unit can further include a spectroscopic unit, for example. Optical parts constituting the spectroscopic unit include, for example, a grating, a prism, and an optical filter. Due to the spectroscopic unit, light of a wavelength to be detected, for example, can be detected in a state of being separated from light of other wavelengths. The detecting unit can convert the detected light into an analog electric signal by photoelectric conversion. The detecting unit can further convert the analog electric signal into a digital electric signal by AD conversion.

The determining step may be performed by a determining unit that performs determination processing as to whether to determine the cell on the basis of the light detected in the detecting step. The processing of the determining unit can be implemented by, for example, an information processing device such as a general-purpose computer, particularly a processing unit included in the information processing device.

(Particle Isolating Step)

The isolating step includes the particle isolating step of isolating a cell into a minute space. The minute space may refer to a space that has such a size as to be able to house one of the above-described ones as analysis targets. The size may be determined as appropriate according to factors such as the size of the cell. The minute space may have such a size as to be able to house two or more cells as analysis targets. However, in this case, a case where two or more cells are housed within one minute space can occur in addition to a case where one cell is housed within one minute space. The cells within the minute space housing the two or more cells may be excluded from destruction targets in a destroying step to be described later or may be excluded from analysis targets in the analyzing step to be described later.

In addition, in the destroying step to be described later, a conjugate of a nucleic acid concatenating portion and a target nucleic acid, for example, can be generated. The multiple minute spaces are preferably separated from each other so that the conjugate generated within one minute space does not move to another minute space. Examples of the minute spaces thus separated from each other include spaces within wells and spaces within emulsion particles. That is, in a preferable embodiment, the minute spaces may be spaces within wells or spaces within emulsion particles. In the following, description will be made of each of an example of the particle isolating step in a case where the minute spaces are spaces within wells and an example of the particle isolating step in a case where the minute spaces are spaces within emulsion particles.

(Case of Spaces Within Wells)

A schematic diagram of an example of wells used to perform the particle isolating step is illustrated in FIG. 12. As illustrated in the figure, multiple wells 400 having such a size as to be able to house one cell, for example, may be formed in the surface of a substrate 401. Liquid including cells set free from the substrate by the above-described cleavage is applied from an optional nozzle 402, for example, to the surface of the substrate 401. A cell 403 is thereby isolated into a space within a well 400, as illustrated in the figure. Thus, one cell may enter a space within one well, that is, the cell may be isolated within the minute space.

In a case where liquid including multiple cells is applied to the substrate in which the wells are formed as in the example illustrated in the figure, the particle isolating step may be performed without the determining step described in the above description being performed, but the particle isolating step may be performed after the determining step is performed.

In addition, in a case where the above-described determining step is performed, a device such as a cell sorter or a single cell dispenser may be used which places one bioparticle in one well. The device can also be used to isolate a cell into the substrate (for example, a plate or the like) in which the multiple wells are formed. A commercially available device may be used as the device. The device can, for example, include a light irradiating unit that irradiates a cell with light, a detecting unit that detects the light from the cell, a determining unit that determines whether to place the cell in a well on the basis of the detected light, and a distributing unit that distributes, to the well, the cell whose placement in the well is determined.

The light irradiating unit and the detecting unit perform the detecting step. Then, the determining unit performs the determining step. The distributing unit, for example, includes a micro fluid chip that has a nozzle for forming a droplet including a cell.

The device operates the position of the micro fluid chip and places one cell containing droplet in a predetermined well according to a determination result of the determining unit. Alternatively, the device controls the traveling direction of the cell containing droplet taken out from the nozzle by using a charge imparted to the droplet according to the determination result of the determining unit. As a result of the control, one cell containing droplet is placed within the predetermined well. Thus, one cell is distributed to one well.

As illustrated in FIG. 13A, for example, a cell containing droplet is taken out from a nozzle 502 provided to the micro fluid chip of the device. A light irradiating unit 504 irradiates a bioparticle included in the droplet with light (for example, laser light L). Then, a detecting unit 505 performs the detecting step, and thereby detects light (fluorescence F). Then, a determining unit (not illustrated) performs a determining step on the basis of the detected light. Then, according to a determination result, the distributing unit controls the traveling direction of the droplet by using a charge imparted to the droplet. As a result of the control, the droplet including the target bioparticle is collected into a predetermined well. One bioparticle is thereby distributed to one well.

By performing the determining step, it is possible, for example, to identify a cell group to which the bioparticle belongs, identify the bioparticle provided with a barcode, or identify the droplet including a singlet bioparticle according to a detection signal. It is thereby possible to collect only droplets including target bioparticles. As a result, it becomes unnecessary to exclude data in the analyzing step to be described later, so that analysis efficiency is improved.

The number of wells provided to one substrate (plate) may, for example, be 1 to 1000, particularly 10 to 800, more particularly 30 to 500. However, the number of wells may be selected as appropriate by those skilled in the art.

(Case of Spaces Within Emulsion Particles)

The emulsion particles can be generated by using a micro-flow passage, for example. The flow passage, for example, includes a flow passage through which a first liquid forming a dispersoid of an emulsion flows and a flow passage through which a second liquid forming a dispersion medium flows. Cells may be included in the first liquid. The device further includes a region in which these two kinds of liquid come into contact with each other to form the emulsion. In the following, an example of the micro-flow passage will be described with reference to FIG. 13B.

The micro-flow passage illustrated in the figure includes a flow passage 601 through which the first liquid including cells flows and flow passages 602-1 and 602-2 through which the second liquid flows. The first liquid forms emulsion particles (dispersoid). The second liquid forms the dispersion medium of the emulsion. The flow passage 601 and the flow passages 602-1 and 602-2 merge with each other. The emulsion particles are formed at this merging point. Then, cells 603 are isolated within the emulsion particles. The size of the emulsion particles can be controlled by, for example, controlling the flow rates of these flow passages.

In order to form the emulsion, the first liquid and the second liquid are immiscible with each other. For example, the first liquid may be a hydrophilic liquid, and the second liquid may be a hydrophobic liquid, or vice versa.

In addition, the micro-flow passage illustrated in the figure can include a flow passage 604 for introducing a cell destroying substance into the emulsion particles. When the micro-flow passage is configured such that the flow passage 604 merges with the flow passage 601 immediately in front of the merging point, the cells can be prevented from being destroyed by the cell destroying substance before the emulsion particles are formed.

Next, an example of a device for forming an emulsion including an emulsion particle including one cell more efficiently will be described with reference to FIG. 14A and FIG. 14B. The emulsion forming device can isolate one cell within one emulsion particle and can thereby reduce the number of empty emulsion particles with a very high probability. Further, the emulsion forming device can also increase the probability of isolating one cell into one emulsion particle.

FIG. 14A is an example of a micro-flow passage chip (hereinafter referred to also as a “microchip”) used to form emulsion particles in the device.
A microchip 150 illustrated in the figure includes a main flow passage 155 through which bioparticles (cells in particular) are flowed and a collection flow passage 159 into which collection target particles among the bioparticles are collected. The microchip 150 is provided with a particle sorting section 157. An enlarged view of the particle sorting section 157 is illustrated in FIG. 15. As illustrated in A of the figure, the particle sorting section 157 includes a connection flow passage 170 that connects the main flow passage 155 and the collection flow passage 159 to each other. Liquid supply flow passages 161 that can supply liquid to the connection flow passage 170 are connected to the connection flow passage 170. As described above, the microchip 150 has a flow passage structure including the main flow passage 155, the collection flow passage 159, the connection flow passage 170, and the liquid supply flow passages 161.
FIG. 14B is a schematic diagram of assistance in explaining the formation of emulsion particles and the isolation of bioparticles within the formed emulsion particles in the microchip 150 illustrated in FIG. 14A.

In addition, as illustrated in FIG. 14A, the microchip 150 constitutes a part of a bioparticle sorting device 200 including a light irradiating unit 191, a detecting unit 192, and a control unit 193 in addition to the microchip. As illustrated in FIG. 16, the control unit 193 can include a signal processing unit 194, a determining unit 195, and a sorting control unit 196. The bioparticle sorting device 200 is used as the emulsion forming device described in the above description.

As illustrated in FIG. 17, in order to form an emulsion including an emulsion particle including one target cell, for example, the following steps can be performed in the microchip 150: a flowing step S201 of feeding the first liquid including the cell into the main flow passage 155, a determining step S202 of determining whether the cell flowing through the main flow passage 155 is a collection target particle, and a collecting step S203 of collecting the collection target particle into the collection flow passage 159. The determining step S202 corresponds to the determining step described in the above description. The collecting step S203 corresponds to the particle isolating step described in the above description.
Each of the steps will be described in the following.

(Flowing Step)

In the flowing step S201, the first liquid including cells is flowed through the main flow passage 155. The first liquid flows within the main flow passage 155 from a merging portion 162 to the particle sorting section 157. The first liquid may be a laminar flow formed by a sample liquid including cells and a sheath liquid, and particularly may be a laminar flow in which the periphery of the sample liquid is surrounded by the sheath liquid. A flow passage structure for forming the laminar flow will be described in the following.
Incidentally, the sheath liquid may include, for example, a cell destroying component, or, for example, a cytolysis component or the like. The component is thereby captured into emulsion particles, so that cells can be destroyed within the emulsion particles in the destroying step to be described later. The cytolysis component may be a cytolysis enzyme and may, for example, be proteinase K or the like. For example, after a cell is captured into an emulsion particle including proteinase K, the emulsion particle is placed at a predetermined temperature (for example, 37°C to 56°C) for, for example, one hour or less, particularly less than one hour. The cell is thereby dissolved. Incidentally, proteinase K has activity even at 37°C or lower. However, in a case where such a lower temperature is used, incubation can be performed overnight, for example, in consideration of a decrease in the cytolysis property of proteinase K. In addition, the sheath liquid may include a surface-active agent (for example, SDS, Sarkosyl, Tween 20, Triton X-100, or the like). The surface-active agent can enhance the activity of proteinase K.
In addition, the sheath liquid may not include the cell destroying component. In this case, the cell may be destroyed physically. Adoptable as a physical destruction method is optical processing (for example, optical cytolysis (Optical lysis)) or thermal processing (for example, cytolysis by heat (Thermal lysis)), for example. The optical processing can be performed by forming plasma or a cavitation bubble within the particle by the irradiation of the emulsion particle with laser light, for example. A thermal particle destruction can be performed by heating the emulsion particle.

The microchip 150 is provided with a sample liquid inlet 151 and a sheath liquid inlet 153. The sample liquid including cells and the sheath liquid not including the cells are introduced from these inlets into a sample liquid flow passage 152 and sheath liquid flow passages 154, respectively.

The microchip 150 has a flow passage structure in which the sample flow passage 152 through which the sample liquid flows and the sheath liquid flow passages 154 through which the sheath liquid flows merge with each other at the merging portion 162 to form the main flow passage 155. The sample liquid and the sheath liquid merge with each other at the merging portion 162 to form, for example, a laminar flow in which the periphery of the sample liquid is surrounded by the sheath liquid. A schematic diagram of the formation of the laminar flow is illustrated in FIG. 14B. As illustrated in the figure, the laminar flow is formed such that the sample liquid introduced from the sample flow passage 152 is surrounded by the sheath liquid introduced from the sheath liquid flow passages 154.
Preferably, cells are arranged substantially in a row in the laminar flow. As illustrated in the figure, for example, cells P may be arranged substantially in a row in the sample liquid. Thus, the flow passage structure in the present disclosure forms a laminar flow including the cells flowing in a state of being arranged substantially in a row.

The laminar flow flows through the main flow passage 155 toward the particle sorting section 157. Preferably, the cells flow in a state of being arranged in a row within the main flow passage 155. Consequently, in light irradiation in a detection region 156 to be described in the following, light produced by the irradiation of one microparticle with light and light produced by the irradiation of another microparticle with light are distinguished from each other easily.

(Determining Step)

The determining step S202 determines whether a cell flowing through the main flow passage 155 is a collection target particle. The determination can be performed by the determining unit 195. The determining unit 195 can make the determination on the basis of light produced by the irradiation of the cell with light by the light irradiating unit 191. An example of the determining step S202 will be described in more detail in the following.

In the determining step S202, the light irradiating unit 191 irradiates a cell flowing through the main flow passage 155 (detection region 156 in particular) in the microchip 150 with light (for example, exciting light or the like), and the detecting unit 192 detects light produced by the light irradiation. According to a characteristic of the light detected by the detecting unit 192, the determining unit 195 included in the control unit 193 determines whether the bioparticle is a collection target particle. For example, the determining unit 195 can make a determination based on scattered light, a determination based on fluorescence, or a determination based on an image (for example, one or more of a dark visual field image, a bright visual field image, and a phase difference image, or the like). In the collecting step S203 to be described later, the control unit 193 controls a flow in the microchip 150, and thereby the collection target particle is collected into the collection flow passage 159.

The light irradiating unit 191 irradiates the cell flowing within a flow passage in the microchip 150 with light (for example, exciting light or the like). The light irradiating unit 191 can include a light source that emits the light, and an objective lens that condenses exciting light onto the microparticle flowing through the detection region. The light source may be selected as appropriate by those skilled in the art according to a purpose of analysis. The light source may, for example, be a laser diode, an SHG laser, a solid-state laser, a gas laser, a high luminance LED, or a halogen lamp, or may be a combination of two or more of these. The light irradiating unit may include another optical element as needed in addition to the light source and the objective lens.

(Collecting Step)

In the collecting step S203, the cell determined to be a collection target particle in the determining step S202 is collected into the collection flow passage 159. In the collecting step S203, the collection target particle is collected, in a state of being included in the first liquid, into the second liquid immiscible with the first liquid within the collection flow passage. Consequently, an emulsion in which the second liquid is a dispersion medium and the first liquid is a dispersoid can be formed within the collection flow passage 159, and one collection target particle is included in each emulsion particle of the emulsion. Thus, the cell set as a target is isolated in a space within the emulsion particle.
As illustrated in FIG. 14B, for example, a collection target particle P is collected, in a state of being included in the first liquid indicated in white, into the second liquid indicated in gray. Consequently, an emulsion particle 190 is formed, and one collection target particle P is isolated in a space within one emulsion particle 190.
The collecting step will be described in more detail in the following.

The collecting step S203 is performed in the particle sorting section 157 in the microchip 150. In the particle sorting section 157, the laminar flow that has flowed through the main flow passage 155 separates and flows into two discarding flow passages 158. While the particle sorting section 157 depicted in FIG. 14A has the two discarding flow passages 158, the number of branched flow passages is not limited to two. The particle sorting section 157 can, for example, be provided with one or multiple (for example, two, three, four, or the like) branched flow passages. The branched flow passages may be configured to branch in a shape of a letter Y on one plane as in FIG. 14A or may be configured to branch three-dimensionally.

In the particle sorting section 157, only in a case where a collection target particle flows in, a flow that enters the collection flow passage 159 from the main flow passage 155 through the connection flow passage 170 is formed, and the collection target particle is collected into the collection flow passage 159. An enlarged view of the particle sorting section 157 is illustrated in FIG. 15. As illustrated in A of the figure, the main flow passage 155 and the collection flow passage 159 are made to communicate with each other via the connection flow passage 170 on the same axis as the main flow passage 155. As illustrated in B of the figure, the collection target particle flows into the collection flow passage 159 through the connection flow passage 170. As illustrated in C of the figure, microparticles that are not the collection target particle flow into the discarding flow passages 158.

An enlarged view of the connection flow passage 170 and the vicinities thereof is illustrated in FIG. 18A and FIG. 18B. FIG. 18A is a schematic perspective view of the connection flow passage 170 and the vicinities thereof. FIG. 18B is a schematic sectional view in a plane passing through the center line of the liquid supply flow passages 161 and the center line of the connection flow passage 170. The connection flow passage 170 includes a flow passage 170a on the detection region 156 side (hereinafter, referred to also as an upstream side connection flow passage 170a), a flow passage 170b on the collection flow passage 159 side (hereinafter, referred to also as a downstream side connection flow passage 170b), and a connecting portion 170c for connection between the connection flow passage 170 and the liquid supply flow passages 161. The liquid supply flow passages 161 are provided in such a manner as to be substantially perpendicular to the flow passage axis of the connection flow passage 170. While the two liquid supply flow passages 161 are provided in such a manner as to face each other at a substantially central position of the connection flow passage 170 in FIGS. 18A and 18B, only one liquid supply flow passage may be provided.

As indicated by arrows in FIG. 18B, the second liquid is supplied from the two liquid supply flow passages 161 to the connection flow passage 170. The second liquid flows from the connecting portion 170c to both the upstream side connection flow passage 170a and the downstream side connection flow passage 170b.

In a case where the collecting step is not performed, the second liquid flows as follows.
The second liquid that has flowed to the upstream side connection flow passage 170a exits from a connection plane of the connection flow passage 170 whose plane is connected to the main flow passage 155, and then separates and flows to the two discarding flow passages 158. Because the second liquid thus exits from the connection plane, the first liquid and microparticles that may not need to be collected into the collection flow passage 159 can be prevented from entering the collection flow passage 159 through the connection flow passage 170.
The second liquid that has flowed to the downstream side connection flow passage 170b flows into the collection flow passage 159. Consequently, the inside of the collection flow passage 159 is filled with the second liquid, and the second liquid, for example, becomes a dispersion medium for forming an emulsion.

Also in a case of performing the collecting step, the second liquid can be supplied from the two liquid supply flow passages 161 to the connection flow passage 170. However, a pressure variation within the collection flow passage 159, particularly a negative pressure generated within the collection flow passage 159, forms a flow that flows from the main flow passage 155 through the connection flow passage 170 to the collection flow passage 159. That is, a flow is formed which flows from the main flow passage 155 to the collection flow passage 159 through the upstream side connection flow passage 170a, the connecting portion 170c, and the downstream side connection flow passage 170b in this order. Consequently, the collection target particle is collected, in a state of being wrapped in the first liquid, into the second liquid within the collection flow passage 159. When the collecting step is performed, an emulsion, for example, can be formed within the collection flow passage 159 or within a container connected to a collection flow passage end 163 via a flow passage, for example.

In the collecting step S203, due to a pressure variation within the collection flow passage 159, the collection target particle is collected into the collection flow passage through the connection flow passage. The collection may, for example, be performed by generating a negative pressure within the collection flow passage 159, as described in the above description. The negative pressure can be generated by the deformation of a wall defining the collection flow passage 159 by, for example, an actuator 197 (particularly a piezoelectric actuator) attached to the outside of the microchip 150. The negative pressure can form the flow that enters the collection flow passage 159. The actuator 197 can be attached to the outside of the microchip 150 to generate the negative pressure, for example, to be able to deform the wall of the collection flow passage 159. The deformation of the wall can change an inner space of the collection flow passage 159, and thereby generate the negative pressure. The actuator 197 can, for example, be a piezoelectric actuator. When the collection target particle is sucked into the collection flow passage 159, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can flow to the collection flow passage 159. Thus, the collection target particle is sorted in the particle sorting section 157 and is collected into the collection flow passage 159.

The collection target particle is collected, in a state of being wrapped in the first liquid, into the second liquid immiscible with the first liquid within the collection flow passage 159. Consequently, as described in the above description, an emulsion in which the second liquid is a dispersion medium and the first liquid is a dispersoid is formed within the collection flow passage 159.

The connection flow passage 170 is provided with the liquid supply flow passages 161 in order to prevent bioparticles that are not the collection target particle from entering the collection flow passage 159 through the connection flow passage 170. The second liquid that is immiscible with the liquid (the sample liquid and the sheath liquid) flowing through the main flow passage 155 is introduced into the connection flow passage 170 from the liquid supply flow passages 161.
A part of the second liquid introduced into the connection flow passage 170 forms a flow going from the connection flow passage 170 to the main flow passage 155. Bioparticles other than the collection target particle are thereby prevented from entering the collection flow passage 159. Due to the flow in which the first liquid that has flowed through the main flow passage 155 flows to the discarding flow passages 158, the second liquid formed by the flow going from the connection flow passage 170 to the main flow passage 155 flows through the discarding flow passages 158 as with the first liquid without flowing within the main flow passage 155.
Incidentally, the rest of the second liquid introduced into the connection flow passage 170 flows to the collection flow passage 159. Consequently, the inside of the collection flow passage 159 can be filled with the second liquid.

The inside of the collection flow passage 159 may be filled with the second liquid that is immiscible with the first liquid. In order to fill the inside of the collection flow passage 159 with the second liquid, the second liquid can be supplied from the liquid supply flow passages 161 to the connection flow passage 170. The supply causes the second liquid to flow from the connection flow passage 170 to the collection flow passage 159. The inside of the collection flow passage 159 can be thereby filled with the second liquid.

The laminar flow that has flowed to the discarding flow passages 158 can be discharged at discarding flow passage ends 160 to the outside of the microchip. In addition, the collection target particle collected into the collection flow passage 159 can be discharged at a collection flow passage end 161 to the outside of the microchip.

A container (not illustrated) can be connected to the collection flow passage end 163 via a flow passage such as a tube. The emulsion in which the first liquid including the collection target particle is a dispersoid and the second liquid is a dispersion medium is collected into the container. Thus, the bioparticle sorting device may include a flow passage for collecting the emulsion including the collection target particle into the container.
In addition, when a collecting operation is performed with the collection flow passage end 163 closed, multiple emulsion particles can be held within the collection flow passage 159. After an end of the collecting operation, it is possible also to continue to perform an assay such as single cell analysis within the collection flow passage 159. For example, the destroying step to be described later may be performed within the collection flow passage 159. Then, with the destroying step, binding between a target capturing molecule and a target substance may be performed.

Incidentally, as illustrated in FIG. 14A, in the microchip, among inlets through which liquid is introduced and outlets through which liquid is discharged, for example, two or more inlets and/or outlets, preferably all of the inlets and/or the outlets may be formed in one surface. As illustrated in the figure, each of the collection flow passage end 163 and two branched flow passage ends 160 is formed in a surface in which the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. Further, an introduction flow passage inlet 164 for introducing liquid into the introduction flow passages 161 is also formed in the surface. Thus, in the microchip, all of the inlets through which liquid is introduced and the outlets through which liquid is discharged are formed in one surface. Consequently, the attachment of the chip to the bioparticle sorting device is facilitated.
Incidentally, in the figure, a part of the sheath liquid flow passages 154 is indicated by a dotted line. The part indicated by the dotted line is located at a position lower than the sample liquid flow passage 152 indicated by a solid line (at a position displaced in an optical axis direction indicated by an arrow). At a position at which the flow passages indicated by the dotted line and the flow passage indicated by the solid line intersect each other, these flow passages do not communicate with each other. This description applies also to a part of the collection flow passage 159 which part is indicated by a dotted line and the branched flow passage 158 that intersects the part.

In addition, the liquid supply flow passage supplies liquid (second liquid in particular) to the connection flow passage. Consequently, a flow that flows from a connection position between the liquid supply flow passage and the connection flow passage to the main flow passage is formed within the connection flow passage. It is thereby possible to prevent the liquid flowing through the main flow passage from entering the connection flow passage and is also possible to prevent microparticles other than the collection target particle from flowing to the collection flow passage through the connection flow passage. As described in the above description, when the collecting step is performed, a negative pressure generated within the collection flow passage, for example, causes the first liquid including one collection target particle to be collected into the second liquid in the collection flow passage through the connection flow passage. Consequently, an emulsion particle including the one collection target particle is formed in the second liquid.

In addition, the hydrophilic solution including the collection target particle is collected into the collection flow passage 159 by, for example, driving the piezoelectric actuator in appropriate timing (for example, at a point in time that a bioparticle determined to be the collection target particle in the determining step reaches the particle sorting section 157). The emulsion particle is thereby formed. When whether the particle is the collection target particle is determined by using, for example, a peak signal and an area signal in the determining step, it is possible also to determine whether the particle is one microparticle (singlet), whether the particle is one in which two bioparticles are bound to each other (doublet), or whether the particle is one in which three bioparticles are bound to each other (triplet). It is therefore possible to avoid the formation of an emulsion particle including two or more cells in the one emulsion particle. Therefore, an emulsion particle including one cell can be formed with a high probability and a high efficiency. In addition, because the formation of an emulsion particle including two or more cells bound to each other can be thus avoided, it is possible to omit an operation of removing a bound substance of two or more cells before an emulsion forming operation by such as a cell sorter.

(4-2) Modification of Single Cell Analysis
In single cell analysis using the analyzing method according to an embodiment of the present disclosure, the circular nucleic acid generating process used for the analyzing method is not particularly limited, and an optional method can be combined with the circular nucleic acid generating process, as long as the circular nucleic acid generating process generates a circular nucleic acid by concatenating the two or more complementary strands generated by the complementary nucleic acid generating step described above via the nucleic acid concatenating portion.

FIG. 6D illustrates that, as a modification of the single cell analysis, the circular nucleic acid generating process in the single cell analysis illustrated in FIG. 5 is performed by the first modification of the circular nucleic acid generating process of (2-3). However, a nucleic acid concatenating portion illustrated in FIG. 6E is used as a nucleic acid concatenating portion to be used in the circular nucleic acid generating process in FIG. 6D. The nucleic acid concatenating portion illustrated in FIG. 6E is different from the nucleic acid concatenating portion illustrated in <2D-α> used in the description of the first modification of the circular nucleic acid generating process of (2-3) in that the nucleic acid concatenating portion illustrated in FIG. 6E has a sequence additional portion (Cell barcode in the figure) between the target nucleic acid capturing portion (poly-T in the figure) and the complementary strand capturing portion (concatenator in the figure).

Also in step S610 of FIG. 6D, as in step S42 in FIG. 5 according to the single cell analysis described above, each cell set free from the substrate is isolated in a minute space on a one-by-one basis while each cell has the nucleic acid concatenating portion bound thereto via the cell capturing portion. Thereafter, each cell is destroyed in the isolated state. As these conditions, conditions similar to those applicable to the single cell analysis described above can be suitably applied also in the present modification. Incidentally, as for the binding of the nucleic acid concatenating portion via the cell capturing portion in this case, the nucleic acid concatenating portion and the cell capturing portion may be directly bound to each other, whereas, as illustrated in FIG. 6D, the nucleic acid concatenating portion may be bound to the cell capturing portion via a nucleic acid having a sequence complementary to the sequence of the nucleic acid concatenating portion. In a case where the nucleic acid concatenating portion is bound to the cell capturing portion via the nucleic acid having the sequence complementary to the sequence of the nucleic acid concatenating portion, it is possible to avoid the cell capturing portion from becoming a steric hindrance in the subsequent circular nucleic acid generating process.

With the destruction, as illustrated in step S611 of FIG. 6D, the target nucleic acid capturing portion included in the nucleic acid concatenating portion illustrated in FIG. 6F captures the 3’ terminus of an mRNA in the destroyed cell. Subsequent steps are similar to those of the first modification of the circular nucleic acid generating process of (2-3), and conditions similar to those applicable to the circular nucleic acid generating process can be suitably applied also in the present modification.

It is to be noted that while an example in which the circular nucleic acid generating process in the single cell analysis is performed by the first modification of the circular nucleic acid generating process of (2-3) has been illustrated as a modification of the single cell analysis, this is a mere example, and in the single cell analysis using the analyzing method according to an embodiment of the present disclosure, as the circular nucleic acid generating process, including examples disclosed in the present specification, methods can be suitably used which can generate the circular nucleic acid by concatenating the two or more complementary strands generated by the complementary nucleic acid generating step to each other via the nucleic acid concatenating portion.

(4-3) Example of Application of Single Cell Analysis
The expression analysis of the mRNA of a single cell by using the analyzing method according to an embodiment of the present disclosure can be used in combination with another optional analysis as long as desired analysis characteristics are not significantly impaired.

For example, at the same time as the analysis of the mRNA within the cell, analysis of a molecule expressed within a cell, a molecule expressed on a cell surface, or a secreted molecule captured on the cell surface can be performed by using an antibody-nucleic acid molecule complex, in which the antibody is bound to the nucleic acid molecule including a sequence as an antibody identifier sequence (Antibody barcode) for identifying the source of the antibody and a poly-A sequence as a sequence to be captured by the target nucleic acid capturing portion included in the nucleic acid concatenating portion, as illustrated in FIG. 14C. In the following, description will be made concretely with reference to drawings. Incidentally, as for the antibody-nucleic acid molecule complex described above, as in an example illustrated in FIG. 14C, the nucleic acid molecule included in the antibody-nucleic acid molecule complex may include a priming sequence (Amp. Primer) in addition to the antibody identifier sequence and the poly-A sequence. In addition, an optional nucleic acid concatenating portion disclosed in the present specification can be suitably used as the nucleic acid concatenating portion used in the present application example.

In the antibody-nucleic acid molecule complex illustrated in FIG. 14C, as the antibody included in the complex, an antibody is used which specifically binds to a molecule expressed within the cell, a molecule expressed on the cell surface, or a secreted molecule captured on the cell surface. In addition, a sequence unique to each antibody is provided as the antibody identifier sequence possessed by the nucleic acid molecule included in the complex. That is, in a case where there are multiple secreted molecules on the cell surface to be analyzed, multiple kinds of antibody-nucleic acid molecule complexes are prepared according to the kinds. Further, the antibody identifier sequence may have the above-described error correction function. In addition, while FIG. 14 illustrates an example of using the antibody-nucleic acid molecule complex, the object that forms the complex with the nucleic acid molecule is not limited to the antibody as long as the object has the function of binding to a molecule expressed within the cell, a molecule expressed on the cell surface, or a secreted molecule captured on the cell surface. For example, an antibody fragment (Fab, scFv, VHH, Minobody, or the like), an aptamer, a molecularly imprinted polymer, or the like can be used. Complexes in which these bind to nucleic acid molecules may be used in the present application example.

The above-described prepared antibody-nucleic acid molecule complex binds to the cell having a molecule as an antigen expressed on the surface or a secreted molecule captured on the surface due to an antigen-antibody reaction. On the other hand, the antibody-nucleic acid molecule complex does not bind to the cell in a case where the molecule as the antigen is not expressed within the cell, expressed on the surface, nor captured on the surface.

The cell bound with the antibody corresponding to a molecule expressed within the cell, a molecule expressed on the cell surface, or a secreted molecule captured on the surface is obtained by subjecting the cell bound with the nucleic acid concatenating portion via the cell capturing portion described above to the antigen-antibody reaction using the above-described prepared antibody-nucleic acid molecule complex.

Next, the above-described cell resulting from the antigen-antibody reaction is isolated into a minute space. FIG. 14D illustrates an example in which the cell is isolated into the minute space by using a chip for forming an emulsion. However, there is no limitation to this method, and optional methods for isolating the cell into the minute space, including the examples disclosed in the present specification, can be suitably adopted.

Incidentally, the example of FIG. 14D represents an example in which one antibody-nucleic acid molecule complex binds to the cell bound with the nucleic acid concatenating portion via the cell capturing portion as a cell isolated in a space within an emulsion particle. However, there are also cases where multiple kinds of antibody-nucleic acid molecule complexes bind to one cell. In addition, as conditions in the case of isolating the cell resulting from the antigen-antibody reaction into the space within the emulsion particle, as illustrated in FIG. 14D, conditions similar to those applicable in the method illustrated in the foregoing (in the case of the space within the emulsion particle) can be suitably applied also in the present application example.

Next, each cell is destroyed under conditions similar to those applicable to the above-described single cell analysis or the modification thereof.

With the destruction, as illustrated in step S1411 of FIG. 14E, the target nucleic acid capturing portion included in the nucleic acid concatenating portion captures a poly-A sequence at the 3’ terminus of an mRNA in the destroyed cell or the poly-A sequence at the 3’ terminus of the nucleic acid molecule included in the antibody-nucleic acid molecule complex. Subsequent steps are similar to those of the circular nucleic acid generating process of (2-2). When the steps are performed, a single-stranded circular nucleic acid is synthesized, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized. Incidentally, conditions similar to those applicable to the circular nucleic acid generating process can be suitably applied also in the present application example.

The single-stranded circular nucleic acid obtained as a result of the foregoing includes the antibody identifier sequence included in the nucleic acid molecule of the antibody-nucleic acid molecule complex in addition to the sequence derived from the mRNA. It is therefore possible to analyze the expression and secretion conditions of the molecule as the antigen to which the antibody identified by the antibody identifier sequence binds by decoding the base sequence of the single-stranded linear nucleic acid obtained with the single-stranded circular nucleic acid as a template. That is, in addition to the expression analysis of the mRNA of the cell isolated in the minute space, the expression and secretion conditions of the molecules expressed and secreted on the cell surface can be simultaneously analyzed.

Incidentally, the identification of the antibody identifying sequence can be made easier by, for example, providing antibody identifier sequences having different base sequence lengths for respective antibodies. In this case, the base sequences of the antibody identifier sequences may be adjusted in a range of 5 to 25 bases, for example.

Incidentally, as for the decoding of the base sequence of the obtained single-stranded linear nucleic acid, a method similar to that applicable in the above-described circular nucleic acid generating process of (2-2), for example, can be suitably applied also in the present modification.

In addition, while an example in which the circular nucleic acid generating process is performed by the circular nucleic acid generating process of (2-2) has been illustrated as an example of application of the single cell analysis, this is a mere example, and in the single cell analysis using the analyzing method according to an embodiment of the present disclosure, as the circular nucleic acid generating process, including examples disclosed in the present specification, methods can be suitably used which can generate the circular nucleic acid by concatenating the two or more complementary strands generated by the complementary nucleic acid generating step to each other via the nucleic acid concatenating portion.

(5) Example 4 (Modification of Nucleic Acid Concatenating Portion by Phosphorylation)

Of the two base sequence strands constituting the double stranded portion of the nucleic acid concatenating portion, the strand coupled to the target nucleic acid capturing portion (strand having a poly-T in particular) may have the 5’ terminus thereof modified by phosphorylation. It is thereby possible to reliably perform ligation processing to be described later more. This will be described with reference to FIG. 7.

As illustrated in A of the figure, a cell is captured by a nucleic acid concatenating portion immobilized on a substrate.

In step S51, as described with reference to FIG. 5 in the above description, the cell having the nucleic acid concatenating portion bound thereto is set free from the substrate and is isolated into a minute space (for example, an emulsion particle). The cell is destroyed in a state of being thus isolated. Consequently, as illustrated in B of the figure, an mRNA is released into the minute space.

In step S52, the mRNA released into the minute space by the destruction of the cell binds to a nucleic acid concatenating portion (enclosed by a broken line) to form a hybrid as illustrated in C of the figure. The hybrid is generated by complementary binding between a target nucleic acid capturing portion (poly-T sequence in particular) of the nucleic acid concatenating portion and a poly-A tail portion of the mRNA.

Here, the 5’ terminus P of the strand coupled to the target nucleic acid capturing portion (strand having a poly-T in particular) among the two base sequence strands of the nucleic acid concatenating portion is modified by phosphorylation. In the figure, being phosphorylated is indicated by a circle shape. The phosphorylation is useful for performing a ligation step in a subsequent stage more reliably.

Step S53 is cDNA synthesis that is the same as that in step S12 in FIG. 2A described in the above description. The description of step S12 applies also to step S53.
When step S53 is performed, a cDNA having a CCC sequence at a terminus of a generated strand is generated, as illustrated in D of FIG. 7.

Step S54 is nucleic acid concatenation that is the same as that in step S13 in FIG. 2A described in the above description. The description of step S13 applies also to step S54.
When step S54 is performed, two or more cDNAs are concatenated to each other via the complementary strand capturing portion, as illustrated in E of FIG. 7.

Step S55 is circular nucleic acid formation, ligation, and primer addition that are the same as those in steps S14 and 15 in FIG. 2A described in the above description. The description of steps S14 and S15 applies also to step S55.
When step S55 is performed, a circular nucleic acid to which a primer is added is formed, as illustrated in F of FIG. 7.
Here, because the 5’ terminus of the nucleic acid concatenating portion is phosphorylated as described above, the ligation is performed more reliably. Consequently, the circular nucleic acid can be formed more efficiently.

Step S56 is nucleic acid amplification that is the same as that in step S16 in FIG. 2A described in the above description. The description of step S16 applies also to step S56.
When step S56 is performed, the nucleic acid is amplified, as illustrated in G of FIG. 7. The nucleic acid thus amplified is used in single cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed by using a result of the sequencing.

(6) Example 5 (Nucleic Acid Concatenating Portion Including Self-Bonding Suppressing Sequence)

The complementary strand capturing portion of the nucleic acid concatenating portion is not limited to the GGG sequence and may include a self-bonding suppressing sequence (for example, Hn and/or Nn) in addition to GGG. Here, H is a base other than G, that is, H is A, T, or C. N is A, T, G, or C. n is the number of Hs or Ns and may, for example, be an integer of 1 or more. n may, for example, be an integer of any of 1 to 5. In a case where n is 2 or more, each H or N constituting the self-bonding suppressing sequence may be selected independently and randomly.
In a case where the complementary strand capturing portion is, for example, a base sequence having a self-bonding suppressing sequence of one base in addition to the GGG sequence, the complementary strand capturing portion may have a base sequence of GGGH or GGGN. In a case where the self-bonding suppressing sequence is two bases, the complementary strand capturing portion may have a base sequence of GGGHN, GGGNH, GGGNN, or GGGHH.
An example of the formation of the circular nucleic acid in a case where such a complementary strand capturing portion is provided is illustrated in FIG. 8.

In step S61, as described with reference to FIG. 5 in the above description, the cell having the nucleic acid concatenating portion bound thereto is set free from the substrate and is isolated into a minute space (for example, an emulsion particle). The cell is destroyed in a state of being thus isolated. Consequently, an mRNA is released into the minute space, as illustrated in B of the figure.

In step S62, the mRNA released into the minute space by the destruction of the cell binds to a nucleic acid concatenating portion to form a hybrid as illustrated in C of the figure. The hybrid is generated by complementary binding between a target nucleic acid capturing portion (poly-T sequence in particular) of the nucleic acid concatenating portion and a poly-A tail portion of the mRNA.

Incidentally, the 5’ terminus of the strand coupled to the target nucleic acid capturing portion (strand having a poly-T in particular) among the two base sequence strands of the nucleic acid concatenating portion is modified by phosphorylation. In the figure, being phosphorylated is indicated by a circle shape. The phosphorylation is useful for performing a ligation step in a subsequent stage more reliably.

Step S63 is cDNA synthesis that is the same as that in step S12 in FIG. 2A described in the above description. The description of step S12 applies also to step S63.
When step S63 is performed, a cDNA having a CCC sequence at a terminus of a generated strand is generated, as illustrated in D of FIG. 8.

In step S64, RNA digestion and nucleic acid concatenation are performed. Consequently, as illustrated in E of the figure, the mRNA is digested, and a single stranded part is formed. The cDNA to be captured by the complementary strand capturing portion 52 is selected on the basis of a sequence H* on the 5’ terminus side of the CCC sequence of 3 at the 3’ terminus of the single stranded part. That is, because there is H*, not all of cDNAs are captured by the complementary strand capturing portion, but in a case where the complementary strand capturing portion is GGGH-3’, for example, a cDNA having 3’-CCCH* is captured. Because H is four kinds of ATGC, a probability of circular formation by only one cDNA is 1/4. As n of Hn (n is an integer of 1 or more) is increased, the probability is decreased and becomes (1/4)ⁿ. Consequently, a nucleic acid is generated in which two or more cDNAs are concatenated to each other, the two or more cDNAs being bound to each other via the nucleic acid concatenating portion.
Thus, a circular nucleic acid including only one cDNA can be prevented from being formed. That is, more circular nucleic acids in which two or more cDNAs are concatenated to each other can be generated.

In step S65, circular nucleic acid formation and ligation are performed. Consequently, as illustrated in F of FIG. 8, a circular nucleic acid is generated.
Here, because the 5’ terminus of the nucleic acid concatenating portion is phosphorylated as described above, the ligation is performed more reliably. Consequently, the circular nucleic acid can be formed more efficiently.

Step S66 is nucleic acid amplification that is the same as that in step S16 in FIG. 2A described in the above description. The description of step S16 applies also to step S66.
When step S66 is performed, the nucleic acid is amplified, as illustrated in G of FIG. 8. The nucleic acid thus amplified is used in single cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed by using a result of the sequencing.

(7) Example 6 (Nucleic Acid Concatenating Portion Including Priming Sequence)

As described in the above description, the double stranded portion of the nucleic acid concatenating portion may have a base sequence different for the respective regions, and the base sequence may, for example, be a random sequence. The double stranded portion may have a priming sequence in addition to the random sequence. The priming sequence may be the same in two or more regions, and, for example, the nucleic acid concatenating portions present in all of the regions may have the same priming sequence. Consequently, in the nucleic acid amplification processing, nucleic acid amplification can be performed by using the common priming sequence. It is therefore unnecessary to add a primer before the nucleic acid amplification processing to be performed. This will be described with reference to FIG. 9.

In step S71, as described with reference to FIG. 5 in the above description, the cell having the nucleic acid concatenating portion bound thereto is set free from the substrate and is isolated into a minute space (for example, an emulsion particle). The cell is destroyed in a state of being thus isolated. Consequently, as illustrated in B of the figure, an mRNA is released into the minute space.

In step S72, the mRNA released into the minute space by the destruction of the cell binds to a nucleic acid concatenating portion to form a hybrid as illustrated in C of FIG. 9. The hybrid is generated by complementary binding between a target nucleic acid capturing portion (poly-T sequence in particular) of the nucleic acid concatenating portion and a poly-A tail portion of the mRNA. The nucleic acid concatenating portion has a priming sequence in the double stranded portion of the nucleic acid concatenating portion.

In addition, the 5’ terminus of the strand coupled to the target nucleic acid capturing portion (strand having a poly-T in particular) among the two base sequence strands of the nucleic acid concatenating portion is modified by phosphorylation. In the figure, being phosphorylated is indicated by a circle shape. The phosphorylation is useful for performing a ligation step in a subsequent stage more reliably. Incidentally, phosphorylation modification may not be performed.

Step S73 is cDNA synthesis that is the same as that in step S12 in FIG. 2A described in the above description. The description of step S12 applies also to step S73.
When step S73 is performed, a cDNA having a CCC sequence at a terminus of a generated strand is generated, as illustrated in D of FIG. 9.

Step S74 is nucleic acid concatenation that is the same as that in step S13 in FIG. 2A described in the above description. The description of step S13 applies also to step S74.
When step S54 is performed, two or more cDNAs are concatenated to each other via the complementary strand capturing portion, as illustrated in E of FIG. 9.

Step S75 is circular nucleic acid formation and ligation that are the same as those in step S14 in FIG. 2A described in the above description. The description of step S14 applies also to step S75.
When step S75 is performed, a circular nucleic acid to which a primer is added is formed, as illustrated in F of FIG. 9.
Here, because the 5’ terminus of the nucleic acid concatenating portion is phosphorylated as described above, the ligation is performed more reliably. Consequently, the circular nucleic acid can be formed more efficiently.
In addition, while primer addition is performed in the method described with reference to FIG. 2A, the primer addition does not have to be performed because the nucleic acid concatenating portion has a priming sequence, as described above.

Step S76 is nucleic acid amplification that is the same as that in step S16 in FIG. 2A described in the above description. The description of step S16 applies also to step S76.
When step S76 is performed, the nucleic acid is amplified, as illustrated in G of FIG. 9. The nucleic acid thus amplified is used in single cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed by using a result of the sequencing.

FIG. 19 is a schematic diagram illustrating an example of a case where priming sequences are provided to a nucleic acid concatenating portion. It can be confirmed that, in the example illustrated in FIG. 19, as opposed to a nucleic acid concatenating portion not provided with the priming sequences as illustrated in <19A>, the double stranded portion of the nucleic acid concatenating portion of <19B> is provided with priming sequences. Incidentally, while the example illustrated in FIG. 19 represents an example in which the priming sequences are provided to the nucleic acid concatenating portion having the double stranded portion, the priming sequences can also be provided to a nucleic acid concatenating portion not having the double stranded portion.

(8) Example 7 (Nucleic Acid Concatenating Portion Including Self-Bonding Suppressing Sequence and Priming Sequence)

The nucleic acid concatenating portion may include both the self-bonding suppressing sequence and the priming sequence described in the above description. Consequently, the formation of circular nucleic acids having only one cDNA is suppressed, and a primer adding step before the nucleic acid amplification processing is rendered unnecessary. An example of the formation of a circular nucleic acid in a case where such a complementary strand capturing portion is provided is illustrated in FIG. 10.

In step S81, as described with reference to FIG. 5 in the above description, the cell having the nucleic acid concatenating portion bound thereto is set free from the substrate and is isolated into a minute space (for example, an emulsion particle). The cell is destroyed in a state of being thus isolated. Consequently, an mRNA is released into the minute space, as illustrated in B of the figure.

In step S82, the mRNA released into the minute space by the destruction of the cell binds to a nucleic acid concatenating portion to form a hybrid as illustrated in C of the figure. The hybrid is generated by complementary binding between a target nucleic acid capturing portion (poly-T sequence in particular) of the nucleic acid concatenating portion and a poly-A tail portion of the mRNA. The nucleic acid concatenating portion has a priming sequence in the double stranded portion of the nucleic acid concatenating portion.

Step S83 is cDNA synthesis that is the same as that in step S12 in FIG. 2A described in the above description. The description of step S12 applies also to step S83.
When step S83 is performed, a cDNA having a CCC sequence at a terminus of a generated strand is generated, as illustrated in D of FIG. 10.

In step S84, RNA digestion and nucleic acid concatenation are performed. Consequently, as illustrated in E of the figure, the mRNA is digested, and a single stranded part is formed. An optional base H* is also exposed in front of the CCC sequence of 3 at the 3’ terminus of the single stranded part. That is, a CCCH* sequence is present at the 3’ terminus of the single stranded part. On the other hand, because the nucleic acid concatenating portion is DNA, the nucleic acid concatenating portion is not digested in the RNA digestion. Consequently, the double stranded portion and GGGH that were coupled to the digested mRNA continue to exist. Then, GGGH binds to complementary CCCH*. Consequently, a nucleic acid is generated in which two or more cDNAs are concatenated to each other, the two or more cDNAs being bound to each other via the nucleic acid concatenating portion.
Thus, a circular nucleic acid including only one cDNA can be prevented from being formed. That is, more circular nucleic acids in which two or more cDNAs are concatenated to each other can be generated.

In step S85, circular nucleic acid formation and ligation are performed. Consequently, as illustrated in F of FIG. 8, a circular nucleic acid is generated.
Here, because the 5’ terminus of the nucleic acid concatenating portion is phosphorylated as described above, the ligation is performed more reliably. Consequently, the circular nucleic acid can be formed more efficiently.
In addition, while primer addition is performed in the method described with reference to FIG. 2A, the primer addition does not have to be performed because the nucleic acid concatenating portion has a priming sequence, as described above.

Step S86 is nucleic acid amplification that is the same as that in step S16 in FIG. 2A described in the above description. The description of step S16 applies also to step S66.
When step S86 is performed, the nucleic acid is amplified, as illustrated in G of FIG. 10. The nucleic acid thus amplified is used in single cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed by using a result of the sequencing.

(9) Example 8 (Example of Analysis of Sequence Result)

In the analyzing method according to an embodiment of the present disclosure, the nucleic acid amplification processing using the circular nucleic acid as a template is performed. Here, in the circular nucleic acid, sequences corresponding to two or more target nucleic acids are concatenated to each other. For example, two or more cDNAs complementary to mRNAs are concatenated to each other. Therefore, a product obtained in the nucleic acid amplification processing has a repetition unit including the sequences corresponding to the two or more target nucleic acids.

For example, as illustrated in a of FIG. 11, in a case where one formed circular nucleic acid has two cDNAs, a nucleic acid in which a repetition unit including the two cDNAs concatenated to each other repeatedly appears is obtained by performing RCA processing on the circular nucleic acid.
In addition, in a case where one formed circular nucleic acid has three cDNAs, a nucleic acid in which a repetition unit including the three cDNAs concatenated to each other repeatedly appears is obtained by performing the RCA processing on the circular nucleic acid.
As illustrated in b of the figure, also in a case where the nucleic acid concatenating portion has a self-bonding suppressing sequence, a product obtained in the nucleic acid amplification processing has a repetition unit including the sequences corresponding to the two or more target nucleic acids.
In the analyzing method according to an embodiment of the present disclosure, analysis based on the repetition unit may be performed. For example, analysis based on the kinds and/or the number of target nucleic acid sequences included in the repetition unit may be performed.
The nucleic acid amplification processing reduces the bias described in the above description. Therefore, an analysis result is obtained in which target nucleic acids present in small numbers within cells are taken into consideration appropriately.

(10) Example 9 (Example of Generation of Circular Nucleic Acid and Amplification Processing on Generated Circular Nucleic Acid)

Nucleic acid concatenating portions having base sequences different for arrayed regions on the substrate are arranged as illustrated in FIG. 5. The double stranded portions of the nucleic acid concatenating portions have a sequence common to all of the regions (for example, the priming sequence described in the above description) in addition to random sequences different for the respective regions. The common sequence may be used as a priming sequence in the nucleic acid amplification processing, as described in the above description, or may be used to bind the cell capturing portion.

The nucleic acid concatenating portions are manufactured by using a synthesizing technology using the oligo pools described in the above description. That is, oligo nucleotides having many kinds of different base sequences are synthesized en bloc, and thereafter PCR processing is performed on the oligo nucleotides, so that many kinds of nucleic acid concatenating portions having different base sequences are obtained at low cost and in large amounts. More specifically, after the PCR processing, IVT and RT (reverse transcription) are performed. In addition, a cell capturing portion such as an antibody or a lipid is bound to the terminuses of the nucleic acid concatenating portions.

Cell capturing processing is performed by using the substrate on which the nucleic acid concatenating portions described above are arrayed. After the capturing of the cells, the cells are set free from the substrate by using the restriction enzyme, and each cell is sealed in a water-in-oil droplet. Cytolysis and the capturing of an mRNA by a poly-T sequence are performed within the droplet.
For the cytolysis, a lysis reagent such as an NP-40 Surfactant-Amps Detergent solution or IGEPAL CA-630 is used.

After the capturing of the mRNA, a reverse transcription reaction is performed, and thereby a cDNA of the mRNA is synthesized. The reverse transcription reaction is performed for 50 minutes and at 50°C under the presence of a dNTP, a 1xThermopol buffer (or a 1x RT buffer), SuperScript III, and RNasin plus (or RNase OUT).

Next, a GGG sequence included in the nucleic acid concatenating portion complementarily binds to a terminus CCC sequence provided at a time of the cDNA synthesis. Two or more cDNAs are thereby concatenated to each other. After the concatenation, the RNA is degraded by RNase H or the like. Then, ligation processing is performed at 37°C and for 30 minutes or at 45°C and for 45 minutes under the presence of a 1x Ampligase buffer, a 50 μM dNTP, 0.5 U/μL Ampligase, and 50 mM KCl. A circular nucleic acid is thus obtained. Incidentally, Formamide may be added at a concentration of approximately 20% to liquid in which the above-described ligation processing is performed.

RCA is performed by using the obtained circular nucleic acid. The RCA is performed at 30°C and for 60 minutes under the presence of a 1 U/μL Phi29 polymerase, a 1xPhi29 polymerase buffer, 0.25 mM dNTPs, 0.2 μg/μL BSA, and 5% glycerol. Consequently, an amplification product having a repetition unit complementary to the circular nucleic acid is obtained. The amplification product is useful for performing single cell analysis. In particular, an analysis result is obtained in which nucleic acids present in small numbers within cells are reflected appropriately.

2. Second Embodiment (Circular Nucleic Acid Manufacturing Method)

The present disclosure also provides a method of manufacturing a circular nucleic acid. The circular nucleic acid is the circular nucleic acid generated in the analyzing method described in the foregoing 1. Therefore, the description of the method for generating the circular nucleic acid applies also to the circular nucleic acid manufacturing method according to an embodiment of the present disclosure.
In one embodiment, the manufacturing method includes a complementary nucleic acid generating step of generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid, and a circular nucleic acid generating step of concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid.
The manufacturing method according to an embodiment of the present disclosure can manufacture the circular nucleic acid that is useful for cell analysis. The effects described in the foregoing 1. are produced by the manufacturing method.

3. Third Embodiment (Nucleic Acid)

The present disclosure also provides a nucleic acid. The nucleic acid is a nucleic acid corresponding to the nucleic acid concatenating portion described in the foregoing 1. Therefore, the description of the nucleic acid concatenating portion applies also to the nucleic acid according to an embodiment of the present disclosure. In addition, the present disclosure also provides a complex including the nucleic acid. The complex is the complex 100 described in the foregoing 1., and the description thereof applies also to the present embodiment.
In one embodiment, the nucleic acid includes a target nucleic acid capturing portion configured to capture a 3’ terminus region of a target nucleic acid, a complementary strand capturing portion configured to capture a 3’ terminus region of a complementary strand produced by complementary strand generation of the target nucleic acid, and a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other. Preferably, the target nucleic acid capturing portion is a single strand, and the complementary strand capturing portion is a single strand. In addition, the nucleic acid may be used to generate a circular nucleic acid.
The nucleic acid according to an embodiment of the present disclosure is useful for generating the circular nucleic acid described in the above description and is further useful also for cell analysis. The effects described in the foregoing 1. are produced by the nucleic acid.

Incidentally, the present disclosure can also adopt the following configurations.
<1>
An analyzing method including:
generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid;
concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid; and
performing analysis using the circular nucleic acid.
<2>
The analyzing method according to <1>, in which
the nucleic acid concatenating portion includes a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid.
<3>
The analyzing method according to <1> or <2>, in which,
in the generating of the complementary strands, target nucleic acid complementary strand generation is performed with the nucleic acid concatenating portion as a primer.
<4>
The analyzing method according to any one of <1> to <3>, in which,
in the generating of the complementary strands, a double strand of each target nucleic acid and the complementary strand of each target nucleic acid is formed.
<5>
The analyzing method according to <4>, in which,
in the generating of the circular nucleic acid, the double strand is concatenated via the nucleic acid concatenating portion.
<6>
The analyzing method according to any one of <1> to <5>, in which
the nucleic acid concatenating portion includes a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand.
<7>
The analyzing method according to any one of <1> to <6>, in which,
in the generating of the circular nucleic acid, a 5’ terminus of one complementary strand and a 3’ terminus of another complementary strand are concatenated to each other, and
the concatenation is performed in a state in which a complementary strand capturing portion of the nucleic acid concatenating portion bound to the one complementary strand and a 3’ terminus region of the other complementary strand are bound to each other.
<8>
The analyzing method according to any one of <1> to <7>, in which,
in the generating of the circular nucleic acid, a single stranded circular nucleic acid in which the complementary strands are concatenated to each other is obtained by forming a double stranded circular nucleic acid and then removing the target nucleic acid from the double stranded circular nucleic acid.
<9>
The analyzing method according to any one of <1> to <8>, in which,
in the analyzing, a nucleic acid amplification reaction using the circular nucleic acid is performed.
<10>
The analyzing method according to <9>, in which
the nucleic acid amplification reaction includes rolling circle amplification or polymerase chain reaction.
<11>
The analyzing method according to any one of <1> to <10>, in which
the nucleic acid concatenating portion includes
a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid,
a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand generated in the generating of the complementary strands, and
a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
<12>
The analyzing method according to <11>, in which
the target nucleic acid capturing portion has a poly-T sequence, and
the complementary strand capturing portion has a base sequence complementary to a base sequence provided to a 3’ terminus at a time of reverse transcription by a reverse transcriptase.
<13>
The analyzing method according to <11> or <12>, in which
the double stranded portion has a restriction enzyme recognition sequence.
<14>
The analyzing method according to any one of <11> to <13>, in which
the double stranded portion has a non-natural base sequence.
<15>
The analyzing method according to any one of <11> to <14>, in which
the double stranded portion has a base sequence having an error correcting function.
<16>
The analyzing method according to any one of <11> to <15>, in which
the analyzing method includes an analyzing method for performing single cell analysis, and
the nucleic acid concatenating portion including the double stranded portion different for each cell is used.
<17>
The analyzing method according to any one of <1> to <16>, in which
the analyzing method includes destroying a cell, and
the generating of the complementary nucleic acid is performed on the target nucleic acid included in the cell.
<18>
The analyzing method according to <16> or <17>, in which
the destroying the cell is performed within a space partitioned for each cell.
<19>
The analyzing method according to any one of <1> to <18>, in which
the nucleic acid concatenating portion is immobilized on a substrate.
<20>
A circular nucleic acid manufacturing method including:
generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid; and
concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid.
<21>
A nucleic acid including:
a target nucleic acid capturing portion configured to capture a 3’ terminus region of a target nucleic acid;
a complementary strand capturing portion configured to capture a 3’ terminus region of a complementary strand produced by complementary strand generation of the target nucleic acid; and
a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
<22>
The nucleic acid according to <21>, in which
the target nucleic acid capturing portion includes a single strand, and the complementary strand capturing portion includes a single strand.
<23>
The nucleic acid according to <21> or <22>, in which
the nucleic acid is used to generate a circular nucleic acid.
It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and alterations may occur depending on design requirements and other factors insofar as they are within the scope of the appended claims or the equivalents thereof.

10: Nucleic acid concatenating portion
11: Target nucleic acid capturing portion
12: Complementary strand capturing portion
13: Double stranded portion
14: Sequence additional portion
100: Complex

Claims

An analyzing method comprising:
generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid;
concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid; and
performing analysis using the circular nucleic acid.
The analyzing method according to claim 1, wherein
the nucleic acid concatenating portion includes a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid.
The analyzing method according to claim 1, wherein,
in the generating of the complementary strands, target nucleic acid complementary strand generation is performed with the nucleic acid concatenating portion as a primer.
The analyzing method according to claim 1, wherein,
in the generating of the complementary strands, a double strand of each target nucleic acid and the complementary strand of each target nucleic acid is formed.
The analyzing method according to claim 4, wherein,
in the generating of the circular nucleic acid, the double strand is concatenated via the nucleic acid concatenating portion.
The analyzing method according to claim 1, wherein
the nucleic acid concatenating portion includes a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand.
The analyzing method according to claim 1, wherein,
in the generating of the circular nucleic acid, a 5’ terminus of one complementary strand and a 3’ terminus of another complementary strand are concatenated to each other, and
the concatenation is performed in a state in which a complementary strand capturing portion of the nucleic acid concatenating portion bound to the one complementary strand and a 3’ terminus region of the other complementary strand are bound to each other.
The analyzing method according to claim 1, wherein,
in the generating of the circular nucleic acid, a single stranded circular nucleic acid in which the complementary strands are concatenated to each other is obtained by forming a double stranded circular nucleic acid and then removing the target nucleic acid from the double stranded circular nucleic acid.
The analyzing method according to claim 1, wherein,
in the analyzing, a nucleic acid amplification reaction using the circular nucleic acid is performed.
The analyzing method according to claim 9, wherein
the nucleic acid amplification reaction is rolling circle amplification or polymerase chain reaction.
The analyzing method according to claim 1, wherein
the nucleic acid concatenating portion includes
a target nucleic acid capturing portion configured to capture a 3’ terminus region of the target nucleic acid,
a complementary strand capturing portion configured to capture a 3’ terminus region of the complementary strand generated in the generating of the complementary strands, and
a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
The analyzing method according to claim 11, wherein
the target nucleic acid capturing portion has a poly-T sequence, and
the complementary strand capturing portion has a base sequence complementary to a base sequence provided to a 3’ terminus at a time of reverse transcription by a reverse transcriptase.
The analyzing method according to claim 11, wherein
the double stranded portion has a restriction enzyme recognition sequence.
The analyzing method according to claim 11, wherein
the double stranded portion has a non-natural base sequence.
The analyzing method according to claim 11, wherein
the double stranded portion has a base sequence having an error correcting function.
The analyzing method according to claim 11, wherein
the analyzing method is an analyzing method for performing single cell analysis, and
the nucleic acid concatenating portion including the double stranded portion different for each cell is used.
The analyzing method according to claim 1, wherein
the analyzing method includes destroying a cell, and
the generating of the complementary nucleic acid is performed on the target nucleic acid included in the cell.
The analyzing method according to claim 17, wherein
the destroying the cell is performed within a space partitioned for each cell.
The analyzing method according to claim 1, wherein
the nucleic acid concatenating portion is immobilized on a substrate.
A circular nucleic acid manufacturing method comprising:
generating complementary strands of one or more kinds of target nucleic acid in a state in which a nucleic acid concatenating portion is bound to one end of each of the one or more kinds of target nucleic acid; and
concatenating the two or more generated complementary strands to each other via the nucleic acid concatenating portion and forming a circular nucleic acid.
A nucleic acid comprising:
a target nucleic acid capturing portion configured to capture a 3’ terminus region of a target nucleic acid;
a complementary strand capturing portion configured to capture a 3’ terminus region of a complementary strand produced by complementary strand generation of the target nucleic acid; and
a double stranded portion that connects the target nucleic acid capturing portion and the complementary strand capturing portion to each other.
The nucleic acid according to claim 21, wherein
the target nucleic acid capturing portion is a single strand, and the complementary strand capturing portion is a single strand.
The nucleic acid according to claim 21, wherein
the nucleic acid is used to generate a circular nucleic acid.