WO2020218553A1 - Digital analysis of microbial flora - Google Patents

Abstract

The present disclosure provides a method for evaluating an absolute number of a microbial flora composition inexpensively, simply, and/or highly accurately. The method for analyzing a microbial flora composition comprises a step for evaluating the microbial flora composition from a specimen including amplified nucleic acids derived from respective cells in the microbial flora. According to this method, in an execution before whole-genome sequencing, the absolute number of the microbial flora can be evaluated inexpensively and simply, and after whole-genome sequencing, it becomes possible to compare with a long gene sequence by narrowing, from digital sequence information, information to be analyzed, thereby increasing the degree of accuracy.

Description

Digital microflora analysis

This disclosure is available in fields such as biological research, medicine, environment, and healthcare regarding the analysis of microbiota composition.

As a method for evaluating the diversity of environmental microorganisms, amplicon sequence analysis targeting the conserved regions of 16S rRNA genes and 18S rRNA genes has been widely used in recent years using a next-generation sequencer. When the 16SrRNA gene is targeted, a storage region of several hundred bases is PCR-amplified on DNA collectively extracted from a sample containing various microorganisms (feces, soil, seawater, etc.), and the storage region is stored by a next-generation sequencer. By comprehensively analyzing the base sequence, the microbiota composition can be evaluated as the type of base sequence = the type of microbial species. On the other hand, there are some problems with this method.

First, the copy number of the 16S rRNA gene on the genome is different for each bacterium. Some bacteria have only a single copy of the 16S rRNA gene, while others have more than 7 genes. Furthermore, since the genomes of most of the environmental microorganisms are undecided, the number of the genes is unknown and cannot be corrected.

Secondly, there is the problem of amplification bias during PCR. In the PCR amplification, amplification is performed for the storage region, but since the internal sequence is different for each microorganism, the amplification efficiency is different due to differences in the base sequence and the like. As a result, as the number of PCR amplification cycles increases, the bias due to the difference in amplification efficiency increases. For the above reasons, the composition ratio of the gene sequence finally detected after PCR amplification does not reflect the actual bacterial composition ratio. Moreover, since what is obtained by such an amplicon sequence analysis is only the composition ratio, the actual increase or decrease in the total amount of microorganisms is unknown. In other words, even if a sample is compared and an event is found in which the number of microorganisms X is smaller than that of other microorganisms, the total amount of the microorganisms X may have decreased, or the number of other microorganisms has simply increased and the number of microorganisms X has not changed. It is also possible.

Furthermore, in many microbiota analyzes, microorganisms with a composition ratio of 1% or less are collectively discussed as "others", but considering the above bias, the microbial composition is estimated to be more or less than the actual one. There is concern that there is. As a solution to this, DNA with a known sequence is used as an internal standard, and this is spiked into a sample for PCR (Non-Patent Document 1 (Stammler et al., 2016 Microbiome, 4 (1), 28)) and bacteria. There is a method of counting and correcting absolute numbers by another method. In the former, it is possible to make a relative comparison of the total number of microorganisms for each sample by comparing the total amount of read sequences with the internal standard, but the problem of PCR amplification still remains, and the absolute numbers of various bacteria are counted. I haven't been able to do that. In the latter, the absolute number of bacteria is counted by flow cytometry in Non-Patent Document 2 (Vandeputte et al. 2017 Nature), and the number of various bacteria is quantified by correcting with the typical 16S rRNA gene copy number of each microbial species. The method of evaluation is introduced. In this method, correction is applied with a typical value, and the problem that there are microorganisms whose accurate copy number cannot be estimated, and the sample for measuring the number of bacteria and the sample for sequence analysis are substantially different. In addition, it is generally difficult to count minute microorganisms separately from non-cellular particles by flow cytometry, and there remains a problem in terms of accuracy.

The present inventors are a method of analyzing the microbiota composition as a result of diligent research, and include a step of evaluating the microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota. By the method, it is possible to evaluate the absolute number of microbiota composition cheaply and easily by executing before decoding the whole genome sequence. After decoding the whole genome sequence, by narrowing down the information to be analyzed from the digital sequence information, a long gene sequence can be obtained. We have completed this disclosure by finding that it is possible to make comparisons with the above and the accuracy is improved.

In one aspect, the present disclosure comprehensively reads the gene sequences that identify microbial species from amplified polynucleotides that are conditioned in parallel for each cell from a variety of microbial species, and digitally counts the microbial species in the sample cell by cell. However, it provides information that is digitally counted with the microbiota as an absolute amount.
Examples of embodiments of the present disclosure include:
(Item 1) A method for analyzing microbiota composition.
A method comprising the step of evaluating microbiota composition from a sample containing an amplified nucleic acid derived from each cell in the microbiota.
(Item 1A) A method for analyzing the microflora.
A method comprising the step of evaluating the microbiota based on information for each nucleic acid derived from each cell in the microbiota.
(Item 2) The amplified nucleic acid derived from each of the cells
A step of encapsulating cells one by one in a droplet using a sample containing a microflora,
The process of gelling the droplets to form gel capsules,
A step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, wherein the genomic DNA of the cell or a polynucleotide containing a portion thereof is eluted into the gel capsule and the genomic DNA or its portion. The process of holding in the gel capsule with the substance bound to the moiety removed,
The method according to any of the above items, which is produced by a method comprising contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule.
(Item 3) The item, which comprises flowing the suspension of the cells into a microchannel and shearing the suspension with oil to produce the droplets encapsulating the cells. The method described in any of.
(Item 4) The method according to any one of the above items, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
(Item 5) The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform , Guanidin Hydrochloride, Urea, 2-Mercaptoethanol, Dithiotreitol, TCEP-HCl, Sodium Colate, Sodium Deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58 , Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-hydrochloride, Nonidet P-40, and Zwittergent 3-12. The method according to any one of the above items.
(Item 6) The method according to any one of the above items, wherein the gel capsule is a hydrogel capsule.
(Item 7) The method according to any one of the above items, further comprising a step of selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell.
(Item 8) The method according to any one of the above items, which comprises a step of detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each cell.
(Item 9) The method according to any one of the above items, wherein the step of detecting the nucleic acid having the specific sequence comprises amplifying and sequencing the nucleic acid having the specific sequence.
(Item 10) The method according to any one of the above items, wherein the step of evaluating the microbiota composition comprises specifying the absolute number of various microorganisms in the microbiota.
(Item 11) The method according to any one of the above items, further comprising a step of obtaining genomic sequence data of each cell from a sample containing amplified nucleic acid derived from each cell in the microbiota.
(Item 12) The method according to any one of the above items, further comprising a step of selecting genome sequence data to be analyzed from the genome sequence data of each cell.
(Item 13) The step of evaluating the microbiota composition includes comparing gene sequences (for example, 16S rRNA gene sequence, 18S rRNA gene sequence, common gene set, etc.) extracted from Denovo assembly data in each microorganism. The method according to any of the above items.
(Item 14) The method according to any one of the above items, wherein the microbiota is a bacterial flora.
(Item 15) The method according to any one of the above items, wherein the microbiota is an intestinal flora.
(Item 16) A kit for use in the methods of items 1 to 15, comprising a sample collection container containing a storage solution.
(Item 17) The kit according to item 16, wherein the preservation solution contains guanidine or ethanol.
(Item 18) A system for analyzing microbiota composition.
A sample providing unit that provides a sample containing an amplified nucleic acid derived from each cell in the microflora, and a sample providing unit.
A system comprising a composition evaluation unit for evaluating the microbiota composition from a sample containing each cell-derived amplified nucleic acid in the microbiota.
(Item 19) The sample providing unit is
A droplet encapsulation part that encloses cells one by one in a droplet using a sample containing a microflora,
A gel capsule generation unit that gels the droplet to generate a gel capsule,
A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, which contains one or more lysis reagents for lysing cells. , The polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted in the gel capsule and retained in the gel capsule with the substance binding to the genomic DNA or the portion removed. There is a cell lysate and
The system according to any of the above items, comprising a reagent for amplifying the polynucleotide for amplifying the polynucleotide in a gel capsule.
(Item 20) The system according to any one of the above items, further comprising a sample collection container containing a storage solution.
(Item 21) The system according to any one of the above items, further comprising a sample selection unit for selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell.
(Item 22) The composition evaluation unit is described in any one of the above items, which includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells. System.
23. The system of claim 5A, wherein the detection reagent or device comprises a nucleic acid amplification sequencing device for amplifying and sequencing nucleic acids.
(Item 24) The system according to any one of the above items, wherein the composition evaluation unit includes a calculation unit that executes a procedure for specifying the absolute number of various microorganisms in the microbiota.
(Item 25) The composition evaluation unit further includes a genome sequence data acquisition unit for genome sequence data of each cell from a sample containing an amplified nucleic acid derived from each cell in the microbiota. The system described in any of the items.
(Item 26) The system according to any one of the above items, wherein the composition evaluation unit further includes a data selection unit that selects genome sequence data to be analyzed from the genome sequence data of each cell.
(Item 27) The system according to any one of the above items, wherein the composition evaluation unit has a function of containing or introducing a gene sequence extracted from De novo assembly data in each microorganism and comparing the gene sequences.

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

According to the present disclosure, it is possible to evaluate the absolute number of the composition of the microbiota by evaluating the microbiota based on the information for each nucleic acid derived from each cell in the microbiota.

In the case of microbiota analysis by PCR, it is possible to use a part of the amplified DNA sample, and by performing sequencing and sequencing and determining the partial sequence, information on a specific gene sequence can be used. The microbiota composition can be evaluated from the above, and a cheap and simple evaluation can be performed. When performing microbiota analysis based on digital sequence data, the gene sequence used for bacterial flora analysis is extracted from the digital sequence data obtained by sequencing, and composition data is created with high accuracy. Analysis can also be performed. With the present disclosure, it is possible to solve the problem of bias caused by the copy number and sequence of the target gene, and at the same time, to obtain the absolute amount ratio instead of the relative composition ratio. Microorganisms with a low composition ratio may be grouped as "other", but where there is concern that the microbial composition is estimated to be higher or lower than it actually is, this disclosure allows for absolute counts. This allows for a more accurate measurement of the actual composition.

Therefore, in the present disclosure, it is possible to evaluate the absolute number of microbiota composition cheaply and easily by executing before decoding the whole genome sequence, and after decoding the whole genome sequence, the information to be analyzed is narrowed down from the digital sequence information. It has the effect of increasing accuracy by making it possible to compare long gene sequences.

FIG. 1 shows a schematic diagram in which steps are taken to prepare amplified DNA. FIG. 2 shows a schematic diagram of wet sequence screening. FIG. 3 is a schematic diagram of the sequence determination after selection. FIG. 4 is a schematic diagram showing a dry sequence screening step. FIG. 5 is a schematic diagram showing the overall flow of analysis of the microbiota in the present disclosure. FIG. 6 is a box-and-whisker plot of the genome decoding rate (complete rate) of the draft genome prepared from the amplified nucleic acid derived from a single cell from a stool sample. The left is the analysis result of the single cell derived from the stool sample not immersed in the preservation solution, and the right is the analysis result of the single cell derived from the stool sample immersed in the preservation solution. FIG. 7 shows the evolutionary phylogenetic classification analysis of bacteria from the genomic data of each sample with reference to the marker gene group detected using CheckM based on the draft genome prepared from the amplified nucleic acid derived from a single cell from the fecal sample. It is a figure which shows the result of having performed. The left is the analysis result of the single cell derived from the stool sample not immersed in the preservation solution, and the right is the analysis result of the single cell derived from the stool sample immersed in the preservation solution.

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

The present disclosure relates to a method for detecting and analyzing a microbial flora (for example, a microbial flora) in a microbial manner.

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

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

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

As used herein, the term "aggregate" refers to an aggregate containing two or more single biological units, cells or structures for cells.

As used herein, the term "subset" refers to a portion of a set having a smaller number of single biological units, cells or cell structures.

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

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

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

As used herein, the term "single biological unit level" refers to genetic information contained in one single biological unit or information on other biomolecules, as opposed to genetic information contained in another single biological unit or other information. It refers to processing in a state that can be distinguished from the information of biomolecules.

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

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

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

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

In the present specification, the "microbiota" refers to the entire set of microorganisms existing in a certain physical range. The "microorganism" includes an organism (in the case of a multicellular organism, it may be an individual cell) whose existence cannot be discriminated by the naked eye and whose size is smaller than that that can be observed with a microscope or the like. A certain physical range includes, for example, a certain range in the intestine, skin, oral cavity, nasal cavity, or vagina of an individual, water area in the environment, soil, biological surface, and internal organism. The microflora may be a combination of microorganisms of a plurality of classifications, for example, a combination of fungi, bacteria, archaea, unicellular animals, viruses, etc., or a collection of microorganisms of some classifications may be extracted. One example of the microflora is the bacterial flora.

As used herein, the "composition" of the microbiota refers to information about what species are contained in the microbiota or the amount of each species contained, and is a part of the microbiota. It also includes information about whether or the amount of microbial species contained in the plant.

(analysis)
In one aspect of the present disclosure, there is provided a method of analyzing the microbiota, comprising the step of evaluating the microbiota based on information for each cell-derived nucleic acid in the microbiota. Will be done. Although the amount of nucleic acid in a certain sequence can be specified from the information on nucleic acids derived from multiple cells, it is possible to measure the absolute amount of a specific type of microorganism because there is a difference in the number of copies for each microorganism. Although not possible, the absolute amount of a particular species of microorganism can be measured based on the information of nucleic acids derived from each cell.

In one embodiment of the present disclosure, a method of analyzing microbiota composition, comprising the step of evaluating microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota. Can be provided. The amount of nucleic acid derived from microorganisms is small per cell, and even when analysis is performed in a state where nucleic acids derived from a plurality of cells are mixed, an amplification reaction is generally used, but a random primer is used. It is known that even when an attempt is made to amplify a nucleic acid as a whole, the degree of amplification is biased for each sequence depending on the GC content and the like. When amplification is biased, it is difficult to measure the relative amount of a particular species of microorganism in the microbiota.

(Single cell analysis)
In the present disclosure, information for each nucleic acid derived from each cell may be obtained by any method, but in order to easily obtain information for each nucleic acid derived from one cell from a large number of cells, a microorganism A step of encapsulating cells one by one in a droplet using a sample containing a flora, a step of gelling the droplet to generate a gel capsule, and a step of immersing the gel capsule in one or more kinds of solubilizing reagents. In the step of lysing the cell, the genomic DNA of the cell or the polynucleotide containing the portion thereof is eluted into the gel capsule, and the substance binding to the genomic DNA or the portion thereof is removed in the gel capsule. It is preferable to obtain each cell-derived amplified nucleic acid by a method including a step of contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule. In some cases.
In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..

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

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

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

Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, At least one can be selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12.

When amplifying or analyzing nucleic acids for each cell of various microorganisms, it is desirable to use a lytic reagent or a combination of lysing reagents that is strong to some extent. For example, Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.

In the present disclosure, as a method for preparing a sample containing an amplified nucleic acid by dividing into individual cells, one cell is divided into tubes using a micromanipulator or flow cytometry, and each tube is subjected to. There are common methods of adding lysis reagents and whole genome amplification reagents. Non-patent literature (Rinke C, Lee J, Nath N, et al. Obtaining genomes from uncultivated denvironmental microorganisms using FACS-based single-cell genomics. Nat Protoc. 2014; 9 (5): 1038-1048. Doi: 10.1038 / pro 2014.067). As a method for evaluating the microbial composition, as reported in various literatures such as non-patent literature (Vandeputteet al. 2017 Nature), the partial sequence or the entire sequence of the 16S rRNA gene is sequenced.

The cells or cell-like structures that can be targeted in the microbiota analysis of the present disclosure are two or more arbitrary numbers, for example, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more. It can be 5000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more. The microbiota analysis of the present disclosure may use individual cell-derived nucleic acid-by-nucleic acid information from a larger number of cells than using conventional single-cell reaction systems, such as 0.2 mL, 1.5 mL microtube reaction systems. ..
(Analysis before genome sequencing)

In the present disclosure, prior to analysis of the total sequence information of individual microorganisms (for example, genome sequencing), the structures and sequences of nucleic acids or other biomolecules prepared in parallel from various microorganisms are used. It may be carried out to detect and select each individual specifically with reference to. That is, the method may include selecting a sample containing the amplified nucleic acid to be analyzed from a sample containing the amplified nucleic acid derived from each cell.

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

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

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

Evaluation of microbiota composition may include identifying the absolute number of various microbiota in the microbiota. The absolute number of various microorganisms can be specified by specifying the microbial species for each of the amplified nucleic acids derived from each cell. The microbial species can be specified, for example, by specifying the presence or absence of a specific gene sequence.

(Analysis with genome sequencing)
The method of the present disclosure may include obtaining genomic sequence data for each cell from a sample containing amplified nucleic acid derived from each cell in the microbiota. By obtaining genome sequence data, it is possible to obtain not only information as a sequence but also information from the viewpoint of the function performed by the sequence for each microorganism in the microflora.

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

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

When genomic sequence data is obtained, evaluation of microbiota composition may be performed by comparing long gene sequences in each microorganism. The long sequence may be, for example, a gene sequence extracted from de novo assembly data (for example, a 16S gene sequence or a set of common genes). Comparison of such long gene sequences can not only improve the accuracy of evaluation, but also enable evaluation of function in the microflora. For example, even if it is known that multiple species exist from sequence information alone, it is not possible to understand the function of the entire microbiota without individual information on each function, but it is based on genetic information. For example, information can be obtained about the amount of species that may have a particular activity (eg, enzymatic activity) within the microbiota.

(Microbiota)
The microbial flora that can be covered in this disclosure is, but is not limited to, eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, racen, gram-negative bacteria, gram-positive bacteria, archaea, fungi. , Or any combination thereof. An example of a microflora is a bacterial flora. Since the microflora can contain multiple microorganisms having different cell properties (eg, the presence or absence of a cell wall), the nucleic acid information for each cell can be obtained or one cell in the same manner regardless of the microbial species. Adopting a means capable of obtaining each amplified nucleic acid may be preferable in the analysis of the present disclosure.

The microbiota is not limited, but is limited to intestinal microbiota, cutaneous microbiota, oral microbiota, nasal microbiota, vaginal microbiota, soil microbiota, root zone microbiota, river / seawater microbiota, and activity. It can be a sludge microbiota, an insect coexisting microbiota, an animal symbiotic microbiota, and the like. The gut flora has been extensively studied for its effects on human health and is one of the preferred analyzes of the present disclosure.

(Combination embodiment)
In the present disclosure, it is possible to further combine and analyze the information of the analysis of nucleic acid sequence information derived from the same microflora and not derived from each cell. Examples of the analysis include metagenomic analysis. Metagenomic analysis is a method of extracting and collecting nucleic acids, genes, and DNA possessed by microorganisms in the environment as a whole and comprehensively examining their structures (base sequences) without going through the process of culturing. Although it is not known which microorganism the individual nucleic acid or gene is derived from, it is possible to obtain information about the gene group of a collection (community) of microorganisms in the environment, and such a method is called metagenomic analysis.

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

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

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

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

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

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

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

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

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

The kit may include means for taking a sample. The kit may also include means for storing the sample taken. Means for collecting a sample include a syringe, a swab, a biopsy punch, a urine collection container, a stool collection container, a saliva collection container, a medical tape, and the like. Examples of the means for storage include a coolant (for example, an ice pack, dry ice, liquid nitrogen), a preservation solution (for example, a solution containing any of guanidine hydrochloride, ammonium sulfate, ethanol, etc.) and the like.

In one aspect of the present disclosure, a kit comprising a sample collection container containing a preservation solution for analyzing microbiota composition may be provided. The storage solution may contain, for example, a guanidine solution (for example, FS-0007 or FS-0008, Technosulgarabo) or ethanol (for example, OMR-200 or OM-501, DNA genomek). By using a kit equipped with such a preservation solution, even if it is stored at room temperature for a certain period of time in a hospital facility or the like, a good genome decoding rate (complete rate) and many in the sample can be obtained in the subsequent single cell level analysis. Effects such as identification of biological strains can be achieved. In addition, it is much more convenient than the usual method of collecting and storing frozen stools.

Preservatives are generally used to increase the stability of nucleic acids, for example, guanidine solutions and alcohols are used to cause protein denaturation and inhibit the action of enzymes that promote nucleic acid degradation. Will be done. In the method of the present disclosure, it may be preferable that the morphological stability as a cell is maintained in the storage of the sample. This is because if the cells are broken, it becomes difficult to enclose them in the droplets, or it becomes difficult to acquire data due to the frequent occurrence of cell debris. Therefore, a preservation solution that causes protein denaturation was expected to be disadvantageous for the stability of cell morphology, but in the examples herein, unexpectedly, after preservation with the preservation solution, the cells Many retain their morphology well and have been found to be particularly suitable for the methods of the present disclosure.

(Preferable embodiment)
A step of encapsulating cells one cell or a structural unit in a droplet, a step of gelling the droplet to generate a gel capsule, and a step of immersing the gel capsule in one or more solubilizing reagents to lyse the cells. A step in which the polynucleotide in the cell is eluted in the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed, and the polynucleotide is brought into contact with the amplification reagent. A gel capsule containing the amplified and retained 1-cell genome-derived polynucleotide is prepared by a method including the step of amplifying the polynucleotide in the gel capsule. This gel capsule is stained with a DNA-binding fluorescent dye or the like, and fluorescence-positive capsules are counted by flow cytometry. The number of fluorescence-positive capsules in a fixed volume corresponds to the total number of introduced microorganisms. In this case, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule can also amplify the polynucleotide while maintaining the gel state in the gel capsule.

Next, select gel capsules that hold genomic DNA amplified above a predetermined level using a flow cytometer or the like, and separate and collect the gel capsules for each cell on a microplate or the like. Further, a whole genome amplification reaction is carried out in the plate using the polynucleotide in each gel capsule as a template, and this is used as a library master plate. Then, a part of the reaction solution in the library master plate is separated to prepare a replica plate. Amplification is performed with a primer set specific to the target gene using this replica as a template. Subsequently, the sequence of the amplified product is specified by a Sanger sequence or the like, and the microbial species of each sample is specified.

More effectively, the above primer set contains a barcode sequence, each sample is subjected to an amplification reaction in which a different barcode is added, and PCR products derived from multiple replica plates are pooled to perform a next-generation sequence. Then, the plate well number is specified by the barcode sequence, and the microbial species of hundreds to thousands of samples are specified at once by specifying the sequence of the PCR product. The primer set may be a mixture of primer sets targeting a plurality of gene regions. More specifically, it is selected from those targeting the v3-v4 and v1-v2 regions of the 16S rRNA gene, and primer sets for distinguishing bacteria, archaea, fungi, etc. such as 18S rRNA and ITS. Bacteria, archaea, and fungi can be detected at the same time by using a plurality of types of the primer sets at the same time.

By any of the above methods, identify the microbial species contained in each gel capsule separated into a well plate or the like, count them, and combine them with the total number of microorganisms measured initially. In this reaction, all PCR reactions are performed separately, and the microbial species are identified and counted for each barcode, so that digital count data reflecting the number of cells can be obtained. That is, data can be obtained that avoids the bias caused by the difference in copy number and sequence, which has been a conventional problem. In addition, it is possible to compare rare cells with a relative ratio of 1% or less in a substantially total number.

(system)
Contains a droplet encapsulation unit that encloses cells one by one in a droplet, a gel capsule generation unit that gels the droplets to generate gel capsules, and one or more lysis reagents for lysing cells. A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, and the cell lysate is a polynucleotide containing genomic DNA of the cells or a portion thereof. In the gel capsule, the cell lysate and the polynucleotide, which are configured to be retained in the gel capsule in a state where the substance that is eluted in the gel capsule and binds to the genomic DNA or a portion thereof is removed, are contained in the gel capsule. A gel capsule containing a single-cell genome-derived polynucleotide that has been amplified and retained is prepared by a system for analyzing the composition of the microflora containing the polynucleotide amplification reagent for amplification.

Using the gel capsule prepared in this way, it is possible to perform a complex analysis of the genetic information and the like of the host itself such as a human and the microflora of the human-derived sample. For example, from each gel capsule obtained by the above method and system for the human intestinal microflora, the microbial species contained in the sample is identified, and the information on the intestinal microbiota and human cells are targeted. The genetic information obtained from each gel capsule obtained by the above method and system is also analyzed. In this case, the composition evaluation unit of the above system can evaluate the human intestinal microflora and the human cell to be evaluated, respectively, to amplify and sequence the nucleic acid, and obtain the genome sequence data of each cell. Can be obtained.
As described above, the system of the present invention can evaluate the effects of the intestinal bacteria and their biotransformers on the host and the functions of the intestinal bacteria themselves, and can find the correlation between them. it can.

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

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

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

(Example 1: Analysis of mouse intestinal flora composition)
In the experiment, feces of male BALB / c mice (7, 9 weeks old) (Nippon Claire Co., Ltd.) were collected in a 1.5 mL tube (1212-10, SSIbio) (n = 5), and 500 μL of phosphate was collected. It was ground in buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) using a crusher until the solids were gone. Centrifuge at 2,000 xg for 2 seconds (himac CF15RX, Koki Holdings), repeat the operation of collecting the supernatant twice, and then centrifuge at 15,000 xg for 3 minutes to intestinal microorganisms in mice. Was collected. The cell pellet was washed twice with PBS by centrifugation and then suspended in PBS to obtain a cell suspension of mouse intestinal microorganisms. Measure the cell concentration in the prepared cell suspension (microscope: CKX41, OLYMPUS, bacterium calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) so that the final concentration is 1.5%. By adding -10G, SIGMA-ALDRICH), an intestinal microbial suspension used for gel capsule preparation was prepared (cell final concentration: 1.5 × 10 ³ cells / μL).

Using a microchannel made by myself using polydimethylsiloxane (Sylgard 184: Dow Corning), microdroplets were prepared and mouse intestinal microbial cells were encapsulated in the microdroplets. In this embodiment, a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, in which adjacent channels are arranged at right angles, is used, but they are connected in a substantially T shape. It is also possible to use a microchannel. The microchannel of this example used had a width of 34 μm and a height of 50 μm, but the size of the microchannel can be appropriately changed depending on the size of the microdroplets to be produced and the size of one cell to be encapsulated.

Next, the intestinal microbial suspension was introduced from the first channel (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) from the second channel and the fourth channel (oil phase inlet). ) (Hereinafter referred to as "oil") is introduced to shear the intestinal microbial suspension to prepare fine droplets having a diameter of 50 μm, and the third flow path 7 is allowed to flow through a tube having a capacity of 0.2 mL. Collected in. Approximately 450,000 microdroplets were produced at a rate of 500 droplets / second. The cell concentration in the microdroplets is 0.1 cells / droplet.

In this embodiment, the diameter of the microdroplets is made uniform to 50 μm, so that each cell can be easily encapsulated in the microdroplets. Considering the size of one cell, the diameter of the microdroplets is, for example, 1 to 250 μm, preferably 20 to 200 μm. The diameter of the droplet may be from about 1 to 250 μm, more preferably from about 10 to 200 μm, for example, the diameter of the droplet is about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about. It may be 30 μm, about 40 μm, about 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm.

Multiple microdroplets and oil are stored in the tube, but the microdroplets have a lighter specific gravity than oil, so they accumulate in the upper layer.

Next, the tube was cooled on ice for 15 minutes, and microdroplets were gelled with ultra-low melting point agarose. The gelled microdroplets are gel capsules. Since the diameter of the microdroplets is 50 μm, the diameter of the gel capsule is also 50 μm. The diameter of the gel capsule may be about 1-250 μm, more preferably about 10-200 μm, eg, about 1 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 40 μm, about. It may be 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm. The diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation. The diameter of the gel capsule is preferably 1 to 250 μm. By making the diameter of the gel capsule uniform, the penetration rate of the lytic reagent described later into each gel capsule can be made more uniform.

Next, 20 μL of 1H, 1H, 2H, 2H-perfluoro-1-octanol (SIGMA-ALDRICH) was added to the tube, the oil in the lower layer was removed, and then acetone (Fujifilm Wako Pure Chemical Industries, Ltd.) (500 μL). , Isopropanol (Fujifilm Wako Pure Chemical Industries, Ltd.) (500 μL) was added in this order and centrifuged to remove the oil. Centrifugal cleaning for removing the oil was performed by the removing part described later. In this example, the oil that has permeated the gel capsule is also included in the contaminants. Further, 500 μL of PBS was added and centrifugation was performed three times to bring the gel capsule into a suspended state in the aqueous layer (PBS). Since the gel capsule has a heavier specific gravity than the aqueous layer, it accumulated in the lower layer.

Subsequently, the gel capsule was sequentially immersed in a lytic reagent as a lysis reagent, and a part other than the object to be collected such as a cell wall of a cell was dissolved inside the gel capsule, and genomic DNA was eluted into the gel capsule.

Specifically, lysozyme (10 U / μL) (R1804M, Epicenter), which is one of the lytic reagents, was added to the tube to lyse the cells. Next, achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemical Industries, Ltd.), which is one of the lytic reagents, was added to the tube. Next, add protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), which are one of the lytic reagents, to the tube to add cells. After lysis, centrifugation was performed 5 times to remove components (contaminants) other than protease and genomic DNA of the lysed cells from the tube. Subsequently, the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA. As described above, the lytic test solution used in this example was lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2. Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it was used as one of the lytic reagents in this example. Since the gel capsule is immersed in the lytic reagent for a short time, the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule. In this example, the lytic reagent permeated into the gel capsule is also included in the contaminants.

In this example, lysozyme, achromopeptidase, and protease K were added in sequence, sodium dodecyl sulfate was added to lyse the cells, and then centrifugation was performed only before adding Buffer D2. Thereby, a sufficient cleaning effect can be obtained. However, centrifugation may be performed after lysing the cells with each lytic reagent.

In this way, the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.

The amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase, was used. Here, the whole genome amplification reaction reagent was immersed in REPLI-g Single Cell Kit (QIAGEN), and the whole genome amplification reaction was carried out for 3 hours (S1000 thermal cycler, Bio-Rad). The amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).

Furthermore, staining is performed with a fluorescent DNA intercalator (SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)), and each feces is stained using a flow cytometer (BD FACSMelody cell sorter, BD Biosciences). From the derived sample (n = 5), 94 fluorescent gel capsules were individually collected on a plate (PCR-96-FS-C, Axygen) (470 in total).

After melting the gel capsules by heating the collected individual gel capsules at 65 ° C (S1000 thermal cycler, Bio-Rad), the MDA method (REPLI-g Single Cell Kit, 150345) is used in the wells of each plate. , QIAGEN) was performed to prepare a library master plate containing 10 μL of DNA amplification product for each well.

Dispense 39 μL of nuclease-free water (UltraPure DNase / RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) into each well of the new plate, add 1 μL of DNA amplification product in the library master plate, and add 1 μL of DNA amplification product to the library master plate. A 40-fold diluted solution was prepared. Next, the DNA concentration was quantified by a Qubit fluorometer (Q33226, Thermo Fisher Scientific) Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 μL of the diluted solution. In addition, PCR was performed on the V3V4 region of the 16S rRNA gene using 1 μL of the diluted solution as a template (6.25 μL PrimeSTAR Max DNA Polymerase (R045B, Takara Bio), 0.5 μL 10 μM Primer Forward (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3). '(SEQ ID NO: 1)), 0.5 μL 10 μM Primer Reverse (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2)), 1.0 μL DNA diluent, 4.25 μL UltraPure DNase / RNase-Free Distilled Water (10977-015, Thermo) Fisher Scientific) (S1000 thermal cycler, Bio-Rad). PCR reaction conditions are 95 ° C, 5 minutes for initial thermal denaturation, 98 ° C, 10 seconds for thermal denaturation, 51 ° C, 15 seconds for annealing, and 72 ° C for elongation reaction. , Performed 27 cycles in 5 seconds, reacted at 72 ° C for 5 minutes, and stored at 4 ° C. Agalose gel electrophoresis (translocation tank: Mupid-exU, EXU-1, Mupid, marker: GeneRuler ^TM 1kb DNA Ladder, # SM0318, Fermentas, Staining: Midori Green Direct, NE-MG06, Nippon Genetics, Loading Buffer: 6 × Loading Buffer, 9157, Takara Bio) (Migration conditions: 100V, 15min) After confirming the presence or absence of PCR products, amplification is achieved. Sequence analysis was performed on the found samples using the Sanger method (DNA sequence outsourced service of Fasmac Co., Ltd.).

(Selection conditions)
Of the 470 samples amplified by MDA, in this example, the criteria were (1) the DNA yield after MDA amplification was 200 ng or more, and (2) amplification was confirmed after PCR of the 16S rRNA gene. Samples were selected that were set and the DNA yield was sufficient for subsequent analysis and met the criteria of bacterial origin. As a result of sample selection, 347 (74%) out of 470 samples were selected. Furthermore, homology search (default condition) using BLAST was performed on the sequence information obtained by sequence analysis of PCR products, and information on the type and number of bacteria contained in the library master plate was obtained. In this example, as a result of homology search in BLAST, many 1-cell amplified genomic libraries classified into Lachnospiraceae (Lachnospiraceae) and Bacteroidaceae (Bacteroides) were confirmed, 147 were Lachnospiraceae bacteria and 68 were Bacteroidaceae bacteria. It was (table below).

(Example 2: Analysis of human intestinal flora composition)
(Sample collection from humans)
Human feces were collected in a stool collection container (FS-0007 or FS-0008, Technosurgarabo). In the stool collection container containing the preservation solution, after centrifugation at 8000xg, 5min, 4 ° C., the preservation solution was removed, and the container was washed once with 1000 μL of DPBS. Frozen stool (in 1.5 ml tube) was naturally thawed by placing it on ice for 30 minutes. Fresh stools that do not contain a preservative solution are not treated before crushing. It was then ground in 500 μL of Phosphate Buffered Saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) using a crusher until the solids were gone. Centrifuge at 2,000 xg for 2 seconds (himac CF15RX, Koki Holdings), repeat the operation of collecting the supernatant twice, and then centrifuge at 15,000 xg for 3 minutes to intestinal microorganisms in mice. Was collected. The cell pellet was washed twice with PBS by centrifugation and then suspended in PBS to obtain a cell suspension of mouse intestinal microorganisms. Measure the cell concentration in the prepared cell suspension (microscope: CKX41, OLYMPUS, bacterium calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) so that the final concentration is 1.5%. By adding -10G, SIGMA-ALDRICH), an intestinal microbial suspension used for gel capsule preparation was prepared (cell final concentration: 1.5 × 10 ³ cells / μL).

(Treatment of recovered sample (wet))
Using a microchannel self-made using polydimethylsiloxane (Sylgard 184: Dow Corning), microdroplets were prepared and human intestinal microbial cells were encapsulated in the microdroplets. In this embodiment, a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, in which adjacent channels are arranged at right angles, is used, but they are connected in a substantially T shape. It is also possible to use a microchannel. The microchannel of this example used had a width of 34 μm and a height of 50 μm, but the size of the microchannel can be appropriately changed depending on the size of the microdroplets to be produced and the size of one cell to be encapsulated.

Next, the intestinal microbial suspension was introduced from the first channel (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) from the second channel and the fourth channel (oil phase inlet). ) (Hereinafter referred to as "oil") is introduced to shear the intestinal microbial suspension to prepare fine droplets having a diameter of 50 μm, and the third flow path 7 is allowed to flow through a tube having a capacity of 0.2 mL. Collected in. Approximately 450,000 microdroplets were produced at a rate of 500 droplets / second. The cell concentration in the microdroplets is 0.1 cells / droplet.

In this embodiment, the diameter of the microdroplets is made uniform to 50 μm, so that each cell can be easily encapsulated in the microdroplets. Considering the size of one cell, the diameter of the microdroplets is, for example, 1 to 250 μm, preferably 10 to 200 μm.

Multiple microdroplets and oil are stored in the tube, but the microdroplets have a lighter specific gravity than oil, so they accumulate in the upper layer.

Next, the tube was cooled on ice for 15 minutes, and microdroplets were gelled with ultra-low melting point agarose. The gelled microdroplets are gel capsules. Since the diameter of the microdroplets is 50 μm, the diameter of the gel capsule is also 50 μm. The diameter of the gel capsule is preferably 1 to 250 μm. By making the diameter of the gel capsule uniform, the penetration rate of the lytic reagent described later into each gel capsule can be made more uniform.

Next, 20 μL of 1H, 1H, 2H, 2H-perfluoro-1-octanol (SIGMA-ALDRICH) was added to the tube, the oil in the lower layer was removed, and then acetone (Fujifilm Wako Pure Chemical Industries, Ltd.) (500 μL). , Isopropanol (Fujifilm Wako Pure Chemical Industries, Ltd.) (500 μL) was added in this order and centrifuged to remove the oil. Centrifugal cleaning for removing the oil was performed by the removing part described later. In this example, the oil that has permeated the gel capsule is also included in the contaminants. Further, 500 μL of PBS was added and centrifugation was performed three times to bring the gel capsule into a suspended state in the aqueous layer (PBS). Since the gel capsule has a heavier specific gravity than the aqueous layer, it accumulated in the lower layer.

Subsequently, the gel capsule was sequentially immersed in a lytic reagent as a lysis reagent, and a part other than the object to be collected such as a cell wall of a cell was dissolved inside the gel capsule, and genomic DNA was eluted into the gel capsule.

Specifically, lysozyme (10 U / μL) (R1804M, Epicenter), which is one of the lytic reagents, was added to the tube to lyse the cells. Next, achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemical Industries, Ltd.), which is one of the lytic reagents, was added to the tube. Next, add protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), which are one of the lytic reagents, to the tube to add cells. After lysis, centrifugation was performed 5 times to remove components (contaminants) other than protease and genomic DNA of the lysed cells from the tube. Subsequently, the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA. As described above, the lytic test solution used in this example was lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2. Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it was used as one of the lytic reagents in this example. Since the gel capsule is immersed in the lytic reagent for a short time, the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule. In this example, the lytic reagent permeated into the gel capsule is also included in the contaminants.

In this example, sufficient cleaning effect can be obtained by sequentially adding lysozyme, achromopeptidase, and protease K, adding sodium dodecyl sulfate to lyse the cells, and then performing centrifugation only before adding Buffer D2. it can. However, centrifugation may be performed after lysing the cells with each lytic reagent.

In this way, the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.

The amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase, was used. Here, the whole genome amplification reaction reagent was immersed in REPLI-g Single Cell Kit (QIAGEN), and the whole genome amplification reaction was carried out for 3 hours (S1000 thermal cycler, Bio-Rad). The amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).

Furthermore, staining is performed with a fluorescent DNA intercalator (SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)), and each feces is stained using a flow cytometer (BD FACSMelody cell sorter, BD Biosciences). From the derived sample (n = 2), 94 fluorescent gel capsules were individually collected on a plate (PCR-96-FS-C, Axygen) (188 in total).

After melting the gel capsules by heating the collected individual gel capsules at 65 ° C (S1000 thermal cycler, Bio-Rad), the MDA method (REPLI-g Single Cell Kit, 150345) is used in the wells of each plate. , QIAGEN) was performed to prepare a library master plate containing 10 μL of DNA amplification product for each well.

Dispense 39 μL of nuclease-free water (UltraPure DNase / RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) into each well of the new plate, add 1 μL of DNA amplification product in the library master plate, and add 1 μL of DNA amplification product to the library master plate. A 40-fold diluted solution was prepared. Next, the DNA concentration was quantified by a Qubit fluorometer (Q33226, Thermo Fisher Scientific) Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 μL of the diluted solution. In addition, PCR was performed on the V3V4 region of the 16S rRNA gene using 1 μL of the diluted solution as a template (6.25 μL PrimeSTAR Max DNA Polymerase (R045B, Takara Bio), 0.5 μL 10 μM Primer Forward (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3). '(SEQ ID NO: 1)), 0.5 μL 10 μM Primer Reverse (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3' (SEQ ID NO: 2)), 1.0 μL DNA diluent, 4.25 μL UltraPure DNase / RNase-Free Distilled Water (10977-015, Thermo) Fisher Scientific) (S1000 thermal cycler, Bio-Rad). PCR reaction conditions are 95 ° C, 5 minutes for initial thermal denaturation, 98 ° C, 10 seconds for thermal denaturation, 51 ° C, 15 seconds for annealing, and 72 ° C for elongation reaction. , Performed 27 cycles in 5 seconds, reacted at 72 ° C for 5 minutes, and stored at 4 ° C. Agalose gel electrophoresis (translocation tank: Mupid-exU, EXU-1, Mupid, marker: GeneRuler ^TM 1kb DNA Ladder, # SM0318, Fermentas, Staining: Midori Green Direct, NE-MG06, Nippon Genetics, Loading Buffer: 6 × Loading Buffer, 9157, Takara Bio) (Migration conditions: 100V, 15 min) After confirming the presence or absence of PCR products, amplification Sequence analysis was performed on the samples in which were found using the Sanger method (DNA sequence outsourced service of Fasmac Co., Ltd.).

(Analysis of data obtained at the single cell level)
(Selection conditions)
96 of the 188 samples amplified by MDA were targeted, and in this example, (1) the DNA yield after MDA amplification was 200 ng or more, and (2) amplification was confirmed after PCR of the 16S rRNA gene. Criteria were set and samples were selected whose DNA yield was sufficient for subsequent analysis and met the criteria of bacterial origin. As a result of sample selection, 95 samples were selected. In addition, homology search (default condition) using BLAST was performed on the sequence information obtained by sequence analysis of PCR products to obtain information on the type and number of bacteria contained in the library master plate.

For the above 95 samples, library preparation was performed using Nextera XT DNA genome prep kit (Illumina, FC-131-1096), and all using Miseq (Illumina, SY-410-1003). A 2 × 75 bp paired-end read was obtained by genome sequencing. Assemble the sequence data using SPAdes (Bankevich A et al., J Comput Biol. 2012 May; 19 (5): 455-77. Http://doi.org/10.1089/cmb.2012.0021) and contig. Made. In addition, checkM (Parks et al., Genome Res. 2015. 25: 1043-1055, doi: 10.1101 / gr.186072.114) was used to evaluate the genome decoding rate (complete rate) and contamination degree under the default conditions. Was done. As a result, the average completion rate was calculated to be 37.8% and the average contamination rate was 1.3% for the fresh stool sample, and the average completion rate was 52.6% and the average contamination rate was 4.8% for the preservative stool sample (Fig. 6).

Furthermore, with reference to the marker gene group detected using CheckM, the evolutionary phylogenetic classification analysis of bacteria was performed from the genomic data of each sample. As a result, it was shown that many data are derived from the phylum Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria, which are the preferred species of gut flora. It is possible to identify and count bacterial species and calculate the composition ratio as shown in FIG. 7 by referring to the index related to such phylogenetic classification and the identity of the 16S rRNA gene. Further, by analyzing the digital sequence information of the bacterium, it is possible to analyze the metabolic function of the bacterium, possess a drug resistance gene, analyze a gene mutation, compare with a known bacterium, and compare with another sample. This process includes, for example, search engines such as blast, analysis websites such as Ensembl, assembly evaluation tools such as QUAST and CheckM, annotation tools such as Prokka, InterProScan, and DFAST, and metabolism / physiology such as MetaCyc and MAPLE. It can be carried out using a functional potential evaluation system. As a series of flow of analysis of genetic information, the effectiveness of the assembly is evaluated by QUAST and the quality of the genome set is evaluated by CheckM for the genetic information obtained by performing the de novo assembly. Then, using an annotation tool such as Prokka, the genes in the genome are identified, and the conservativeness as a metabolic pathway is evaluated by MAPLE. Using the Pfam and COG databases, the conservativeness and identity of each gene will be evaluated, and comparative genome analysis with related species will be performed.

More specifically, when the purpose is to evaluate gene mutations on a single cell basis, (1) homology search of gene sequences derived from a single organism using BLAST or hmmer, or homology search for target genes, or (2) By mapping the sequence data derived from a single organism using BWA and bowtie2, the difference between the gene sequence derived from a single organism and the target gene sequence is detected, and the gene mutation is identified.

For the purpose of comparison with known bacteria, comparison with other samples, and analysis of subspecies, genomes derived from allogeneic microorganisms based on the single copy marker gene sequences detected by Average Nucleotide Identity, CheckM, and GTDB-tk. Create a genome set containing genetic information derived from a single organism that is presumed to be and genetic information of known microorganisms. Subsequently, differences between genomes such as the presence or absence of a specific gene, a locus, and a mutation in a common gene sequence are identified and extracted by a homology search tool such as BLAST or hmmer. By performing clustering based on characteristic intergenomic differences, classification of allogeneic microorganisms at the strain level or substrain level of homologous microorganisms is performed.

(Example 3: When it is desired to selectively acquire data of cells having specific characteristics from various cells)
A gel containing a polynucleotide when the purpose is to obtain genomic data of one or more specific microorganisms of interest to those skilled in the art among animal symbiotic microorganisms such as gut flora and marine / soil microorganisms. By confirming in advance the presence or absence of a gene fragment of the target microorganism in the amplified nucleic acid recovered from the capsule or gel capsule, it is possible to reduce unnecessary gene sequence data acquisition and the cost associated therewith. Examples of measurement targets include comparative analysis of microorganisms of the same strain (for example, analysis of subspecies in a group of microorganisms related to diseases, etc.) and microorganisms having a specific gene (for example, secondary metabolites and enzymes produced by microorganisms). It is assumed that the purpose is to search for or analyze bacteria, archaea, fungi, and other eukaryotic cells individually from among various species. In particular, for the purpose of evaluating the intestinal environment, oral cavity, and skin environment of the host human, intestinal bacteria and oral bacteria responsible for specific metabolic functions can be specifically detected from genes, and variants of skin bacteria can be detected. It is expected to be detected.

(Example 4: When a large amount of host DNA or the like is mixed)
When the analysis sample is feces, saliva, sputum or skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning solution, tissue extract or blood, and the microorganisms contained in the sample are analyzed. Contains cells, intracellular small organs, and nucleic acids from many host animals in the sample. Some of these can also be encapsulated inside the gel capsule to perform polynucleotide amplification. Therefore, by confirming in advance the presence or absence of the gene fragment of the target microorganism or the gene fragment derived from the host in the gel capsule containing the amplified polynucleotide, unnecessary gene sequence data containing the polynucleotide derived from the host can be confirmed. You can avoid acquisition and reduce the cost of it. As a measurement example, detection of microorganisms from a human-derived sample, for example, search for pathogenic microorganisms from blood or sputum, and analysis of microorganisms symbiotic inside tissues are assumed.

Alternatively, by actively analyzing the polynucleotide derived from the host, it can be applied to the complex analysis of the microflora.

For example, the intestinal flora and host animals present in the gastrointestinal tract have a symbiotic relationship in which the host provides an anaerobic environment for colonization of the gastrointestinal tract, while the intestinal flora affects the health of the host. It is known to have. For example, the major effects on the health status of the host include the production of nutrients, the defense against infectious diseases, and the development of the immune system. In addition, to give another example, inflammatory bowel disease is a disease caused by an abnormality of intestinal environmental factors such as intestinal bacteria in addition to a genetic predisposition.

Regarding such pathological conditions, analysis of the genetic information of the host itself, integrated analysis including the intestinal microflora, that is, bacteria, viruses, and fungi, and the mechanism of action on the host physiological function will be clarified. It is necessary, and the techniques of the present disclosure may contribute to these. In addition, the relationship between the intestinal microflora and the pathophysiology of various diseases and the functional analysis centered on metabolites can be further analyzed by analyzing the genetic information of the host itself.

In addition, in the intestinal tract, the intestinal bacteria and the living body side compete for nutrients, while the intestinal bacteria also metabolize and decompose nutrients for the living body for more integrated analysis. It can be carried out. In this way, the function of biotransformers derived from gut flora is analyzed in a complex manner by host-side genetic analysis, metabolome analysis, biochemical analysis, etc., and the gut flora Regarding the function, the function can be inferred from the genome sequence information of the bacterium, and the correlation with the produced metabolome can be seen.

(Example 5: Evaluation of gene mutation in a single cell unit)
Mutant diversity on the genome sequence can be evaluated on a single cell basis to track the genomic heterogeneity of each microorganism in the microbiota and the development and progression of mutant strains. By selectively amplifying and detecting the target gene mutation site of the target cell for the gel capsule containing the amplified polynucleotide, only the gene sequence data to be analyzed can be specifically acquired, and the cost for it can be reduced. can do. It can be used to detect genetically mutated microorganisms and plasmid infections.

(Example 6: Evaluation of preservation of specific species)
When the purpose is to prove or reject a specific organism in a drug, pesticide, food, etc., the disclosed technology is used to amplify the specific organism from a gel capsule containing an amplified polynucleotide. By detecting and selecting, the content rate can be shown. In addition, by analyzing the obtained genetic information in detail, it is possible to evaluate the degree of coincidence with the standard at the genome level, preservation, and the like. It is expected to be used for quality assurance of microbial preparations.

Specific procedures include (1) homology search of single organism-derived gene sequences using BLAST and hmmer for target genes, or (2) single organism-derived sequence data using BWA and bowtie2. By mapping, the difference between the gene sequence derived from a single organism and the target gene sequence is detected, and the gene mutation is identified.

(Example 7: Detection of specific species)
Analysis samples are feces, saliva, sputum and skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical lavage fluid, tissue extract, blood, etc., and detect the presence of one or more specific microorganisms. It may determine drug response, drug resistance, and evaluate the metabolic capacity of foods. By using the disclosed technology, a specific organism can be detected and selected from a gel capsule containing an amplified polynucleotide to indicate its content. In addition, by analyzing the obtained genetic information in detail, it is possible to evaluate the functional estimation and conservativeness at the genome level.

For example, when single cell-derived sequence data of a mouse intestinal microorganism is obtained, a homology search for a single organism-derived gene sequence is performed based on a gene that metabolizes dietary fiber inulin or a gene marker that identifies the microbial Bacteroides species. As a result, the microorganisms and gene groups responsible for the degradation of the dietary inulin could be identified, and their functions could be estimated from the homology of known genes and the prediction of the three-dimensional structure based on the amino acid sequence. In addition, it was possible to evaluate up to the ratio (content rate) of the microorganism species in the single cell-derived sequence data of a plurality of intestinal microorganisms.

(Example 8: Evaluation of soil bacteria)
When soil or seawater is used as an analysis sample, various specific organisms inhabiting soil or seawater are detected and selected from gel capsules containing amplified polynucleotide using the technology disclosed in the present disclosure. It is possible to identify the area and indicate the abundance. Further, by analyzing the obtained genetic information in detail, it is possible to evaluate cultivars, livestock or aquaculture varieties suitable for the soil or seawater at the genome level. Furthermore, it is expected to be used for pesticide-free manufacturing methods using soil bacteria or marine bacteria.

Specifically, 10 g of soil was collected in a 50 mL volume tube (2342-050, Iwaki) (n = 3) and phosphate buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) was added until the total volume was 40 mL and suspended sufficiently. After allowing the suspended tube to stand on ice for 5 minutes, the supernatant was transferred to a new 50 mL tube. The supernatant was filtered using a filter having a diameter of 5 μm (SMWP04700, Sigma-Aldrich) and then centrifuged at 10,000 × g for 5 minutes (75004263, Thermo Fisher Scientific) to remove the supernatant. The pellets are resuspended in 10 mL PBS, dispensed in 1 mL each in a 1.5 mL tube (MCT-150-C, Axygen), and then centrifuged at 10,000 xg for 5 minutes to collect soil bacteria. Bacteria. Bacterial pellets were combined into a single 1.5 mL tube, washed twice by centrifugation with PBS, and then suspended in PBS to obtain a cell suspension of soil bacteria. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%. -10G, Sigma-Aldrich) was added to prepare a soil bacterial suspension used for gel capsule preparation (cell final concentration: 1.5 × 10 ³ cells / μL). Subsequently, the analysis was advanced and evaluated in the same procedure as the method described in Example 1 or 2.

(Example 9: Analysis of aquatic microbial composition)
In the experiment, 4 L of seawater or fresh water was collected in a sterilized plastic tank, filtered using a 5 μm diameter filter (SMWP04700, Sigma-Aldrich), and then filtered through a 0.22 μm diameter filter (GSWP04700, Sigma-Aldrich). By doing so, the bacterial fraction was trapped on the filter. A 0.22 μm diameter filter in a 25 mL tube (2362-025, Iwaki) containing 10 mL Phosphate Buffered Saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific). And suspended well. Bacteria derived from seawater or freshwater are collected by dispensing 1 mL of a 10 mL bacterial suspension into a 1.5 mL tube (MCT-150-C, Axygen) and centrifuging at 10,000 xg for 5 minutes. Bacteria. Bacterial cell suspensions derived from seawater or freshwater are obtained by collecting the bacterial cell pellets in a single 1.5 mL tube, washing them by centrifugation twice with PBS, and then suspending them in PBS. did. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%. -10G, Sigma-Aldrich) was added to prepare seawater or freshwater-derived bacterial suspensions used for gel capsule preparation (cell final concentration: 1.5 × 10 ³ cells / μL). Subsequently, the analysis was advanced and evaluated in the same procedure as the method described in Example 1 or 2.

(Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but the present disclosure should not be construed as being limited to this embodiment. It is understood that the present disclosure should be construed only by the claims. It will be understood from those skilled in the art that the description of the specific preferred embodiments of the present disclosure will enable equivalent scope to be implemented based on the description of the present disclosure and common general technical knowledge. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood. The present application claims priority to the patent application 2019-85836 filed with the Japan Patent Office on April 26, 2019, and the content of the application itself is specifically described in the present specification. It is understood that the content should be incorporated as a reference to this specification as well as.

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

SEQ ID NO: 1: Forward primer SEQ ID NO: 2: Reverse primer

Claims (27)

1. A method of analyzing microbiota composition
   A method comprising the step of evaluating microbiota composition from a sample containing an amplified nucleic acid derived from each cell in the microbiota.
2. Amplified nucleic acid derived from each of the above cells
   A step of encapsulating cells one by one in a droplet using a sample containing a microflora,
   The process of gelling the droplets to form gel capsules,
   A step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, wherein the genomic DNA of the cell or a polynucleotide containing a portion thereof is eluted into the gel capsule and the genomic DNA or its portion. The process of holding in the gel capsule with the substance bound to the moiety removed,
   The method of claim 1, wherein the method is produced by a method comprising contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule.
3. The second aspect of the present invention, wherein the droplets containing the cells are produced by flowing the suspension of the cells into a microchannel and shearing the suspension with oil. Method.
4. The method according to any one of claims 2 to 3, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
5. The solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride. , Urea, 2-mercaptoethanol, dithioreagent, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Claimed, wherein at least one is selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12. The method according to any one of 2 to 4.
6. The method according to any one of claims 2 to 5, wherein the gel capsule is a hydrogel capsule.
7. The method according to any one of claims 1 to 6, further comprising a step of selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell.
8. The method according to any one of claims 1 to 7, which comprises a step of detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells.
9. The method of claim 8, wherein the step of detecting the nucleic acid having the specific sequence comprises amplifying and sequencing the nucleic acid having the specific sequence.
10. The method according to any one of claims 1 to 9, wherein the step of evaluating the microbiota composition comprises specifying the absolute number of various microorganisms in the microbiota.
11. The method according to any one of claims 1 to 10, further comprising a step of obtaining genomic sequence data of each cell from a sample containing amplified nucleic acid derived from each cell in the microbiota. ..
12. The method according to claim 11, further comprising a step of selecting genome sequence data to be analyzed from the genome sequence data of each cell.
13. The method according to any one of claims 1 to 12, wherein the step of evaluating the microbiota composition comprises comparing gene sequences extracted from De novo assembly data in each microorganism.
14. The method according to any one of claims 1 to 13, wherein the microbiota is a bacterial flora.
15. The method according to claim 14, wherein the microbiota is an intestinal flora.
16. A kit for use in the methods of claims 1 to 15, which includes a sample collection container containing a storage solution.
17. The kit according to claim 16, wherein the preservation solution contains guanidine or ethanol.
18. A system for analyzing microbiota composition
    A sample providing unit that provides a sample containing an amplified nucleic acid derived from each cell in the microflora, and a sample providing unit.
    A system comprising a composition evaluation unit for evaluating the microbiota composition from a sample containing each cell-derived amplified nucleic acid in the microbiota.
19. The sample providing unit
    A droplet encapsulation part that encloses cells one by one in a droplet using a sample containing a microflora,
    A gel capsule generation unit that gels the droplet to generate a gel capsule,
    A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, which contains one or more lysis reagents for lysing cells. , The polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted in the gel capsule and retained in the gel capsule with the substance binding to the genomic DNA or the portion removed. There is a cell lysate and
    The system of claim 18, comprising a reagent for amplifying the polynucleotide for amplifying the polynucleotide in a gel capsule.
20. The system according to claim 18 or 19, further comprising a sample collection container containing a storage solution.
21. The system according to any one of claims 18 to 20, further comprising a sample selection unit for selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each of the cells.
22. The composition evaluation unit comprises any one of claims 18 to 21, which includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells. Described system.
23. 22. The system of claim 22, wherein the detection reagent or device comprises a nucleic acid amplification sequencing device for amplifying and sequencing nucleic acids.
24. The system according to any one of claims 18 to 23, wherein the composition evaluation unit includes a calculation unit that executes a procedure for specifying the absolute number of various microorganisms in the microbiota.
25. The composition evaluation unit further includes a genome sequence data acquisition unit for collecting genome sequence data of each cell from a sample containing an amplified nucleic acid derived from each cell in the microbiota, claims 18 to 24. The system according to any one of the above.
26. The system according to claim 25, wherein the composition evaluation unit further includes a data selection unit that selects genome sequence data to be analyzed from the genome sequence data of each cell.
27. The system according to any one of claims 18 to 26, wherein the composition evaluation unit has a function of including or introducing a gene sequence extracted from De novo assembly data in each microorganism and comparing them.

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