JP7584800B2 - Digital Microbiome Analysis - Google Patents

Description

This disclosure relates to the analysis of microbiome composition and can be used in fields such as biological research, medicine, the environment, and healthcare.

Amplicon sequencing analysis using next-generation sequencers to target conserved regions of 16SrRNA genes and 18SrRNA genes has been widely used in recent years as a method for evaluating the diversity of environmental microorganisms. When targeting the 16SrRNA gene, conserved regions of several hundred bases are PCR-amplified from DNA extracted en bloc from samples containing a variety of microorganisms (feces, soil, seawater, etc.), and the base sequences of the conserved regions are comprehensively analyzed using a next-generation sequencer, allowing the composition of the microbial flora to be evaluated in terms of the type of base sequence = the type of microbial species. However, there are several problems with this method.

First, the number of copies of the 16S rRNA gene on the genome varies from bacteria to bacteria. Some bacteria have only a single copy of the 16S rRNA gene, while others have seven or more copies. Furthermore, because the genomes of most environmental microorganisms have yet to be determined, the number of genes in question is unknown and cannot be corrected.

Secondly, there is the problem of amplification bias during PCR. In the PCR amplification, the amplification is performed on the conserved region, but since the internal sequence differs for each microorganism, the amplification efficiency differs for each microorganism due to differences in base sequence, etc. As a result, the bias resulting from the difference in amplification efficiency increases as the number of PCR amplification cycles 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. In addition, since such amplicon sequence analysis only obtains the composition ratio, the actual increase or decrease in the total amount of microorganisms is unknown. In other words, even if a phenomenon is found in which microorganism X is less than other types of microorganisms by comparing certain samples, it is possible that the total amount of microorganism X has decreased, or that the number of other microorganisms has simply increased and the number of microorganism X has not changed.

Furthermore, in many microbiome analyses, microorganisms with a composition ratio of 1% or less are grouped together and discussed as "others," but considering the above bias, there is a concern that the microbial composition is estimated to be higher or lower than it actually is. As a solution to this problem, there is a method of using DNA with a known sequence as an internal standard, spiking this into the sample and performing PCR (Non-Patent Document 1 (Stammler et al., 2016 Microbiome, 4(1), 28)), and a method of counting and correcting the absolute number of bacteria using a different method. In the former, it is possible to compare the total number of microorganisms in each sample relatively by comparing the total amount of read sequences with the internal standard, but problems with PCR amplification remain, and it is not possible to count the absolute number of various types of bacteria. In the latter, Non-Patent Document 2 (Vandeputte et al. 2017 Nature) introduces a method of quantitatively evaluating the number of various types of bacteria by counting the absolute number of bacteria using flow cytometry and correcting for the representative 16S rRNA gene copy number of each microbial species. This method involves correction using a representative value, and there are problems with the accuracy of the method, such as the existence of microorganisms for which the copy number cannot be accurately estimated, the fact that the samples used to measure the bacterial count and the samples used for sequence analysis are essentially separate, and the fact that it is generally difficult to count tiny microorganisms separately from non-cellular particles using flow cytometry.

Stammler et al., 2016 Microbiome, 4(1), 28 Vandeputte et al. 2017 Nature, 551(7681), 507-511

As a result of intensive research, the inventors have discovered that a method for analyzing microbiota composition, which includes a step of evaluating the microbiota composition from a sample containing amplified nucleic acid derived from each individual cell in the microbiota, enables absolute numerical evaluation of the microbiota composition cheaply and easily when performed prior to whole genome sequencing, and after whole genome sequencing, narrowing down the information to be analyzed from the digital sequence information enables comparisons of long gene sequences, etc., thereby increasing accuracy, and have completed the present disclosure.

In one aspect, the present disclosure provides information on the digital counting of the absolute amount of the microbiome by comprehensively reading gene sequences that identify microbial species from amplified polynucleotides prepared in parallel on a cell-by-cell basis from a variety of microorganisms, and digitally counting the microbial species in a sample on a cell-by-cell basis.
Examples of embodiments of the present disclosure include the following:
(Item 1) A method for analyzing a microbiota composition, comprising:
The method comprises a step of assessing the microbiota composition from a sample comprising amplified nucleic acid from each cell in the microbiota.
(Item 1A) A method for analyzing a microbiome, comprising:
A method comprising a step of evaluating the microbiome based on information from each nucleic acid derived from each cell in the microbiome.
(Item 2) The amplified nucleic acid derived from each of the cells is
Using a sample containing a microbiota, encapsulating cells one by one in a droplet;
gelling the droplets to form gel capsules;
immersing said gel capsule in one or more lysis reagents to lyse said cells, wherein a polynucleotide comprising said genomic DNA or a portion thereof is dissolved into said gel capsule and is retained within said gel capsule in a state in which substances binding to said genomic DNA or a portion thereof have been removed;
and a. contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule.
(Item 3) The method according to any one of the above items, characterized in that the droplets encapsulating the cells are created by flowing a suspension of the cells through a microchannel and shearing the suspension with oil.
(Item 4) The method according to any of the preceding items, characterized in that the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, Matrigel, collagen or a photocurable resin.
(Item 5) The method according to any one of the preceding items, characterized in that the lysis reagent is at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12.
(Item 6) The method according to any of the preceding items, wherein the gel capsule is a hydrogel capsule.
(Item 7) The method according to any one of the preceding items, further comprising the step of selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells.
(Item 8) The method according to any of the preceding items, further comprising the step of detecting a nucleic acid having a specific sequence in a sample containing amplified nucleic acid derived from each of the cells.
(Item 9) The method according to any of the preceding items, wherein the step of detecting a nucleic acid having a specific sequence comprises amplifying and sequencing the nucleic acid having a specific sequence.
(Item 10) The method according to any of the preceding items, wherein the step of assessing the microbiota composition comprises determining the absolute number of each type of microorganism in the microbiota.
(Item 11) The method according to any of the preceding items, further comprising the step of obtaining genomic sequence data for each of the cells in the microbiota from a sample containing amplified nucleic acid from the cells.
(Item 12) The method according to any one of the preceding items, further comprising the step of selecting genome sequence data to be analyzed from the genome sequence data of each of the cells.
(Item 13) The method according to any of the preceding items, wherein the step of evaluating the microbiota composition includes comparing gene sequences (e.g., 16S rRNA gene sequences, 18S rRNA gene sequences, or a set of common genes) extracted from de novo assembly data for each microorganism.
(Item 14) The method according to any of the preceding items, wherein the microbiota is a bacterial flora.
(Item 15) The method according to any of the above items, wherein the microbiota is an intestinal microbiota.
(Item 16) A kit for use in the method of items 1 to 15, comprising a specimen collection container containing a preservation solution.
(Item 17) The kit according to item 16, wherein the preservation solution comprises guanidine or ethanol.
18. A system for analyzing a microbiota composition, comprising:
A sample providing unit that provides a sample containing amplified nucleic acid derived from each cell in the microbiota;
and a composition evaluation unit that evaluates the microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota.
(Item 19) The sample providing unit includes:
A droplet encapsulation unit that encapsulates cells one by one in a droplet using a sample containing a microbiota;
a gel capsule forming unit that gels the droplets to form gel capsules;
a cell lysis unit that lyses the cells by immersing a gel capsule in one or more lysis reagents, the gel capsule storing one or more lysis reagents for lysing the cells, the cell lysis unit being configured such that a polynucleotide containing the genomic DNA of the cell or a portion thereof is dissolved in the gel capsule and a substance that binds to the genomic DNA or a portion thereof is removed and the polynucleotide is retained in the gel capsule;
and a reagent for amplifying the polynucleotide within the gel capsule.
(Item 20) The system of any of the preceding items, further comprising a specimen collection container containing a preservation fluid.
(Item 21) The system according to any of the preceding items, further comprising a sample selection unit for selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells.
(Item 22) The system according to any one of the above items, wherein the composition evaluation unit includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing amplified nucleic acid derived from each of the cells.
23. The system of claim 5A, wherein the detection reagent or device comprises a nucleic acid amplification and sequencing device for amplifying and sequencing a nucleic acid.
(Item 24) The system according to any of the preceding items, wherein the composition evaluation unit includes a calculation unit that performs a procedure for identifying the absolute number of each type of microorganism in the microbiota.
(Item 25) The system according to any of the above items, wherein the composition evaluation unit further includes a genome sequence data acquisition unit that acquires genome sequence data of each of the cells in the microbiota from a sample containing amplified nucleic acid derived from each of the cells.
(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 of the cells.
(Item 27) The system according to any one of the above items, wherein the composition evaluation unit includes or is introduced with a function of comparing gene sequences extracted from de novo assembly data for each microorganism.

In the present disclosure, it is intended that one or more of the above features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

The present disclosure makes it possible to assess the microbiome based on information from each nucleic acid derived from each cell in the microbiome, thereby enabling an absolute numerical assessment of the composition of the microbiome.

In the case of PCR-based microbiome analysis, it is possible to use a portion of the amplified DNA sample, and by performing PCR and then sequencing to determine the partial sequence, the microbiome composition can be evaluated from the information on a specific gene sequence, making it possible to perform a cheap and simple evaluation. When performing microbiome analysis based on digital sequence data, the digital sequence data obtained by sequencing can be used as the target, and the gene sequence to be used in the microbiome analysis can be extracted to create composition data, allowing for highly accurate analysis. This disclosure makes it possible to eliminate the problem of bias caused by the copy number and sequence of the target gene, while at the same time determining the absolute amount ratio rather than the relative composition ratio. Microorganisms with low composition ratios are sometimes grouped together as "others," but there is a concern that the microbial composition is estimated to be higher or lower than it actually is. The present disclosure enables more accurate measurement of the actual composition by enabling absolute number counting.

Therefore, the present disclosure enables cheap and easy evaluation of absolute numbers of microbiota composition when performed prior to whole genome sequencing, and after whole genome sequencing, it has the effect of improving accuracy by narrowing down the information to be analyzed from the digital sequence information, making it possible to compare long gene sequences.

FIG. 1 shows a schematic diagram of the steps taken to prepare amplified DNA. FIG. 2 shows a schematic diagram of wet sequence screening. FIG. 3 is a schematic diagram of sequencing after selection. FIG. 4 is a schematic diagram showing the dry sequence screening steps. FIG. 5 is a schematic diagram showing the overall flow of the analysis of the microbiome in the present disclosure. Figure 6 shows box plots of genome sequencing rates (completion rates) of draft genomes created from amplified nucleic acids derived from single cells from fecal samples. The left graph shows the analysis results of single cells derived from fecal samples that were not immersed in a preservation solution, and the right graph shows the analysis results of single cells derived from fecal samples that were immersed in a preservation solution. 7 is a diagram showing the results of bacterial evolutionary phylogenetic analysis performed from genome data of each sample based on a draft genome created from amplified nucleic acid derived from a single cell from a fecal sample and with reference to a group of marker genes detected using CheckM. The left side shows the analysis result of a single cell derived from a fecal sample that was not immersed in a preservation solution, and the right side shows the analysis result of a single cell derived from a fecal sample that was immersed in a preservation solution.

Hereinafter, the present disclosure will be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the concept of the plural form, unless otherwise specified. Therefore, singular articles (e.g., in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of a conflict, the present specification (including definitions) will take precedence.

The present disclosure relates to methods for microbe-specific detection and analysis of microbiomes (e.g., bacterial biota).

(Definitions, etc.)
The following provides definitions of terms particularly used in this specification and/or provides basic technical information as appropriate.

As used herein, a "cell" refers to any particle that contains molecules containing genetic information and is capable of replicating (whether independently or not). As used herein, a "cell" includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi, viruses, etc.

As used herein, a "biomolecule" refers to a molecule possessed by any organism or virus. Biological molecules may include nucleic acids, proteins, glycans, lipids, and the like. As used herein, a "biomolecule analog" refers to a natural or non-natural variant of a biomolecule. Biological molecule analogs may include modified nucleic acids, modified amino acids, modified lipids, modified glycans, and the like.

As used herein, "assembly" refers to an association containing two or more single biological units, cells or cell-like structures.

As used herein, a "subpopulation" refers to a portion of a population that has a smaller number of single biological units, cells, or cell-like structures than the population.

In this specification, "gel" refers to a state in which polymeric substances or colloidal particles in a colloidal solution (sol) form a network structure as a whole through their interactions, and the solution loses its fluidity while still containing a large amount of the liquid phase, which is the solvent or dispersion medium. In this specification, "gelation" refers to changing the solution into a "gel" state.

As used herein, "gel capsule" refers to a gel-like particulate structure capable of holding cells or cell-like structures therein.

In this specification, "genetic analysis" refers to examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample. In one embodiment, the genetic analysis may be one that utilizes a nucleic acid amplification reaction. Examples of genetic analysis, including the above, include sequencing, genotyping/polymorphism analysis (SNP analysis, copy number polymorphism, restriction fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe (Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR, etc.

As used herein, "single-biological unit level" refers to processing the genetic information or other biomolecular information contained in one single biological unit in a state in which it can be distinguished from the genetic information or other biomolecular information contained in other single biological units.

As used herein, the term "single-cell level" refers to processing the genetic information contained in one cell or cell-like structure in a state where it is distinguished from the genetic information contained in other cells or cell-like structures. For example, when amplifying polynucleotides at the "single-cell level," amplification is performed in a state where the polynucleotides in a certain cell and the polynucleotides in other cells can be distinguished from each other.

As used herein, "single-cell analysis" refers to the analysis of the genetic information contained in a single cell or cell-like structure while distinguishing it from the genetic information contained in other cells or cell-like structures.

As used herein, "nucleic acid information" refers to information about the nucleic acids contained in a cell or cell-like structure, including the presence or absence of a particular gene sequence, the yield of a particular gene, or the total nucleic acid yield.

As used herein, "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 this specification, the term "microbiota" refers to the entire collection of microorganisms present in a certain physical range. "Microorganisms" include organisms (or individual cells in the case of multicellular organisms) whose presence cannot be discerned by the naked eye and whose size is small enough to be observed by a microscope or the like. Examples of a certain physical range include a certain range in the intestines, skin, oral cavity, nasal cavity, or vagina of an individual, or in water bodies, soil, the surface of an organism, or inside an organism in the environment. The microbiota may be a collection of microorganisms across multiple classifications, such as a combination of fungi, bacteria, archaea, unicellular animals, viruses, etc., or a collection of microorganisms from some classifications. One example of a microbiota is a bacterial flora.

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

(analysis)
In one aspect of the present disclosure, a method for analyzing a microbiota is provided, comprising a step of evaluating the microbiota based on information on each nucleic acid derived from each cell in the microbiota.Although the amount of a nucleic acid of a certain sequence can be specified from information on nucleic acids derived from multiple cells, the absolute amount of a specific species of microorganism cannot be measured because the copy number varies between microorganisms, but the absolute amount of a specific species of microorganism can be measured based on information on nucleic acids derived from each cell.

In one embodiment of the present disclosure, a method for analyzing a microbiota composition may be provided, comprising a step of evaluating the microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota. Since the amount of nucleic acid derived from microorganisms per cell is small, it is common to use an amplification reaction even when analyzing nucleic acids derived from multiple cells in a mixed state. However, even when attempting to amplify the nucleic acid as a whole, for example, using random primers, it is known that the degree of amplification for each sequence can be biased due to GC content, etc. When amplification is biased, it is difficult to measure the relative amount of a specific species of microorganism in the microbiota.

(Single cell analysis)
In the present disclosure, information on each nucleic acid derived from each cell may be obtained by any method, but in order to easily obtain information on each nucleic acid derived from each cell from a large number of cells, it may be preferable to obtain amplified nucleic acids derived from each cell by a method including the following steps: using a sample containing a microbiome, encapsulating cells one by one in a droplet; gelling the droplet to produce a gel capsule; and immersing the gel capsule in one or more types of lysis reagent to lyse the cells, wherein a polynucleotide containing the genomic DNA of the cell or a part thereof is dissolved into the gel capsule and is retained in the gel capsule in a state in which substances binding to the genomic DNA or part thereof have been removed; and contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule.
In one embodiment of the present disclosure, the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule can also amplify the polynucleotide while maintaining the polynucleotide in a gel state within the gel capsule.

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

Materials for the gel capsules may include agarose, acrylamide, photocurable resins (e.g., PEG-DA), PEG, gelatin, sodium alginate, Matrigel, collagen, and the like. The droplets can be gelled by forming the droplets so that they contain the gel capsule material and then cooling the droplets. Alternatively, gelling can be achieved by applying a stimulus such as light to the droplets. The gel capsule material can be included in the droplets, 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, "hydrogel" refers to a capsule in which the solvent or dispersion medium is water and is held in place by a network structure of polymeric substances or colloidal particles.

The lysis reagent may be at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12.

When amplifying or analyzing nucleic acids on a cell-by-cell basis for a variety of microorganisms, it is desirable to use a lysis reagent or combination of lysis reagents that are relatively strong. For example, gram-positive bacteria have cell walls with a thick peptidoglycan layer, so cells may not be sufficiently lysed with a mild lysis reagent alone.

In this disclosure, a method for preparing a sample containing amplified nucleic acid by dividing cells into individual cells is a general method in which a single cell is separated into a tube using a micromanipulator or flow cytometry, and a lysis reagent or a whole genome amplification reagent is added to each tube. Non-patent literature (Rinke C, Lee J, Nath N, et al. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics. Nat Protoc.2014;9(5):1038-1048. doi:10.1038/nprot.2014.067). As a method for evaluating the microbial composition, as reported in various literature such as non-patent literature (Vandeputte et al.2017 Nature), it is performed by sequencing the partial or entire sequence of the 16S rRNA gene.

The number of cells or cell-like structures that may be targeted in the microbiome analysis of the present disclosure may be any number of 2 or more, for example, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 5000 or more, 10,000 or more, 50,000 or more, 10,000 or more, 500,000 or more, 1 million or more, 5 million or more, or 10 million or more. The microbiome analysis of the present disclosure may use information from each nucleic acid derived from each individual cell from a larger number of cells than using a conventional single-cell reaction system, for example, a 0.2 mL or 1.5 mL microtube reaction system.
(Pre-genome sequencing analysis)

In the present disclosure, prior to performing an analysis of the total sequence information of each microorganism (e.g., genome sequencing), individual-specific detection and selection may be performed from structures or sequences of nucleic acids or other biomolecules prepared in parallel from various microorganisms, with reference to the structures or sequences. That is, the method may include a step of selecting a sample containing amplified nucleic acid to be analyzed from samples containing amplified nucleic acid derived from individual cells.

In one embodiment, selection may be 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, selection may be made for the presence or absence of a particular gene sequence. In some embodiments, selection may be made for a yield of a particular gene that is greater than or less than a reference yield. In some embodiments, selection may be made for a total nucleic acid yield that is greater than or less than a reference yield.

In certain embodiments, the presence or absence of a specific gene sequence is detected by a means selected from the group consisting of a reagent that specifically detects a specific gene sequence, agarose gel electrophoresis, microchip electrophoresis, PCR, qPCR, and gene sequencing (Sanger sequencing, NGS). In some embodiments, the reagent that specifically detects a specific gene sequence includes an antibody, a probe, a DNA-binding fluorescent dye, and a fluorescent dye-binding nucleotide.

In certain embodiments, the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorometry, agarose gel electrophoresis, or microchip electrophoresis. The method can include detecting a nucleic acid having a specific sequence in a sample containing amplified nucleic acid from each cell. Detecting a nucleic acid having a specific sequence can include amplifying and sequencing the nucleic acid having a specific sequence.

Assessment of the microbiota composition may include determining the absolute number of each type of microorganism in the microbiota. The absolute number of each type of microorganism may be determined by identifying the microbial species for each amplified nucleic acid from each individual cell. Identification of the microbial species may be performed, for example, by determining the presence or absence of a particular gene sequence.

(Analysis involving genome sequencing)
The disclosed method may include obtaining genomic sequence data for each cell in the microbiota from a sample containing amplified nucleic acid from the cell. Obtaining genomic sequence data may provide information about the individual microorganisms in the microbiota, not just in terms of their sequences, but also in terms of the functions of the sequences.

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

The selection may include evaluating the presence or absence of a specific gene sequence and/or identity with a specific gene sequence. In some embodiments, the identity with a specific gene sequence can be evaluated by using BLAST or the like. In certain embodiments, the selection may be based on the presence or absence of a specific gene sequence, and the nucleic acid information may be sorted for each cell. In other embodiments, the selection may be based on the identity between a specific gene sequence and nucleic acid information derived from two or more cells, and the nucleic acid information may be sorted for each cell. In some embodiments, the selection may be performed when the identity is equal to or greater than a certain level, or when the identity is equal to or less than a certain level. In certain embodiments, the identity may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. In other embodiments, the identity may be 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, 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 genome sequence data is available, the evaluation of the 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 (e.g., a 16S gene sequence or a set of common genes). Comparison of such long gene sequences may not only increase the accuracy of the evaluation, but may also enable evaluation of the function in the microbiota. For example, even if it is known that multiple species exist, the function of the entire microbiota cannot be understood without individual information on each function, even if sequence information alone is used. However, based on gene information, information can be obtained about the amount of species that may have a specific activity (e.g., enzyme activity) in the microbiota.

(Microbiota)
The microbiome that may be the subject of the present disclosure may include, but is not limited to, eubacteria, Escherichia coli, Bacillus subtilis, cyanobacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, fungi, or any combination thereof. An example of a microbiome is a bacterial flora. Since a microbiome may include multiple microorganisms with different cellular properties (e.g., the presence or absence of a cell wall, etc.), it may be preferable in the analysis of the present disclosure to employ a means that can obtain nucleic acid information for each cell or amplified nucleic acid for each cell in the same manner regardless of the microbial species.

The microbiota may be, but is not limited to, the intestinal microbiota, the skin microbiota, the oral microbiota, the nasal microbiota, the vaginal microbiota, the soil microbiota, the rhizosphere microbiota, the river/seawater microbiota, the activated sludge microbiota, the insect symbiotic microbiota, the animal symbiotic microbiota, etc. The intestinal microbiota has been widely studied for its effect on human health, and is one of the preferred subjects for analysis in the present disclosure.

(Combination embodiment)
In the present disclosure, it is possible to perform analysis by further combining information from analysis of nucleic acid sequence information derived from the same microflora, but not from individual cells. Examples of analysis include metagenomic analysis. Metagenomic analysis is a method of extracting and collecting nucleic acids, genes, and DNA from microorganisms in the environment as a whole, without going through the process of culturing, and comprehensively examining their structures (base sequences). Although it is not known which microorganism each 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 present disclosure, a system for analyzing microbiota composition may be provided. The system may comprise means for implementing a method or steps thereof comprising any of the features described in other sections herein.

The system may include an apparatus for amplifying polynucleotides in cells. The apparatus may be capable of amplifying polynucleotides in cells at the single-cell level, among others. The apparatus may include a droplet generator that encapsulates cells or cell-like structures in droplets one cell or structure at a time; a gel capsule generator that gels the droplets to generate gel capsules; a lysis reagent immersion unit that immerses the gel capsules in a lysis reagent; a removal unit that removes contaminants from the gel capsules; and/or an amplification reagent immersion unit that immerses the gel capsules in an amplification reagent. The system or apparatus may further include a sorting unit that sorts the gel capsules and stores the gel capsules in a storage container.

The system or device may optionally include reagents such as a medium for encapsulating the cells or cell-like structures, a gel encapsulant, lysis reagents, amplification reagents, reagents for nucleic acid sequencing (e.g., polymerase, primer sets (which may include barcode sequences), etc.). Reagents may be those known in the art, in addition to those described elsewhere herein.

The device or system may further include a sequencing unit that performs sequencing of the nucleic acid sequence in the polynucleotide amplified in the amplification reagent immersion unit. The sequencing unit may be provided integrally with the device or as a separate device in the system. Sequencing units may include Sanger sequencing, Maxam-Gilbert sequencing, single molecule real-time sequencing (e.g., Pacific Biosciences, Menlo Park, California), ion semiconductor sequencing (e.g., Ion Torrent, South San Francisco, California), sequencing by synthesis, pyrosequencing (e.g., 454, Branford, Connecticut), sequencing by ligation (e.g., SOLiD sequencing from Life Technologies, Carlsbad, California), sequencing by synthesis and reversible terminator (e.g., Illumina, San In one embodiment, the instrument may be an imaging device (such as one located at San Diego, California) for performing nucleic acid imaging techniques such as transmission electron microscopy, nanopore sequencing, and the like.

The system or device may include a means for detecting and measuring the amplified genes. For example, a flow cytometry instrument suitable for handling the gel capsule shape may be provided integrally with the device or as a separate device in the system. The means for detecting and measuring the amplified genes may include a means for performing a detection reaction (e.g., a thermal cycler and appropriate reagents) and/or a means for detecting a signal (e.g., a light sensor, a camera, and appropriate analytical means).

The system or device may include a computing unit that may be configured to perform any of the information processing described elsewhere in this specification. The computing unit may be provided integrally with the device or as a separate device (computer) in the system. In another aspect of the present disclosure, a program for causing the computing unit to perform the information processing described elsewhere in this specification and to implement the method of the present disclosure and a storage medium having the program recorded thereon may also be provided. The computing unit may include such a program and/or a storage medium having the program recorded thereon, as necessary.

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

A kit for analyzing microbiota composition may include a lysis reagent. The lysis reagent may comprise at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12. The lysis reagent is useful for obtaining amplified polynucleotides at the single cell level, particularly genome-wide amplification products.

The kit may include reagents for amplifying nucleic acids. Examples of the amplification reagents include polymerase, a primer set (which may include a barcode sequence), a base mix, and a suitable buffer. The kit may include reagents used for sequencing nucleic acids (e.g., polymerase, a primer set (which may include a barcode sequence), etc.). For example, the kit may include reagents for amplifying and deciphering specific genes in Sanger sequencing or NGS (polymerase, a primer set (which may include a barcode sequence), etc.). The kit may also include reagents that may be used for detecting and measuring specific sequences, such as nucleic acid binding dyes and fluorescently labeled probes. Using these reagents, the presence or absence of specific sequences can be measured using an instrument (such as a flow cytometer) that detects and measures amplified genes.

The kit may include a means for collecting a sample. The kit may also include a means for storing the collected sample. Examples of 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, and medical tape. Examples of means for storing the sample include a cooling agent (e.g., an ice pack, dry ice, liquid nitrogen) and a preservation solution (e.g., a solution containing any of guanidine hydrochloride, ammonium sulfate, ethanol, etc.).

In one aspect of the present disclosure, a kit for analyzing the composition of a microbiota may be provided, which includes a specimen collection container containing a preservation solution. The preservation solution may include, for example, a guanidine solution (e.g., FS-0007 or FS-0008, Techno Suruga Lab) or ethanol (e.g., OMR-200 or OM-501, DNA genotek). By using a kit containing such a preservation solution, even if the sample is stored at room temperature for a certain period of time in a hospital facility or the like, effects such as a good genome sequencing rate (completion rate) and identification of many biological lineages in the sample can be achieved in subsequent single-cell level analysis. In addition, the kit is much more convenient than the usual method of collecting and preserving stool as frozen stool.

Preservation solutions are generally used to enhance 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 the decomposition of nucleic acids. In the method of the present disclosure, it may be preferable to maintain the morphological stability of cells in the preservation of samples. This is because collapsed cells make it difficult to encapsulate them in droplets, or because frequent occurrence of cell debris makes it difficult to obtain data. For this reason, it was expected that preservation solutions that cause protein denaturation would be disadvantageous to the stability of cell morphology, but in the examples of this specification, it was unexpectedly found that many of the cells maintained their morphology well after storage in the preservation solution, and that this is particularly suitable for the method of the present disclosure.

Preferred Embodiments
A gel capsule containing a polynucleotide derived from a single cell genome that has been amplified and retained is prepared by a method including the steps of: encapsulating a cell in a droplet at a cell or structure unit; gelling the droplet to produce a gel capsule; immersing the gel capsule in one or more lysis reagents to dissolve the cell, in which the polynucleotide in the cell is dissolved in the gel capsule and retained in the gel capsule in a state in which substances that bind to the polynucleotide have been removed; and contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. The gel capsule is stained with a DNA-binding fluorescent dye or the like, and fluorescent positive capsules are counted by flow cytometry. The number of fluorescent positive capsules in a given volume corresponds to the total number of microorganisms introduced. 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, gel capsules that hold genomic DNA that has been amplified to a predetermined level are selected using a flow cytometer or the like, and the gel capsules are collected separately for each cell onto a microplate or the like. Furthermore, a whole genome amplification reaction is carried out within the plate using the polynucleotides in each gel capsule as a template, and this is used as a library master plate. Next, a portion of the reaction solution in the library master plate is taken out to prepare a replica plate. Using this replica as a template, amplification is carried out with a primer set specific to the target gene. Next, the sequence of the amplified product is identified by Sanger sequencing or the like, and the microbial species of each sample is identified.

More effectively, the primer set contains a barcode sequence, an amplification reaction is performed in which a different barcode is assigned to each sample, and the PCR products from multiple replica plates are pooled and next-generation sequencing is performed. After that, the plate/well number is identified by the barcode sequence, and the sequence of the PCR product is identified to identify the microbial species of hundreds to thousands of samples at once. The primer set may be a mixture of primer sets targeting multiple gene regions. More specifically, the primer set is selected from primer sets targeting the v3-v4 and v1-v2 regions of the 16S rRNA gene, and primer sets such as 18S rRNA and ITS that distinguish bacteria, archaea, fungi, etc. If multiple types of the primer sets are used simultaneously, bacteria, archaea, and fungi can be detected simultaneously.

Using one of the above methods, the microbial species contained in each gel capsule dispensed onto a well plate or similar are identified and counted, and the number is added to the total number of microorganisms measured initially. In this reaction, all PCR reactions are performed separately, and the microbial species is identified and counted for each barcode, resulting in digital count data that reflects the number of cells. In other words, data is obtained that avoids bias due to differences in copy number and sequence, a problem that has existed in the past. It also makes it possible to compare the actual total number of rare cells with a relative ratio of 1% or less.

(system)
A gel capsule containing an amplified and retained single-cell genome-derived polynucleotide is prepared by a system for analyzing a microbiota composition, the system comprising a cell lysis unit comprising a droplet encapsulation unit which encapsulates cells one by one in a droplet, a gel capsule generation unit which gels the droplet to generate a gel capsule, and a cell lysis unit which stores one or more lysis reagents for lysing the cells and immerses the gel capsule in the one or more lysis reagents, the cell lysis unit being configured so that a polynucleotide containing the genomic DNA of the cell or a part thereof is dissolved into the gel capsule and is retained in the gel capsule in a state in which substances binding to the genomic DNA or a part thereof have been removed, and a polynucleotide amplification reagent for amplifying the polynucleotide within the gel capsule.

Using the gel capsules prepared in this way, it is possible to perform a composite analysis of the genetic information of the host itself, such as humans, and the microbiota of a human-derived sample. For example, the microbial species contained in the sample is identified from each gel capsule obtained by the above-mentioned method and system for the human intestinal microbiota, and the information of this intestinal microbiota is analyzed together with the genetic information obtained from each gel capsule obtained by the above-mentioned method and system for human cells. In this case, the composition evaluation unit of the above-mentioned system can evaluate the human intestinal microbiota and human cells that are the evaluation targets, respectively, to amplify and sequence the nucleic acid, and obtain the genome sequence data of each cell.
In this way, the system of the present invention can evaluate the effects of intestinal bacteria and metabolites derived from them on the host, as well as the function of the intestinal bacteria themselves, and can find correlations between them.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth herein.

The present disclosure has been described above by showing preferred embodiments for ease of understanding. Below, the present disclosure will be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided 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 this specification, but is limited only by the scope of the claims.

Examples of the present disclosure are described below.
The specific reagents used were those described in the Examples, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science Inc., etc.) can also be used.

Example 1: Analysis of mouse intestinal microbiota composition
In the experiment, feces from male BALB/c mice (7 and 9 weeks old) (CLEA Japan, Inc.) were collected in 1.5 mL tubes (1212-10, SSIbio) (n=5) and crushed in 500 μL of phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) using a grinder until no solid matter remained. The mouse intestinal microorganisms were collected by centrifuging at 2,000 × g for 2 seconds (himac CF15RX, Koki Holdings) and collecting the supernatant twice, followed by centrifugation at 15,000 × g for 3 minutes. The bacterial pellet was washed twice with PBS by centrifugation, and then suspended in PBS to obtain a cell suspension of mouse intestinal microorganisms. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting chamber A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, SIGMA-ALDRICH) was added to a final concentration of 1.5% to prepare an intestinal microbial suspension for use in producing gel capsules (final cell concentration: 1.5 x ¹⁰³ cells/μL).

Using a microchannel made from polydimethylsiloxane (Sylgard 184: Dow Corning), we created microdroplets and encapsulated mouse intestinal microbial cells in the microdroplets. In this example, we used a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, with adjacent channels arranged at right angles to each other, but it is also possible to use microchannels connected in an approximately T-shape. In this example, we used a microchannel with a width of 34 μm and a height of 50 μm, but the size of the microchannel can be changed appropriately depending on the size of the microdroplets to be created and the size of a single cell to be encapsulated.

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

In this embodiment, the diameter of the microdroplets is uniformly set to 50 μm, making it easier for cells to be encapsulated in the microdroplets one by one. Considering the size of a single cell, the diameter of the microdroplets is, for example, 1 to 250 μm, and preferably 20 to 200 μm. The diameter of the droplets may be about 1 to 250 μm, more preferably about 10 to 200 μm. For example, the diameter of the droplets may be 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 50 μm, about 80 μm, about 100 μm, about 150 μm, about 200 μm, or about 250 μm.

The tube contains multiple microdroplets and oil, but the microdroplets have a lighter specific gravity than the oil and so accumulate in the upper layer.

Next, the tube was cooled on ice for 15 minutes, and the 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 to 250 μm, more preferably about 10 to 200 μm, for example, 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 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 droplets to be produced, but the diameter may change during gelling. In addition, the diameter of the gel capsule is preferably 1 to 250 μm. By making the diameter of the gel capsules uniform, the rate of penetration of the lysis reagent, which will be 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, and the oil in the bottom layer was removed. Then, acetone (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) and isopropanol (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) were added in that order, and the tube was centrifuged to remove the oil. The centrifugal washing to remove the oil was performed by the removal unit described below. In this example, the oil that has permeated the gel capsules is also considered to be included in the impurities. Furthermore, 500 μL of PBS was added and centrifugal washing was performed three times, leaving the gel capsules suspended in the aqueous layer (PBS). The gel capsules accumulated in the bottom layer because they have a higher specific gravity than the aqueous layer.

Next, the gel capsules were successively immersed in a lysis reagent as a dissolution reagent, which dissolved parts of the cells other than the target material to be collected, such as the cell walls, inside the gel capsule, and eluted genomic DNA into the gel capsule.

Specifically, lysozyme (10 U/μL) (R1804M, Epicentre), a type of lysis reagent, was added to the tube to lyse the cells. Next, achromopeptidase (850 U/mL) (015-09951, Fujifilm Wako Pure Chemical) was added to the tube, a type of lysis reagent. Next, protease K (1 mg/mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), a type of lysis reagent, were added to the tube to lyse the cells, and then centrifugal washing was performed five times to remove components (contaminants) other than the protease and the genomic DNA of the lysed cells from the tube. Next, the gel capsule was immersed in Buffer D2 (QIAGEN), an aqueous solution containing potassium hydroxide, which is a type of lysis reagent, to dissolve the remaining components and denature the genomic DNA. As described above, the lysis reagent used in this example was lysozyme, achromopeptidase, protease K, sodium dodecyl sulfate, and Buffer D2. Potassium hydroxide is also used in a normal DNA amplification reaction process, but since it also has a lysis effect, it was used as one of the lysis reagents in this example. Since the gel capsule was immersed in the lysis reagent for a short time, the eluted genomic DNA was not discharged outside the gel capsule by the lysis reagent, but was retained in the gel capsule. In this example, the lysis reagent that penetrated the gel capsule was also included in the impurities.

In this example, lysozyme, achromopeptidase, and protease K were added in sequence, sodium dodecyl sulfate was added to lyse the cells, and then centrifugation washing was performed only before adding Buffer D2. This allows for a sufficient washing effect. However, centrifugation washing may also be performed after the cells are lysed with each lysis reagent.

In this way, the desired genomic DNA can be extracted by lysing the cells using multiple types of lysis reagents, and by immersing the cells in the lysis reagent and then centrifugal washing, contaminants such as the lysis reagent and components other than the polynucleotides of the lysed cells can be removed, allowing the genomic DNA to be purified without inhibiting the subsequent genomic DNA amplification reaction.

Amplification reagent was added to a tube containing a gel capsule that holds genomic DNA denatured in potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Displacement Amplification) method using phi29 DNA polymerase, a strand-displacement DNA polymerase, was used. Here, the gel capsule was immersed in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN) and the whole genome amplification reaction was performed 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).

In addition, the samples were stained with a fluorescent DNA intercalator (SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)), and fluorescent gel capsules were individually collected from each fecal sample (n=5) (94 pieces in total) onto a plate (PCR-96-FS-C, Axygen) using a flow cytometer (BD FACSMelody cell sorter, BD Biosciences) (470 pieces in total).

The collected gel capsules were each dissolved by heating at 65°C (S1000 Thermal Cycler, Bio-Rad), and secondary amplification was then performed in the wells of each plate using the MDA method (REPLI-g Single Cell Kit, 150345, QIAGEN), to create a library master plate containing 10 μL of DNA amplified product per well.

39 μL of nuclease-free water (UltraPure DNase/RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) was dispensed into each well of a new plate, and 1 μL of the DNA amplified product in the library master plate was added to prepare a 40-fold dilution of the library master plate. Next, DNA concentration was quantified using a Qubit fluorometer (Q33226, Thermo Fisher Scientific) and Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 μL of the dilution. In addition, PCR was performed targeting 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 diluted solution, 4.25 μL UltraPure DNase/RNase-Free Distilled Water (10977-015, Thermo Fisher Scientific) (S1000 Thermal Cycler, Bio-Rad). The PCR reaction conditions were initial heat denaturation at 95°C, The mixture was incubated at 98°C for 5 min, denatured at 98°C for 10 sec, annealed at 51°C for 15 sec, and extended at 72°C for 5 sec for 27 cycles, and then incubated at 72°C for 5 min and stored at 4°C. The presence or absence of PCR products was confirmed by agarose gel electrophoresis (electrophoresis 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) (electrophoresis conditions: 100V, 15min), and samples in which amplification was observed were subjected to sequence analysis using the Sanger method (DNA sequencing outsourcing service of FASMAC Co., Ltd.).

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

(Example 2: Analysis of human intestinal microbiota composition)
(Sample collection from humans)
Human feces were collected in a collection container (FS-0007 or FS-0008, Techno Suruga Lab). For collection containers containing preservative solution, the preservative solution was removed after centrifugation at 8000xg, 5 min, and 4°C, and the samples were washed once with 1000 μL of DPBS. Frozen feces (in 1.5 ml tubes) were left on ice for 30 minutes to thaw naturally. For fresh feces not containing preservative solution, no special treatment was required before crushing. The samples were then crushed in 500 μL of phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) using a crusher until no solid matter remained. The mouse intestinal microorganisms were collected by centrifugation at 2,000×g for 2 seconds (himac CF15RX, Koki Holdings) and the operation of collecting the supernatant was repeated twice, followed by centrifugation at 15,000×g for 3 minutes. The bacterial pellet was washed twice with PBS by centrifugation, and then suspended in PBS to obtain a cell suspension of mouse intestinal microorganisms. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting chamber A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, SIGMA-ALDRICH) was added to a final concentration of 1.5%, to prepare an intestinal microorganism suspension to be used for gel capsule production (final cell concentration: 1.5×10 ³ cells/μL).

(Processing of collected samples (wet))
Using a microchannel made by ourselves using polydimethylsiloxane (Sylgard 184: Dow Corning), we prepared microdroplets and encapsulated human intestinal microbial cells in the microdroplets. In this example, we used a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, with adjacent channels arranged at right angles, but it is also possible to use a microchannel connected in a roughly T-shape. In this example, we used a microchannel with a width of 34 μm and a height of 50 μm, but the size of the microchannel can be changed appropriately depending on the size of the microdroplets to be prepared and the size of a single cell to be encapsulated.

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

In this embodiment, the diameter of the microdroplets is made uniform at 50 μm, making it easier for cells to be encapsulated in the microdroplets one by one. Considering the size of a single cell, the diameter of the microdroplets is, for example, 1 to 250 μm, and preferably 10 to 200 μm.

The tube contains multiple microdroplets and oil, but the microdroplets have a lighter specific gravity than the oil and so accumulate in the upper layer.

Next, the tube was cooled on ice for 15 minutes and the microdroplets were gelled using ultra-low melting point agarose. The gelled microdroplets are gel capsules. As the diameter of the microdroplets is 50 μm, the diameter of the gel capsules is also 50 μm. It is preferable that the diameter of the gel capsules is 1 to 250 μm. By making the diameter of the gel capsules uniform, the penetration rate of the lysis reagent, described below, 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, and the oil in the bottom layer was removed. Then, acetone (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) and isopropanol (FUJIFILM Wako Pure Chemical Industries, Ltd.) (500 μL) were added in that order, and the tube was centrifuged to remove the oil. The centrifugal washing to remove the oil was performed by the removal unit described below. In this example, the oil that has permeated the gel capsules is also considered to be included in the impurities. Furthermore, 500 μL of PBS was added and centrifugal washing was performed three times, leaving the gel capsules suspended in the aqueous layer (PBS). The gel capsules accumulated in the bottom layer because they have a higher specific gravity than the aqueous layer.

Next, the gel capsules were successively immersed in a lysis reagent as a dissolution reagent, which dissolved parts of the cells other than the target material to be collected, such as the cell walls, inside the gel capsule, and eluted genomic DNA into the gel capsule.

Specifically, lysozyme (10 U/μL) (R1804M, Epicentre), a type of lysis reagent, was added to the tube to lyse the cells. Next, achromopeptidase (850 U/mL) (015-09951, Fujifilm Wako Pure Chemical) was added to the tube, a type of lysis reagent. Next, protease K (1 mg/mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), a type of lysis reagent, were added to the tube to lyse the cells, and then centrifugal washing was performed five times to remove components (contaminants) other than the protease and the genomic DNA of the lysed cells from the tube. Next, the gel capsule was immersed in Buffer D2 (QIAGEN), an aqueous solution containing potassium hydroxide, which is a type of lysis reagent, to dissolve the remaining components and denature the genomic DNA. As described above, the lysis reagent used in this example was lysozyme, achromopeptidase, protease K, sodium dodecyl sulfate, and Buffer D2. Potassium hydroxide is also used in a normal DNA amplification reaction process, but since it also has a lysis effect, it was used as one of the lysis reagents in this example. Since the gel capsule was immersed in the lysis reagent for a short time, the eluted genomic DNA was not discharged outside the gel capsule by the lysis reagent, but was retained in the gel capsule. In this example, the lysis reagent that penetrated the gel capsule was also included in the impurities.

In this embodiment, lysozyme, achromopeptidase, and protease K are added in sequence, sodium dodecyl sulfate is added to lyse the cells, and then centrifugation is performed only before adding Buffer D2, so that a sufficient washing effect can be obtained. However, centrifugation may be performed after the cells are lysed with each lysis reagent.

In this way, the desired genomic DNA can be extracted by lysing the cells using multiple types of lysis reagents, and by immersing the cells in the lysis reagent and then centrifugal washing, contaminants such as the lysis reagent and components other than the polynucleotides of the lysed cells can be removed, allowing the genomic DNA to be purified without inhibiting the subsequent genomic DNA amplification reaction.

Amplification reagent was added to a tube containing a gel capsule that holds genomic DNA denatured in potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent. Specifically, the MDA (Multiple Displacement Amplification) method using phi29 DNA polymerase, a strand-displacement DNA polymerase, was used. Here, the gel capsule was immersed in the whole genome amplification reaction reagent REPLI-g Single Cell Kit (QIAGEN) and the whole genome amplification reaction was performed 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).

In addition, the samples were stained with a fluorescent DNA intercalator (SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)), and fluorescent gel capsules were individually collected from each fecal sample (n=2) (94 gel capsules in total) onto a plate (PCR-96-FS-C, Axygen) using a flow cytometer (BD FACSMelody cell sorter, BD Biosciences) (188 gel capsules in total).

The collected gel capsules were each dissolved by heating at 65°C (S1000 Thermal Cycler, Bio-Rad), and secondary amplification was then performed in the wells of each plate using the MDA method (REPLI-g Single Cell Kit, 150345, QIAGEN), to create a library master plate containing 10 μL of DNA amplified product per well.

39 μL of nuclease-free water (UltraPure DNase/RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) was dispensed into each well of a new plate, and 1 μL of the DNA amplified product in the library master plate was added to prepare a 40-fold dilution of the library master plate. Next, DNA concentration was quantified using a Qubit fluorometer (Q33226, Thermo Fisher Scientific) and Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 μL of the dilution. In addition, PCR was performed targeting 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 diluted solution, 4.25 μL UltraPure DNase/RNase-Free Distilled Water (10977-015, Thermo Fisher Scientific) (S1000 Thermal Cycler, Bio-Rad). The PCR reaction conditions were initial heat denaturation at 95°C, The mixture was incubated at 98°C for 5 min, denatured at 98°C for 10 sec, annealed at 51°C for 15 sec, and extended at 72°C for 5 sec for 27 cycles. After 5 min of incubation at 72°C, the mixture was stored at 4°C. The presence or absence of PCR products was confirmed by agarose gel electrophoresis (electrophoresis 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) (electrophoresis conditions: 100V, 15 min), and samples in which amplification was observed were subjected to sequence analysis using the Sanger method (DNA sequencing service provided by FASMAC Co., Ltd.).

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

For the above 95 samples, libraries were prepared using Nextera XT DNA sample prep kit (Illumina, FC-131-1096), and 2 × 75 bp paired-end reads were obtained by whole genome sequencing using Miseq (Illumina, SY-410-1003). Sequence data were assembled using SPAdes (Bankevich A et al., J Comput Biol. 2012 May;19(5):455-77. http://doi.org/10.1089/cmb.2012.0021) to create contigs. In addition, the genome sequencing rate (completion rate) and the degree of contamination were evaluated using CheckM (Parks et al., Genome Res. 2015. 25: 1043-1055, doi:10.1101/gr.186072.114) under default conditions. As a result, the average completion rate was 37.8% and the average contamination rate was 1.3% for fresh stool samples, and the average completion rate was 52.6% and the average contamination rate was 4.8% for preserved liquid stool samples (Figure 6).

Furthermore, the marker gene group detected using CheckM was used to perform an evolutionary phylogenetic analysis of bacteria from the genome data of each sample. As a result, it was shown that most of the data was derived from the phyla Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria, which are predominant species of enterobacteria. By referring to such phylogenetic indicators and the identity of the 16SrRNA gene, it is possible to identify and count bacterial species and calculate the composition ratio as shown in Figure 7. In addition, by analyzing the digital sequence information of the bacteria, it is possible to analyze the metabolic function of the bacteria, whether they possess drug resistance genes, analyze gene mutations, compare them with known bacteria, and compare them with other samples. This process can be performed using, 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 metabolic and physiological function potential evaluation systems such as MetaCyc and MAPLE. As a series of steps in the analysis of genetic information, the effectiveness of the assembly is evaluated using QUAST for the genetic information obtained by performing denovo assembly, and the quality of the genome set is evaluated using CheckM. Then, annotation tools such as Prokka are used to identify genes in the genome, and the conservation as a metabolic pathway is evaluated using MAPLE. The conservation and identity of each gene are evaluated using the Pfam and COG databases, and comparative genome analysis with closely related organisms is performed.

More specifically, when the purpose is to evaluate gene mutations on a single-cell basis, (1) a homology search of gene sequences from a single organism using BLAST or hmmer is performed on the target gene, or (2) single-organism sequence data is mapped using BWA or bowtie2 to detect differences between the gene sequence from a single organism and the target gene sequence, and identify gene mutations.

When the purpose is comparison with known bacteria, comparison with other samples, or subspecies analysis, a genome set is created that contains genetic information from a single organism presumed to be genomes from the same species of microorganism and genetic information from known microorganisms based on average nucleotide identity, CheckM, and single copy marker gene sequences detected by GTDB-tk. Next, differences between genomes, such as the presence or absence of specific genes, gene loci, and mutations in common gene sequences, are identified and extracted using homology search tools such as BLAST and hmmer. Clustering based on characteristic genome differences allows classification at the strain level of the same species of microorganism or at the substrain level of the same strain of microorganism.

(Example 3: When it is desired to selectively obtain data on cells having specific characteristics from among a variety of cells)
When the purpose is to obtain genome data of one or more specific microorganisms that are of interest to a person skilled in the art among animal symbiotic microorganisms such as enterobacteria and marine/soil microorganisms, the presence or absence of gene fragments of the target microorganism in advance can be confirmed in advance for gel capsules containing polynucleotides or amplified nucleic acids recovered from the gel capsules, thereby reducing the acquisition of unnecessary gene sequence data and the associated costs. Examples of measurement targets include comparative analysis of microorganisms of the same lineage (e.g., analysis of subspecies in a microbial lineage related to a disease, etc.), searching for microorganisms having specific genes (e.g., secondary metabolites or enzymes produced by microorganisms), or individually selecting and analyzing bacteria, archaea, fungi, and other eukaryotic cells from among organisms of multiple lineages. In particular, specific detection of enterobacteria and oral bacteria that perform specific metabolic functions from genes, and detection of subspecies of skin bacteria, etc., are expected for the purpose of evaluating the intestinal environment, oral cavity, and skin environment of the host human.

(Example 4: When a large amount of host DNA, etc. is present)
When the analysis sample is feces, saliva, sputum, swabs from the skin, oral cavity, nasal cavity, ears, genitals, etc., surgical washings, or tissue extracts or blood, and the microorganisms contained in the sample are the subject of analysis, the sample contains many cells, intracellular organelles, and nucleic acids derived from the host animal. Some of these may also be encapsulated inside the gel capsule to perform polynucleotide amplification. For this reason, by confirming in advance the presence or absence of gene fragments of the target microorganism or gene fragments derived from the host in the gel capsule containing the amplified polynucleotide, it is possible to avoid obtaining unnecessary gene sequence data including polynucleotides derived from the host, and to reduce the costs involved. Examples of measurements include the detection of microorganisms from human samples, such as the search for pathogenic microorganisms from blood or sputum, and the analysis of microorganisms coexisting inside tissues.

Alternatively, by actively analyzing host-derived polynucleotides, it can be applied to comprehensive analysis of the microbiome.

For example, it is known that the intestinal flora present in the digestive tract and the host animal have a symbiotic relationship in which the host provides an anaerobic environment for the colonization of the gut flora, while the intestinal flora influences the health of the host. For example, the major influences on the host's health include the production of nutrients and the development of the immune system and defense against infectious diseases. Another example is inflammatory bowel disease, which is a disease caused by a combination of genetic predisposition and abnormalities in intestinal environmental factors, including intestinal bacteria.

For such pathologies, it is necessary to analyze the genetic information of the host itself, conduct an integrated analysis of the intestinal microbiota, including bacteria, viruses, and fungi, and clarify the mechanism of action on the host's physiological functions, and the technology disclosed herein can contribute to these. Furthermore, by analyzing the genetic information of the host itself, it is possible to perform more in-depth analysis of the relationship between the intestinal microbiota and the pathologies of various diseases and functional analysis centered on metabolic products.

In the intestinal tract, while the intestinal bacteria and the host compete for nutrients, the intestinal bacteria also metabolize and break down nutrients for the host, allowing for more integrated analysis. In this way, the functions of metabolites derived from intestinal bacteria can be analyzed in a comprehensive manner that also includes genetic analysis, metabolomic analysis, and biochemical analysis on the host side, and the functions of intestinal bacteria can be inferred from bacterial genome sequence information and a correlation can be seen with the metabolites produced.

Example 5: Evaluation of gene mutations in single cells
It is possible to evaluate the mutation diversity in genome sequences at the single-cell level, and to track the genomic heterogeneity of each microorganism in the microbiome and the occurrence and progression of mutation lineages. By selectively amplifying and detecting the target gene mutation site in the target cell using a gel capsule containing amplified polynucleotides, it is possible to specifically obtain only the gene sequence data to be analyzed, reducing the costs involved. It can be used to detect genetically mutated microorganisms and plasmid infections.

(Example 6: Evaluation of conservation of specific biological species)
When the purpose is to prove or reject the inclusion of a specific biological species in a drug, pesticide, food, etc., the disclosed technology can be used to detect and select a specific organism from a gel capsule containing an amplified polynucleotide, thereby indicating its content. In addition, by analyzing the obtained genetic information in detail, it is possible to evaluate the degree of identity with a standard at the genome level, storage stability, etc. It is expected to be used for quality assurance of microbial preparations, etc.

The specific procedure involves (1) performing a homology search of gene sequences derived from a single organism using BLAST or hmmer for the target gene, or (2) mapping of sequence data derived from a single organism using BWA or bowtie2 to detect differences between gene sequences derived from a single organism and the target gene sequence, and identifying genetic mutations.

(Example 7: Detection of specific biological species)
The analysis sample may be feces, saliva, sputum, swabs of the skin, oral cavity, nasal cavity, ears, genitals, etc., surgical washings, tissue extracts, blood, etc., and the presence of one or more specific microorganisms may be detected to determine drug efficacy and drug resistance or evaluate the metabolic ability of food. By using the disclosed technology, a specific organism can be detected and selected from a gel capsule containing an amplified polynucleotide, and its content can be indicated. In addition, by analyzing the obtained genetic information in detail, it is possible to estimate the function and storage stability at the genome level.

For example, when single-cell sequence data of mouse intestinal microorganisms was obtained, a homology search of gene sequences derived from a single organism was performed based on genes that metabolize the dietary fiber inulin or gene markers that identify the microbial Bacteroides species, allowing the microorganisms and gene groups responsible for breaking down the food inulin to be identified, and their functions to be estimated from the homology of known genes and prediction of three-dimensional structure based on amino acid sequences. It was also possible to assess the proportion (content) of a given microbial species in single-cell sequence data of multiple intestinal microorganisms.

Example 8: Evaluation of soil bacteria
When soil or seawater is used as an analytical sample, the technology disclosed herein can be used to detect and select various specific organisms that live in soil or seawater from gel capsules containing amplified polynucleotides, thereby identifying their habitat and indicating their population. In addition, detailed analysis of the obtained genetic information can be used to evaluate at the genome level cultivated varieties, livestock or aquaculture varieties, etc. that are suitable for the soil or seawater. It is also expected that the technology can be used in pesticide-free manufacturing methods using soil bacteria or marine bacteria.

Specifically, 10 g of soil was collected in a 50 mL tube (2342-050, Iwaki) (n=3), and phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) was added to a total volume of 40 mL to thoroughly suspend the soil. The tube after suspension was left on ice for 5 minutes, and the supernatant was transferred to a new 50 mL tube. The supernatant was filtered using a 5 μm filter (SMWP04700, Sigma-Aldrich), then centrifuged at 10,000 × g for 5 minutes (75004263, Thermo Fisher Scientific), and the supernatant was removed. The pellet was suspended again in 10 mL of PBS, dispensed into 1.5 mL tubes (MCT-150-C, Axygen) in 1 mL portions, and then centrifuged at 10,000×g for 5 minutes to collect the soil bacteria. The bacterial pellets were collected in one 1.5 mL tube, washed twice with PBS by centrifugation, and then suspended in PBS to obtain a soil bacterial cell suspension. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting board A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, Sigma-Aldrich) was added to a final concentration of 1.5%, to prepare a soil bacterial suspension to be used for gel capsule production (final cell concentration: 1.5×10 ³ cells/μL). Subsequently, the analysis was carried out and evaluated in the same manner as in Example 1 or 2.

(Example 9: Analysis of aquatic microbial composition)
In the experiment, 4 L of seawater or freshwater was collected in a sterilized plastic tank, filtered through a 5 μm filter (SMWP04700, Sigma-Aldrich), and then filtered through a 0.22 μm filter (GSWP04700, Sigma-Aldrich) to trap the bacterial fraction on the filter. The 0.22 μm filter was placed in a 25 mL tube (2362-025, Iwaki) containing 10 mL of phosphate-buffered saline (DPBS) (Dulbecco's Phosphate-Buffered Saline, 14190-144, Thermo Fisher Scientific) and thoroughly suspended. The 10 mL of bacterial suspension was dispensed into 1.5 mL tubes (MCT-150-C, Axygen) at 1 mL each, and the bacteria from the seawater or freshwater were collected by centrifugation at 10,000 × g for 5 minutes. The bacterial pellets were collected in a single 1.5 mL tube, washed twice with PBS by centrifugation, and then suspended in PBS to obtain a cell suspension of bacteria derived from seawater or freshwater. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial counting chamber A161, 2-5679-01, AS ONE), and ultra-low melting point agarose (A5030-10G, Sigma-Aldrich) was added to a final concentration of 1.5% to prepare a bacterial suspension derived from seawater or freshwater for use in gel capsule production (final cell concentration: 1.5 x 10 ³ cells/μL). Subsequently, analysis was carried out and evaluated in the same manner as in Example 1 or 2.

(Note)
As described above, the present disclosure has been illustrated using a preferred embodiment of the present disclosure, but the present disclosure should not be interpreted as being limited to this embodiment. It is understood that the scope of the present disclosure should be interpreted only by the scope of the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present disclosure and technical common sense from the description of the specific preferred embodiment of the present disclosure. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification as if the contents themselves were specifically described in this specification. This application claims priority to patent application 2019-85836 filed on April 26, 2019 at the Japan Patent Office, and it is understood that the contents of the application itself should be incorporated by reference to this specification as if the contents were specifically described in this specification.

This disclosure can be used in fields such as biological research, medicine, the environment, and healthcare.

SEQ ID NO: 1: forward primer SEQ ID NO: 2: reverse primer

Claims (22)

1. A method for analyzing a microbiota composition, comprising:
1. A method comprising assessing microbiota composition from a sample comprising amplified nucleic acid from each cell in the microbiota,
amplified nucleic acid from each of the cells,
Using a sample containing a microbiota, encapsulating cells one by one in a droplet;
gelling the droplets to form gel capsules;
immersing said gel capsule in one or more lysis reagents to lyse said cells, wherein a polynucleotide comprising said genomic DNA or a portion thereof is dissolved in said gel capsule and is retained in said gel capsule in a state where substances binding to said genomic DNA or a portion thereof have been removed;
contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule;
and
The method comprises flowing a suspension of the cells through a microchannel and shearing the suspension with oil to create the droplets encapsulating the cells . 2. The method of claim 1 , wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen, or a photocurable resin. 3. The method of claim 1, wherein the lysis reagent is at least one selected from the group consisting of lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octylglucoside, octylthioglucoside, CHAPS, CHAPSO, dodecyl-β-D-maltoside, Nonidet P-40, and Zwittergent 3-12. The method according to any one of claims 1 to 3 , characterized in that the gel capsules are hydrogel capsules. The method according to any one of claims 1 to 4 , further comprising the step of selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells. The method according to any one of claims 1 to 5 , comprising the step of detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells. 7. The method of claim 6 , wherein the step of detecting a nucleic acid having a specific sequence comprises amplifying and sequencing the nucleic acid having a specific sequence. The method according to any one of claims 1 to 7 , wherein the step of assessing the microbiota composition comprises determining the absolute number of each type of microorganism in the microbiota. The method of any one of claims 1 to 8 , further comprising the step of obtaining genomic sequence data for each of the cells in the microbiota from a sample containing amplified nucleic acid from the cells. The method of claim 9 , further comprising the step of selecting genomic sequence data to analyze from the genomic sequence data of each of the cells. The method according to any one of claims 1 to 10 , wherein the step of evaluating the microbiota composition comprises comparing gene sequences extracted from de novo assembly data for each microorganism. The method according to any one of claims 1 to 11 , wherein the microflora is a bacterial flora. The method of claim 12 , wherein the microbiota is the intestinal microbiota. A kit for use in the method of claims 1 to 13 , comprising a specimen collection container containing a preservation solution. The kit of claim 14 , wherein the preservation solution comprises guanidine or ethanol. 1. A system for analyzing a microbiota composition, comprising:
A sample providing unit that provides a sample containing amplified nucleic acid derived from each cell in the microbiota;
a composition evaluation unit that evaluates a microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota;
The sample providing unit includes:
a droplet encapsulation unit that uses a sample containing a microflora and encapsulates cells one by one in a droplet, the droplet encapsulation unit being configured to flow a suspension of the cells into a microchannel and shear the suspension with oil to produce the droplets in which the cells are encapsulated;
a gel capsule forming unit that gels the droplets to form gel capsules;
a cell lysis unit that lyses cells by immersing the gel capsule in one or more lysis reagents, the gel capsule storing one or more lysis reagents for lysing the cells, the cell lysis unit being configured such that a polynucleotide containing the genomic DNA of the cell or a portion thereof is dissolved in the gel capsule and is retained in the gel capsule in a state in which a substance that binds to the genomic DNA or a portion thereof has been removed;
a polynucleotide amplification reagent for amplifying the polynucleotide in a gel capsule;
Including,
the composition evaluation unit includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells;
The detection reagent or device includes a nucleic acid amplification and sequencing device for amplifying and sequencing nucleic acids.
system. The system of claim 16 , further comprising a specimen collection container containing a preservation fluid. The system according to claim 16 or 17 , further comprising a sample selection unit for selecting a sample containing amplified nucleic acid to be analyzed from the samples containing amplified nucleic acid derived from each of the cells. The system according to claim 16 , wherein the composition evaluation unit includes a calculation unit that performs a procedure for determining the absolute number of each type of microorganism in the microbiome. The system according to any one of claims 16 to 19 , wherein the composition evaluation unit further includes a genome sequence data acquisition unit for acquiring genome sequence data of each of the cells in the microbiome from a sample containing amplified nucleic acid derived from each of the cells . The system according to claim 20 , 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 of the cells. The system according to any one of claims 16 to 21 , wherein the composition evaluation unit includes or introduces a gene sequence extracted from de novo assembly data for each microorganism and has a function of comparing the gene sequences.