WO2020218553A1 - Analyse numérique de la flore microbienne - Google Patents

Analyse numérique de la flore microbienne Download PDF

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WO2020218553A1
WO2020218553A1 PCT/JP2020/017792 JP2020017792W WO2020218553A1 WO 2020218553 A1 WO2020218553 A1 WO 2020218553A1 JP 2020017792 W JP2020017792 W JP 2020017792W WO 2020218553 A1 WO2020218553 A1 WO 2020218553A1
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cell
nucleic acid
microbiota
gel capsule
cells
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Japanese (ja)
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正人 細川
春子 竹山
西川　洋平
小川　雅人
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ｂｉｔＢｉｏｍｅ株式会社
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Publication of WO2020218553A1 publication Critical patent/WO2020218553A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/26—Inoculator or sampler
    • C12M1/28—Inoculator or sampler being part of container
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • This disclosure is available in fields such as biological research, medicine, environment, and healthcare regarding the analysis of microbiota composition.
  • amplicon sequence analysis targeting the conserved regions of 16S rRNA genes and 18S rRNA genes has been widely used in recent years using a next-generation sequencer.
  • 16SrRNA gene When the 16SrRNA gene is targeted, a storage region of several hundred bases is PCR-amplified on DNA collectively extracted from a sample containing various microorganisms (feces, soil, seawater, etc.), and the storage region is stored by a next-generation sequencer.
  • there are some problems with this method are some problems with this method.
  • the copy number of the 16S rRNA gene on the genome is different for each bacterium. Some bacteria have only a single copy of the 16S rRNA gene, while others have more than 7 genes. Furthermore, since the genomes of most of the environmental microorganisms are undecided, the number of the genes is unknown and cannot be corrected.
  • the total amount of the microorganisms X may have decreased, or the number of other microorganisms has simply increased and the number of microorganisms X has not changed. It is also possible.
  • the present inventors are a method of analyzing the microbiota composition as a result of diligent research, and include a step of evaluating the microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota.
  • a step of evaluating the microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota.
  • the present disclosure comprehensively reads the gene sequences that identify microbial species from amplified polynucleotides that are conditioned in parallel for each cell from a variety of microbial species, and digitally counts the microbial species in the sample cell by cell. However, it provides information that is digitally counted with the microbiota as an absolute amount.
  • Examples of embodiments of the present disclosure include: (Item 1) A method for analyzing microbiota composition. A method comprising the step of evaluating microbiota composition from a sample containing an amplified nucleic acid derived from each cell in the microbiota. (Item 1A) A method for analyzing the microflora.
  • a method comprising the step of evaluating the microbiota based on information for each nucleic acid derived from each cell in the microbiota.
  • (Item 2) The amplified nucleic acid derived from each of the cells A step of encapsulating cells one by one in a droplet using a sample containing a microflora, The process of gelling the droplets to form gel capsules, A step of immersing the gel capsule in one or more solubilizing reagents to lyse the cell, wherein the genomic DNA of the cell or a polynucleotide containing a portion thereof is eluted into the gel capsule and the genomic DNA or its portion.
  • the process of holding in the gel capsule with the substance bound to the moiety removed The method according to any of the above items, which is produced by a method comprising contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule.
  • the item which comprises flowing the suspension of the cells into a microchannel and shearing the suspension with oil to produce the droplets encapsulating the cells.
  • the method described in any of. (Item 4) The method according to any one of the above items, wherein the gel capsule is formed from agarose, acrylamide, PEG, gelatin, sodium alginate, matrigel, collagen or a photocurable resin.
  • the solubilizing reagents are lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform , Guanidin Hydrochloride, Urea, 2-Mercaptoethanol, Dithiotreitol, TCEP-HCl, Sodium Colate, Sodium Deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58 , Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl- ⁇ -D-hydrochloride, Nonide
  • the method according to any one of the above items (Item 6) The method according to any one of the above items, wherein the gel capsule is a hydrogel capsule. (Item 7) The method according to any one of the above items, further comprising a step of selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell. (Item 8) The method according to any one of the above items, which comprises a step of detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each cell.
  • the method according to any one of the above items, wherein the step of detecting the nucleic acid having the specific sequence comprises amplifying and sequencing the nucleic acid having the specific sequence.
  • the step of evaluating the microbiota composition comprises specifying the absolute number of various microorganisms in the microbiota.
  • the step of evaluating the microbiota composition includes comparing gene sequences (for example, 16S rRNA gene sequence, 18S rRNA gene sequence, common gene set, etc.) extracted from Denovo assembly data in each microorganism.
  • the method according to any of the above items. (Item 14) The method according to any one of the above items, wherein the microbiota is a bacterial flora.
  • the microbiota is an intestinal flora.
  • (Item 16) A kit for use in the methods of items 1 to 15, comprising a sample collection container containing a storage solution.
  • (Item 17) The kit according to item 16, wherein the preservation solution contains guanidine or ethanol.
  • (Item 18) A system for analyzing microbiota composition.
  • a sample providing unit that provides a sample containing an amplified nucleic acid derived from each cell in the microflora, and a sample providing unit.
  • a system comprising a composition evaluation unit for evaluating the microbiota composition from a sample containing each cell-derived amplified nucleic acid in the microbiota.
  • the sample providing unit is A droplet encapsulation part that encloses cells one by one in a droplet using a sample containing a microflora, A gel capsule generation unit that gels the droplet to generate a gel capsule, A cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, which contains one or more lysis reagents for lysing cells.
  • the polynucleotide containing the genomic DNA of the cell or a portion thereof is eluted in the gel capsule and retained in the gel capsule with the substance binding to the genomic DNA or the portion removed.
  • a cell lysate comprising a reagent for amplifying the polynucleotide for amplifying the polynucleotide in a gel capsule.
  • a sample collection container containing a storage solution.
  • a sample selection unit for selecting a sample containing the amplified nucleic acid to be analyzed from the sample containing the amplified nucleic acid derived from each cell.
  • composition evaluation unit is described in any one of the above items, which includes a detection reagent or a detection device for detecting a nucleic acid having a specific sequence in a sample containing the amplified nucleic acid derived from each of the cells.
  • System. 23 The system of claim 5A, wherein the detection reagent or device comprises a nucleic acid amplification sequencing device for amplifying and sequencing nucleic acids.
  • the composition evaluation unit includes a calculation unit that executes a procedure for specifying the absolute number of various microorganisms in the microbiota.
  • the composition evaluation unit further includes a genome sequence data acquisition unit for genome sequence data of each cell from a sample containing an amplified nucleic acid derived from each cell in the microbiota. The system described in any of the items.
  • the composition evaluation unit further includes a data selection unit that selects genome sequence data to be analyzed from the genome sequence data of each cell.
  • the composition evaluation unit has a function of containing or introducing a gene sequence extracted from De novo assembly data in each microorganism and comparing the gene sequences.
  • the present disclosure it is possible to evaluate the absolute number of the composition of the microbiota by evaluating the microbiota based on the information for each nucleic acid derived from each cell in the microbiota.
  • microbiota analysis by PCR it is possible to use a part of the amplified DNA sample, and by performing sequencing and sequencing and determining the partial sequence, information on a specific gene sequence can be used.
  • the microbiota composition can be evaluated from the above, and a cheap and simple evaluation can be performed.
  • the gene sequence used for bacterial flora analysis is extracted from the digital sequence data obtained by sequencing, and composition data is created with high accuracy. Analysis can also be performed.
  • Microorganisms with a low composition ratio may be grouped as "other", but where there is concern that the microbial composition is estimated to be higher or lower than it actually is, this disclosure allows for absolute counts. This allows for a more accurate measurement of the actual composition.
  • FIG. 1 shows a schematic diagram in which steps are taken to prepare amplified DNA.
  • FIG. 2 shows a schematic diagram of wet sequence screening.
  • FIG. 3 is a schematic diagram of the sequence determination after selection.
  • FIG. 4 is a schematic diagram showing a dry sequence screening step.
  • FIG. 5 is a schematic diagram showing the overall flow of analysis of the microbiota in the present disclosure.
  • FIG. 6 is a box-and-whisker plot of the genome decoding rate (complete rate) of the draft genome prepared from the amplified nucleic acid derived from a single cell from a stool sample. The left is the analysis result of the single cell derived from the stool sample not immersed in the preservation solution, and the right is the analysis result of the single cell derived from the stool sample immersed in the preservation solution.
  • FIG. 6 is a box-and-whisker plot of the genome decoding rate (complete rate) of the draft genome prepared from the amplified nucleic acid derived from a single cell from a stool sample. The left
  • FIG. 7 shows the evolutionary phylogenetic classification analysis of bacteria from the genomic data of each sample with reference to the marker gene group detected using CheckM based on the draft genome prepared from the amplified nucleic acid derived from a single cell from the fecal sample. It is a figure which shows the result of having performed. The left is the analysis result of the single cell derived from the stool sample not immersed in the preservation solution, and the right is the analysis result of the single cell derived from the stool sample immersed in the preservation solution.
  • the present disclosure relates to a method for detecting and analyzing a microbial flora (for example, a microbial flora) in a microbial manner.
  • a microbial flora for example, a microbial flora
  • cell refers to a particle that contains a molecule that carries the genetic information and is any particle that can be replicated (whether or not it is possible alone).
  • the term “cell” as used herein includes cells of unicellular organisms, bacteria, cells derived from multicellular organisms, fungi, viruses and the like.
  • biomolecule refers to a molecule possessed by any organism or virus.
  • In vivo molecules can include nucleic acids, proteins, sugar chains, lipids, and the like.
  • biomolecular analog refers to a natural or non-natural variant of a biomolecule.
  • Analogs of in vivo molecules can include modified nucleic acids, modified amino acids, modified lipids or modified sugar chains.
  • aggregate refers to an aggregate containing two or more single biological units, cells or structures for cells.
  • subset refers to a portion of a set having a smaller number of single biological units, cells or cell structures.
  • gel refers to a colloidal solution (sol) in which a polymer substance or colloidal particles interact with each other to form a network structure as a whole and contain a large amount of a liquid phase as a solvent or a dispersion medium. A state in which fluidity is lost.
  • gelling means changing a solution into a “gel” state.
  • the "gel capsule” refers to a gel-like fine particle structure capable of holding a cell or a cell-like structure therein.
  • gene analysis means examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample.
  • the gene analysis can include those that utilize a nucleic acid amplification reaction.
  • Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction enzyme fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe: Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, digital PCR and the like can be mentioned.
  • single biological unit level refers to genetic information contained in one single biological unit or information on other biomolecules, as opposed to genetic information contained in another single biological unit or other information. It refers to processing in a state that can be distinguished from the information of biomolecules.
  • single cell level means that the genetic information contained in one cell or cell-like structure is processed in a state of being distinguished from the genetic information contained in another cell or cell-like structure. To do. For example, when amplifying a polynucleotide at the "single cell level", the amplification is performed in a state in which the polynucleotide in one cell and the polynucleotide in another cell can be distinguished from each other.
  • single cell analysis refers to the analysis of genetic information contained in one cell or cell-like structure in a state of being distinguished from the genetic information contained in another cell or cell-like structure. Point to.
  • nucleic acid information refers to information on nucleic acids contained in one cell or cell-like structure, and includes the presence or absence of a specific gene sequence, the yield of a specific gene, or the total nucleic acid yield.
  • 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.
  • microbiota refers to the entire set of microorganisms existing in a certain physical range.
  • the "microorganism” includes an organism (in the case of a multicellular organism, it may be an individual cell) whose existence cannot be discriminated by the naked eye and whose size is smaller than that that can be observed with a microscope or the like.
  • a certain physical range includes, for example, a certain range in the intestine, skin, oral cavity, nasal cavity, or vagina of an individual, water area in the environment, soil, biological surface, and internal organism.
  • the microflora may be a combination of microorganisms of a plurality of classifications, for example, a combination of fungi, bacteria, archaea, unicellular animals, viruses, etc., or a collection of microorganisms of some classifications may be extracted.
  • a combination of fungi, bacteria, archaea, unicellular animals, viruses, etc. or a collection of microorganisms of some classifications may be extracted.
  • One example of the microflora is the bacterial flora.
  • composition refers to information about what species are contained in the microbiota or the amount of each species contained, and is a part of the microbiota. It also includes information about whether or the amount of microbial species contained in the plant.
  • a method of analyzing the microbiota comprising the step of evaluating the microbiota based on information for each cell-derived nucleic acid in the microbiota. Will be done.
  • the amount of nucleic acid in a certain sequence can be specified from the information on nucleic acids derived from multiple cells, it is possible to measure the absolute amount of a specific type of microorganism because there is a difference in the number of copies for each microorganism.
  • the absolute amount of a particular species of microorganism can be measured based on the information of nucleic acids derived from each cell.
  • a method of analyzing microbiota composition comprising the step of evaluating microbiota composition from a sample containing amplified nucleic acid derived from each cell in the microbiota.
  • the amount of nucleic acid derived from microorganisms is small per cell, and even when analysis is performed in a state where nucleic acids derived from a plurality of cells are mixed, an amplification reaction is generally used, but a random primer is used. It is known that even when an attempt is made to amplify a nucleic acid as a whole, the degree of amplification is biased for each sequence depending on the GC content and the like. When amplification is biased, it is difficult to measure the relative amount of a particular species of microorganism in the microbiota.
  • information for each nucleic acid derived from each cell may be obtained by any method, but in order to easily obtain information for each nucleic acid derived from one cell from a large number of cells, a microorganism
  • the genomic DNA of the cell or the polynucleotide containing the portion thereof is eluted into the gel capsule, and the substance binding to the genomic DNA or the portion thereof is removed in the gel capsule. It is preferable to obtain each cell-derived amplified nucleic acid by a method including a step of contacting the polynucleotide with an amplification reagent and amplifying the polynucleotide in a gel capsule. In some cases.
  • the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule can also amplify the polynucleotide while maintaining a gel state in the gel capsule. ..
  • droplets can be made by encapsulating one cell by flowing one cell into a microchannel and shearing the suspension with oil.
  • the gel capsule may be a hydrogel capsule.
  • the material of the gel capsule may include agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • Gelation of the droplets can be performed by configuring the droplets to contain the material of the gel capsule and cooling the prepared droplets. Alternatively, gelation can be performed by giving a stimulus such as light to the droplet.
  • the inclusion of the gel capsule material in the droplets can be done, for example, by including the gel capsule material in a suspension of cells or cell-like structures.
  • the gel capsule may be a hydrogel capsule.
  • hydrogel refers to one in which the solvent or dispersion medium held by the network structure of the polymer substance or colloidal particles is water.
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • a lytic reagent or a combination of lysing reagents that is strong to some extent.
  • Gram-positive bacteria have a cell wall with a thick peptidoglycan layer, so mild ones alone may not be sufficient to lyse cells.
  • Non-patent literature (Rinke C, Lee J, Nath N, et al. Obtaining genomes from uncultivated denvironmental microorganisms using FACS-based single-cell genomics. Nat Protoc. 2014; 9 (5): 1038-1048. Doi: 10.1038 / pro 2014.067).
  • a method for evaluating the microbial composition as reported in various literatures such as non-patent literature (Vandeputteet al. 2017 Nature), the partial sequence or the entire sequence of the 16S rRNA gene is sequenced.
  • the cells or cell-like structures that can be targeted in the microbiota analysis of the present disclosure are two or more arbitrary numbers, for example, 10 or more, 50 or more, 100 or more, 500 or more, 1000 or more. It can be 5000 or more, 10,000 or more, 50,000 or more, 100,000 or more, 500,000 or more, 1 million or more, 5 million or more, 10 million or more.
  • the microbiota analysis of the present disclosure may use individual cell-derived nucleic acid-by-nucleic acid information from a larger number of cells than using conventional single-cell reaction systems, such as 0.2 mL, 1.5 mL microtube reaction systems. .. (Analysis before genome sequencing)
  • the methods prior to analysis of the total sequence information of individual microorganisms (for example, genome sequencing), the structures and sequences of nucleic acids or other biomolecules prepared in parallel from various microorganisms are used. It may be carried out to detect and select each individual specifically with reference to. That is, the method may include selecting a sample containing the amplified nucleic acid to be analyzed from a sample containing the amplified nucleic acid derived from each cell.
  • selection can be made based on the presence or absence of a particular gene sequence, the yield of a particular gene or the total nucleic acid yield. In some embodiments, it may be selected when there is a specific gene sequence, or it may be selected when there is no specific gene sequence. In some embodiments, it may be selected if the yield of the particular gene is greater than or equal to the baseline yield. In some embodiments, it may be selected if the total nucleic acid yield is greater than or equal to the reference yield.
  • reagents that specifically detect the presence or absence of a particular gene sequence include antibodies, probes, DNA-binding fluorescent dyes, fluorescent dye-binding nucleotides.
  • the yield of a specific gene or the total nucleic acid yield can be measured by absorbance measurement, fluorescence measurement, agarose gel electrophoresis, or microchip electrophoresis.
  • the method may include detecting nucleic acid having a particular sequence in a sample containing amplified nucleic acid derived from each cell.
  • the step of detecting a nucleic acid having a specific sequence may include amplifying and sequencing the nucleic acid having a specific sequence.
  • Evaluation of microbiota composition may include identifying the absolute number of various microbiota in the microbiota.
  • the absolute number of various microorganisms can be specified by specifying the microbial species for each of the amplified nucleic acids derived from each cell.
  • the microbial species can be specified, for example, by specifying the presence or absence of a specific gene sequence.
  • the method of the present disclosure may include obtaining genomic sequence data for each cell from a sample containing amplified nucleic acid derived from each cell in the microbiota.
  • obtaining genomic sequence data it is possible to obtain not only information as a sequence but also information from the viewpoint of the function performed by the sequence for each microorganism in the microflora.
  • Genome sequence data It is possible to select the genome sequence data to be analyzed from the genome sequence data of each cell. Genome sequence data has a large amount of information, and limiting the amount to be processed leads to a reduction in labor and time.
  • Selection may include assessing the presence or absence of a particular gene sequence and / or identity with a particular gene sequence. In some embodiments, identity with a particular gene sequence can be assessed by using BLAST or the like. In certain embodiments, the selection may be cell-by-cell selection of nucleic acid information based on the presence or absence of a particular gene sequence. In other embodiments, the selection may be cell-by-cell selection of nucleic acid information based on the identity of a particular gene sequence with nucleic acid information derived from two or more cells. In some embodiments, it may be selected when it has a certain level of identity or more, or it may be selected when it has a certain level of identity or less.
  • the identity is 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, It may be 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 100%.
  • the identity is 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, It may be 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.
  • evaluation of microbiota composition may be performed by comparing long gene sequences in each microorganism.
  • the long sequence may be, for example, a gene sequence extracted from de novo assembly data (for example, a 16S gene sequence or a set of common genes). Comparison of such long gene sequences can not only improve the accuracy of evaluation, but also enable evaluation of function in the microflora. For example, even if it is known that multiple species exist from sequence information alone, it is not possible to understand the function of the entire microbiota without individual information on each function, but it is based on genetic information. For example, information can be obtained about the amount of species that may have a particular activity (eg, enzymatic activity) within the microbiota.
  • the microbial flora that can be covered in this disclosure is, but is not limited to, eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, racen, gram-negative bacteria, gram-positive bacteria, archaea, fungi. , Or any combination thereof.
  • An example of a microflora is a bacterial flora. Since the microflora can contain multiple microorganisms having different cell properties (eg, the presence or absence of a cell wall), the nucleic acid information for each cell can be obtained or one cell in the same manner regardless of the microbial species. Adopting a means capable of obtaining each amplified nucleic acid may be preferable in the analysis of the present disclosure.
  • the microbiota is not limited, but is limited to intestinal microbiota, cutaneous microbiota, oral microbiota, nasal microbiota, vaginal microbiota, soil microbiota, root zone microbiota, river / seawater microbiota, and activity. It can be a sludge microbiota, an insect coexisting microbiota, an animal symbiotic microbiota, and the like.
  • the gut flora has been extensively studied for its effects on human health and is one of the preferred analyzes of the present disclosure.
  • Metagenomic analysis is a method of extracting and collecting nucleic acids, genes, and DNA possessed by microorganisms in the environment as a whole and comprehensively examining their structures (base sequences) without going through the process of culturing. Although it is not known which microorganism the individual nucleic acid or gene is derived from, it is possible to obtain information about the gene group of a collection (community) of microorganisms in the environment, and such a method is called metagenomic analysis.
  • a system for analyzing microbiota composition may be provided.
  • the system may be provided with a method or means for implementing a process thereof that comprises any of the features described in the other items herein.
  • the system may include a device for amplifying polynucleotides in cells.
  • the device can be, among other things, capable of amplifying polynucleotides in cells at the single cell level.
  • the device is a droplet preparation unit that encapsulates cells or cell-like structures in droplets one cell or structure at a time; a gel capsule generation unit that gels droplets to generate gel capsules; a reagent for dissolving gel capsules. It may be provided with a dissolving reagent dipping part to be immersed in; a removing part for removing contaminants from the gel capsule; and / or an amplification reagent dipping part for immersing the gel capsule in the amplification reagent.
  • the system or device may further comprise a sorting unit that sorts the gel capsules and houses the gel capsules in a storage container.
  • the system or device may optionally include media for encapsulating cells or cell-like structures, gel capsule materials, lysis reagents, amplification reagents, reagents used for sequencing nucleic acids (eg, polymerases, primer sets (eg, polymerases, primer sets). Barcode sequences may be included), etc.) and other reagents.
  • reagents in addition to those described elsewhere in the present specification, reagents known in the art may be used.
  • the device or system may further include a sequencing section for sequencing the nucleic acid sequence in the amplified polynucleotide in the amplification reagent immersion section.
  • the sequencing unit may be provided integrally with the above device or as another device in the system.
  • the sequencing unit includes the Sanger method, the Maxam-Gilbird method, single molecule real-time sequencing (for example, Pacific Biosciences, Menlo Park, California), and ion semiconductor sequencing (for example, Ion Torrent, South San Francisco, California).
  • Bisynthesis pyrosequencing (eg, 454, Brandored, Connecticut), ligation sequencing (eg, Life Technologies, Carlsbad, California SOLiD sequencing), synthetic and reversible terminator sequencing (eg, Illumina) , California), nucleic acid imaging techniques such as transmission electron microscopy, nanopore sequencing, and the like.
  • ligation sequencing eg, Life Technologies, Carlsbad, California SOLiD sequencing
  • synthetic and reversible terminator sequencing eg, Illumina
  • nucleic acid imaging techniques such as transmission electron microscopy, nanopore sequencing, and the like.
  • the system or device may be equipped with means for detecting and measuring the amplified gene.
  • a flow cytometry device suitable for handling the shape of a gelul capsule may be provided integrally with the above device or as another device in the system.
  • Means for detecting and measuring the amplified gene include means for performing a detection reaction (eg, thermal cycler and suitable reagents) and / or means for detecting signals (optical sensors, cameras, and suitable means for analysis). Can be included.
  • the system or device may include a calculator that may be configured to perform any information processing described elsewhere herein.
  • the calculation unit may be provided integrally with the above-mentioned device, or may be provided as another device (computer) in the system.
  • a calculation unit may also provide a program for performing information processing described elsewhere in the specification to implement the method of the present disclosure and a storage medium on which it is recorded.
  • the calculator may optionally include such a program and / or a storage medium on which it is recorded.
  • kits for analyzing microbiota composition may be provided.
  • the kit may include the material of the gel capsule, and the use of the gel capsule is advantageous for amplifying nucleic acids in cells or cell-like structures at the single cell level, as described elsewhere herein. And can be used as described herein for analysis of microbial composition.
  • the kit may include, for example, the material of the gel capsule and, optionally, one or more reagents. As the reagent, in addition to those described elsewhere in the present specification, reagents known in the art may be used.
  • Kits for analyzing microbiota composition may include reagents for lysis.
  • Reagents for lysis include lysoteam, labiase, yatarase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolaicin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride.
  • Tween 20 Urea, 2-mercaptoethanol, dithiotreitol, TCEP-HCl, sodium cholate, sodium deoxycholate, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, It may include at least one selected from the group consisting of Tween 80, octyl glucoside, octyl thioglucoside, CHAPS, CHAPSO, dodecyl- ⁇ -D-maltoside, Nonidet P-40, Zwittergent 3-12. Dissolving reagents are useful for obtaining amplified polynucleotides at the single cell level, especially genomic-wide amplification products.
  • the kit may include reagents for amplifying nucleic acids.
  • Amplification reagents include, for example, polymerases, primer sets (which may include barcode sequences), base mixes, suitable buffers and the like.
  • the kit may include reagents such as reagents used for sequencing nucleic acids (eg, polymerases, primer sets (which may include barcode sequences), etc.).
  • reagents polymerase, primer set (which may include a barcode sequence), etc.) for amplifying / decoding a specific gene by a Sanger sequence or NGS may be included.
  • the kit may also include reagents that can be used to detect and measure specific sequences, such as nucleic acid binding dyes, fluorescently labeled probes, and the like. Using these reagents, the presence or absence of a specific sequence can be measured by an instrument that detects and measures the amplified gene (flow cytometry, etc.).
  • specific sequences such as nucleic acid binding dyes, fluorescently labeled probes, and the like.
  • the kit may include means for taking a sample.
  • the kit may also include means for storing the sample taken.
  • Means for collecting a sample include a syringe, a swab, a biopsy punch, a urine collection container, a stool collection container, a saliva collection container, a medical tape, and the like.
  • Examples of the means for storage include a coolant (for example, an ice pack, dry ice, liquid nitrogen), a preservation solution (for example, a solution containing any of guanidine hydrochloride, ammonium sulfate, ethanol, etc.) and the like.
  • a kit comprising a sample collection container containing a preservation solution for analyzing microbiota composition
  • the storage solution may contain, for example, a guanidine solution (for example, FS-0007 or FS-0008, Technosulgarabo) or ethanol (for example, OMR-200 or OM-501, DNA genomek).
  • a guanidine solution for example, FS-0007 or FS-0008, Technosulgarabo
  • ethanol for example, OMR-200 or OM-501, DNA genomek.
  • Preservatives are generally used to increase the stability of nucleic acids, for example, guanidine solutions and alcohols are used to cause protein denaturation and inhibit the action of enzymes that promote nucleic acid degradation. Will be done.
  • a preservation solution that causes protein denaturation was expected to be disadvantageous for the stability of cell morphology, but in the examples herein, unexpectedly, after preservation with the preservation solution, the cells Many retain their morphology well and have been found to be particularly suitable for the methods of the present disclosure.
  • a gel capsule containing the amplified and retained 1-cell genome-derived polynucleotide is prepared by a method including the step of amplifying the polynucleotide in the gel capsule.
  • This gel capsule is stained with a DNA-binding fluorescent dye or the like, and fluorescence-positive capsules are counted by flow cytometry.
  • the number of fluorescence-positive capsules in a fixed volume corresponds to the total number of introduced microorganisms.
  • 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.
  • the above primer set contains a barcode sequence
  • each sample is subjected to an amplification reaction in which a different barcode is added, and PCR products derived from multiple replica plates are pooled to perform a next-generation sequence.
  • the plate well number is specified by the barcode sequence
  • the microbial species of hundreds to thousands of samples are specified at once by specifying the sequence of the PCR product.
  • the primer set may be a mixture of primer sets targeting a plurality of gene regions. More specifically, it is selected from those targeting the v3-v4 and v1-v2 regions of the 16S rRNA gene, and primer sets for distinguishing bacteria, archaea, fungi, etc. such as 18S rRNA and ITS. Bacteria, archaea, and fungi can be detected at the same time by using a plurality of types of the primer sets at the same time.
  • system Contains a droplet encapsulation unit that encloses cells one by one in a droplet, a gel capsule generation unit that gels the droplets to generate gel capsules, and one or more lysis reagents for lysing cells.
  • a cell lysate that lyses the cells by immersing the gel capsule in one or more lysis reagents, and the cell lysate is a polynucleotide containing genomic DNA of the cells or a portion thereof.
  • the cell lysate and the polynucleotide which are configured to be retained in the gel capsule in a state where the substance that is eluted in the gel capsule and binds to the genomic DNA or a portion thereof is removed, are contained in the gel capsule.
  • a gel capsule containing a single-cell genome-derived polynucleotide that has been amplified and retained is prepared by a system for analyzing the composition of the microflora containing the polynucleotide amplification reagent for amplification.
  • the gel capsule prepared in this way it is possible to perform a complex analysis of the genetic information and the like of the host itself such as a human and the microflora of the human-derived sample.
  • the microbial species contained in the sample is identified, and the information on the intestinal microbiota and human cells are targeted.
  • the genetic information obtained from each gel capsule obtained by the above method and system is also analyzed.
  • the composition evaluation unit of the above system can evaluate the human intestinal microflora and the human cell to be evaluated, respectively, to amplify and sequence the nucleic acid, and obtain the genome sequence data of each cell. Can be obtained.
  • the system of the present invention can evaluate the effects of the intestinal bacteria and their biotransformers on the host and the functions of the intestinal bacteria themselves, and can find the correlation between them. it can.
  • Example 1 Analysis of mouse intestinal flora composition
  • DPBS buffered saline
  • microdroplets were prepared and mouse intestinal microbial cells were encapsulated in the microdroplets.
  • a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, in which adjacent channels are arranged at right angles, is used, but they are connected in a substantially T shape. It is also possible to use a microchannel.
  • the microchannel of this example used had a width of 34 ⁇ m and a height of 50 ⁇ m, but the size of the microchannel can be appropriately changed depending on the size of the microdroplets to be produced and the size of one cell to be encapsulated.
  • the intestinal microbial suspension was introduced from the first channel (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) from the second channel and the fourth channel (oil phase inlet). ) (Hereinafter referred to as "oil") is introduced to shear the intestinal microbial suspension to prepare fine droplets having a diameter of 50 ⁇ m, and the third flow path 7 is allowed to flow through a tube having a capacity of 0.2 mL. Collected in. Approximately 450,000 microdroplets were produced at a rate of 500 droplets / second. The cell concentration in the microdroplets is 0.1 cells / droplet.
  • the diameter of the microdroplets is made uniform to 50 ⁇ m, so that each cell can be easily encapsulated in the microdroplets.
  • the diameter of the microdroplets is, for example, 1 to 250 ⁇ m, preferably 20 to 200 ⁇ m.
  • the diameter of the droplet may be from about 1 to 250 ⁇ m, more preferably from about 10 to 200 ⁇ m, for example, the diameter of the droplet is about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about. It may be 30 ⁇ m, about 40 ⁇ m, about 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • microdroplets and oil are stored in the tube, but the microdroplets have a lighter specific gravity than oil, so they accumulate in the upper layer.
  • the tube was cooled on ice for 15 minutes, and microdroplets were gelled with ultra-low melting point agarose.
  • the gelled microdroplets are gel capsules. Since the diameter of the microdroplets is 50 ⁇ m, the diameter of the gel capsule is also 50 ⁇ m.
  • the diameter of the gel capsule may be about 1-250 ⁇ m, more preferably about 10-200 ⁇ m, eg, about 1 ⁇ m, about 5 ⁇ m, about 10 ⁇ m, about 15 ⁇ m, about 20 ⁇ m, about 25 ⁇ m, about 30 ⁇ m, about 40 ⁇ m, about. It may be 50 ⁇ m, about 80 ⁇ m, about 100 ⁇ m, about 150 ⁇ m, about 200 ⁇ m, or about 250 ⁇ m.
  • the diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation.
  • the diameter of the gel capsule is preferably 1 to 250 ⁇ m.
  • the gel capsule was sequentially immersed in a lytic reagent as a lysis reagent, and a part other than the object to be collected such as a cell wall of a cell was dissolved inside the gel capsule, and genomic DNA was eluted into the gel capsule.
  • lysozyme (10 U / ⁇ L) (R1804M, Epicenter), which is one of the lytic reagents, was added to the tube to lyse the cells.
  • achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemical Industries, Ltd.), which is one of the lytic reagents, was added to the tube.
  • protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), which are one of the lytic reagents, to the tube to add cells.
  • the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA.
  • Buffer D2 QIAGEN
  • the lytic test solution used in this example was lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2.
  • Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it was used as one of the lytic reagents in this example. Since the gel capsule is immersed in the lytic reagent for a short time, the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule. In this example, the lytic reagent permeated into the gel capsule is also included in the contaminants.
  • lysozyme, achromopeptidase, and protease K were added in sequence, sodium dodecyl sulfate was added to lyse the cells, and then centrifugation was performed only before adding Buffer D2. Thereby, a sufficient cleaning effect can be obtained.
  • centrifugation may be performed after lysing the cells with each lytic reagent.
  • the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.
  • the amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent.
  • the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase was used.
  • the whole genome amplification reaction reagent was immersed in REPLI-g Single Cell Kit (QIAGEN), and the whole genome amplification reaction was carried out for 3 hours (S1000 thermal cycler, Bio-Rad).
  • the amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).
  • a fluorescent DNA intercalator SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)
  • flow cytometer BD FACSMelody cell sorter, BD Biosciences
  • the MDA method (REPLI-g Single Cell Kit, 150345) is used in the wells of each plate.
  • QIAGEN QIAGEN was performed to prepare a library master plate containing 10 ⁇ L of DNA amplification product for each well.
  • Dispense 39 ⁇ L of nuclease-free water (UltraPure DNase / RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) into each well of the new plate, add 1 ⁇ L of DNA amplification product in the library master plate, and add 1 ⁇ L of DNA amplification product to the library master plate.
  • a 40-fold diluted solution was prepared.
  • the DNA concentration was quantified by a Qubit fluorometer (Q33226, Thermo Fisher Scientific) Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 ⁇ L of the diluted solution.
  • PCR was performed on the V3V4 region of the 16S rRNA gene using 1 ⁇ L of the diluted solution as a template (6.25 ⁇ L PrimeSTAR Max DNA Polymerase (R045B, Takara Bio), 0.5 ⁇ L 10 ⁇ M Primer Forward (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3).
  • PCR reaction conditions are 95 ° C, 5 minutes for initial thermal denaturation, 98 ° C, 10 seconds for thermal denaturation, 51 ° C, 15 seconds for annealing, and 72 ° C for elongation reaction.
  • the criteria were (1) the DNA yield after MDA amplification was 200 ng or more, and (2) amplification was confirmed after PCR of the 16S rRNA gene. Samples were selected that were set and the DNA yield was sufficient for subsequent analysis and met the criteria of bacterial origin. As a result of sample selection, 347 (74%) out of 470 samples were selected. Furthermore, homology search (default condition) using BLAST was performed on the sequence information obtained by sequence analysis of PCR products, and information on the type and number of bacteria contained in the library master plate was obtained.
  • Example 2 Analysis of human intestinal flora composition
  • Example collection from humans Human feces were collected in a stool collection container (FS-0007 or FS-0008, Technosurgarabo).
  • the preservation solution was removed, and the container was washed once with 1000 ⁇ L of DPBS.
  • Frozen stool in 1.5 ml tube was naturally thawed by placing it on ice for 30 minutes. Fresh stools that do not contain a preservative solution are not treated before crushing.
  • DPBS Phosphate Buffered Saline
  • microdroplets were prepared and human intestinal microbial cells were encapsulated in the microdroplets.
  • a microchannel consisting of a first channel, a second channel, a third channel, and a fourth channel, in which adjacent channels are arranged at right angles, is used, but they are connected in a substantially T shape. It is also possible to use a microchannel.
  • the microchannel of this example used had a width of 34 ⁇ m and a height of 50 ⁇ m, but the size of the microchannel can be appropriately changed depending on the size of the microdroplets to be produced and the size of one cell to be encapsulated.
  • the intestinal microbial suspension was introduced from the first channel (aqueous phase inlet), and Pico-Surf1 (2% in Novec7500) (Sphere Fluidics) from the second channel and the fourth channel (oil phase inlet). ) (Hereinafter referred to as "oil") is introduced to shear the intestinal microbial suspension to prepare fine droplets having a diameter of 50 ⁇ m, and the third flow path 7 is allowed to flow through a tube having a capacity of 0.2 mL. Collected in. Approximately 450,000 microdroplets were produced at a rate of 500 droplets / second. The cell concentration in the microdroplets is 0.1 cells / droplet.
  • the diameter of the microdroplets is made uniform to 50 ⁇ m, so that each cell can be easily encapsulated in the microdroplets.
  • the diameter of the microdroplets is, for example, 1 to 250 ⁇ m, preferably 10 to 200 ⁇ m.
  • microdroplets and oil are stored in the tube, but the microdroplets have a lighter specific gravity than oil, so they accumulate in the upper layer.
  • the tube was cooled on ice for 15 minutes, and microdroplets were gelled with ultra-low melting point agarose.
  • the gelled microdroplets are gel capsules. Since the diameter of the microdroplets is 50 ⁇ m, the diameter of the gel capsule is also 50 ⁇ m. The diameter of the gel capsule is preferably 1 to 250 ⁇ m. By making the diameter of the gel capsule uniform, the penetration rate of the lytic reagent described later into each gel capsule can be made more uniform.
  • the gel capsule was sequentially immersed in a lytic reagent as a lysis reagent, and a part other than the object to be collected such as a cell wall of a cell was dissolved inside the gel capsule, and genomic DNA was eluted into the gel capsule.
  • lysozyme (10 U / ⁇ L) (R1804M, Epicenter), which is one of the lytic reagents, was added to the tube to lyse the cells.
  • achromopeptidase (850 U / mL) (015-09951, Fujifilm Wako Pure Chemical Industries, Ltd.), which is one of the lytic reagents, was added to the tube.
  • protease K (1 mg / mL) (MC5005, Promega) and sodium dodecyl sulfate (SDS) 0.5% (71736-100ML, SIGMA-ALDRICH), which are one of the lytic reagents, to the tube to add cells.
  • the gel capsule was immersed in Buffer D2 (QIAGEN), which is an aqueous solution containing potassium hydroxide, which is one of the lytic reagents, to dissolve the residual components and denature the genomic DNA.
  • Buffer D2 QIAGEN
  • the lytic test solution used in this example was lysozyme, achromopeptidase, proteinase K, sodium dodecyl sulfate, and Buffer D2.
  • Potassium hydroxide is also used in a normal DNA amplification reaction step, but since it also has a lytic effect, it was used as one of the lytic reagents in this example. Since the gel capsule is immersed in the lytic reagent for a short time, the eluted genomic DNA is not discharged from the gel capsule by the lytic reagent and is retained in the gel capsule. In this example, the lytic reagent permeated into the gel capsule is also included in the contaminants.
  • sufficient cleaning effect can be obtained by sequentially adding lysozyme, achromopeptidase, and protease K, adding sodium dodecyl sulfate to lyse the cells, and then performing centrifugation only before adding Buffer D2. it can.
  • centrifugation may be performed after lysing the cells with each lytic reagent.
  • the target genomic DNA can be collected by lysing the cells with a plurality of types of lytic reagents, and the lytic reagent and the poly of the lysed cells can be collected by centrifugation after immersion in the lytic reagent. Contaminants such as components other than nucleotides can be removed, and genomic DNA can be purified without inhibiting the subsequent genomic DNA amplification reaction.
  • the amplification reagent was added to the tube containing the gel capsule holding the denatured genomic DNA in the potassium hydroxide solution (Buffer D2), and the gel capsule was immersed in the amplification reagent.
  • the MDA (Multiple Replication Replication) method using phi29 DNA polymerase, which is a strand-substitution type DNA synthase was used.
  • the whole genome amplification reaction reagent was immersed in REPLI-g Single Cell Kit (QIAGEN), and the whole genome amplification reaction was carried out for 3 hours (S1000 thermal cycler, Bio-Rad).
  • the amplification reagent (REPLI-g Single Cell Kit) contains a component that neutralizes the potassium hydroxide solution (Buffer D2).
  • a fluorescent DNA intercalator SYBR Green I nucleic acid gel stain 10,000 in DMSO, (S7563, Thermo Fisher Scientific)
  • flow cytometer BD FACSMelody cell sorter, BD Biosciences
  • the MDA method (REPLI-g Single Cell Kit, 150345) is used in the wells of each plate.
  • QIAGEN QIAGEN was performed to prepare a library master plate containing 10 ⁇ L of DNA amplification product for each well.
  • Dispense 39 ⁇ L of nuclease-free water (UltraPure DNase / RNase-Free Distilled Water, 10977-015, Thermo Fisher Scientific) into each well of the new plate, add 1 ⁇ L of DNA amplification product in the library master plate, and add 1 ⁇ L of DNA amplification product to the library master plate.
  • a 40-fold diluted solution was prepared.
  • the DNA concentration was quantified by a Qubit fluorometer (Q33226, Thermo Fisher Scientific) Qubit dsDNA HS Assay Kit (Q32854, Thermo Fisher Scientific) using 1 ⁇ L of the diluted solution.
  • PCR was performed on the V3V4 region of the 16S rRNA gene using 1 ⁇ L of the diluted solution as a template (6.25 ⁇ L PrimeSTAR Max DNA Polymerase (R045B, Takara Bio), 0.5 ⁇ L 10 ⁇ M Primer Forward (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3).
  • PCR reaction conditions are 95 ° C, 5 minutes for initial thermal denaturation, 98 ° C, 10 seconds for thermal denaturation, 51 ° C, 15 seconds for annealing, and 72 ° C for elongation reaction.
  • analyzing the digital sequence information of the bacterium it is possible to analyze the metabolic function of the bacterium, possess a drug resistance gene, analyze a gene mutation, compare with a known bacterium, and compare with another sample.
  • This process includes, for example, search engines such as blast, analysis websites such as Ensembl, assembly evaluation tools such as QUAST and CheckM, annotation tools such as Prokka, InterProScan, and DFAST, and metabolism / physiology such as MetaCyc and MAPLE. It can be carried out using a functional potential evaluation system.
  • 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
  • metabolism / physiology such as MetaCyc and MAPLE.
  • the genes in the genome are identified, and the conservativeness as a metabolic pathway is evaluated by MAPLE.
  • the conservativeness and identity of each gene will be evaluated, and comparative genome analysis with related species will be performed.
  • homology search of gene sequences derived from a single organism using BLAST or hmmer, or homology search for target genes, or (2) By mapping the sequence data derived from a single organism using BWA and bowtie2, the difference between the gene sequence derived from a single organism and the target gene sequence is detected, and the gene mutation is identified.
  • genomes derived from allogeneic microorganisms based on the single copy marker gene sequences detected by Average Nucleotide Identity, CheckM, and GTDB-tk. Create a genome set containing genetic information derived from a single organism that is presumed to be and genetic information of known microorganisms. Subsequently, differences between genomes such as the presence or absence of a specific gene, a locus, and a mutation in a common gene sequence are identified and extracted by a homology search tool such as BLAST or hmmer. By performing clustering based on characteristic intergenomic differences, classification of allogeneic microorganisms at the strain level or substrain level of homologous microorganisms is performed.
  • Example 3 When it is desired to selectively acquire data of cells having specific characteristics from various cells, A gel containing a polynucleotide when the purpose is to obtain genomic data of one or more specific microorganisms of interest to those skilled in the art among animal symbiotic microorganisms such as gut flora and marine / soil microorganisms. By confirming in advance the presence or absence of a gene fragment of the target microorganism in the amplified nucleic acid recovered from the capsule or gel capsule, it is possible to reduce unnecessary gene sequence data acquisition and the cost associated therewith.
  • measurement targets include comparative analysis of microorganisms of the same strain (for example, analysis of subspecies in a group of microorganisms related to diseases, etc.) and microorganisms having a specific gene (for example, secondary metabolites and enzymes produced by microorganisms). It is assumed that the purpose is to search for or analyze bacteria, archaea, fungi, and other eukaryotic cells individually from among various species. In particular, for the purpose of evaluating the intestinal environment, oral cavity, and skin environment of the host human, intestinal bacteria and oral bacteria responsible for specific metabolic functions can be specifically detected from genes, and variants of skin bacteria can be detected. It is expected to be detected.
  • Example 4 When a large amount of host DNA or the like is mixed, the analysis sample is feces, saliva, sputum or skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical cleaning solution, tissue extract or blood, and the microorganisms contained in the sample are analyzed. Contains cells, intracellular small organs, and nucleic acids from many host animals in the sample. Some of these can also be encapsulated inside the gel capsule to perform polynucleotide amplification.
  • polynucleotide derived from the host can be applied to the complex analysis of the microflora.
  • the intestinal flora and host animals present in the gastrointestinal tract have a symbiotic relationship in which the host provides an anaerobic environment for colonization of the gastrointestinal tract, while the intestinal flora affects the health of the host. It is known to have.
  • the major effects on the health status of the host include the production of nutrients, the defense against infectious diseases, and the development of the immune system.
  • inflammatory bowel disease is a disease caused by an abnormality of intestinal environmental factors such as intestinal bacteria in addition to a genetic predisposition.
  • the intestinal bacteria and the living body side compete for nutrients, while the intestinal bacteria also metabolize and decompose nutrients for the living body for more integrated analysis. It can be carried out.
  • the function of biotransformers derived from gut flora is analyzed in a complex manner by host-side genetic analysis, metabolome analysis, biochemical analysis, etc., and the gut flora Regarding the function, the function can be inferred from the genome sequence information of the bacterium, and the correlation with the produced metabolome can be seen.
  • Mutant diversity on the genome sequence can be evaluated on a single cell basis to track the genomic heterogeneity of each microorganism in the microbiota and the development and progression of mutant strains.
  • By selectively amplifying and detecting the target gene mutation site of the target cell for the gel capsule containing the amplified polynucleotide only the gene sequence data to be analyzed can be specifically acquired, and the cost for it can be reduced. can do. It can be used to detect genetically mutated microorganisms and plasmid infections.
  • Example 6 Evaluation of preservation of specific species
  • the disclosed technology is used to amplify the specific organism from a gel capsule containing an amplified polynucleotide. By detecting and selecting, the content rate can be shown.
  • the degree of coincidence with the standard at the genome level, preservation, and the like. It is expected to be used for quality assurance of microbial preparations.
  • Specific procedures include (1) homology search of single organism-derived gene sequences using BLAST and hmmer for target genes, or (2) single organism-derived sequence data using BWA and bowtie2. By mapping, the difference between the gene sequence derived from a single organism and the target gene sequence is detected, and the gene mutation is identified.
  • Example 7 Detection of specific species
  • Analysis samples are feces, saliva, sputum and skin, oral cavity, nasal cavity, ears, genital lavage fluid, surgical lavage fluid, tissue extract, blood, etc., and detect the presence of one or more specific microorganisms. It may determine drug response, drug resistance, and evaluate the metabolic capacity of foods.
  • a specific organism can be detected and selected from a gel capsule containing an amplified polynucleotide to indicate its content.
  • a homology search for a single organism-derived gene sequence is performed based on a gene that metabolizes dietary fiber inulin or a gene marker that identifies the microbial Bacteroides species.
  • the microorganisms and gene groups responsible for the degradation of the dietary inulin could be identified, and their functions could be estimated from the homology of known genes and the prediction of the three-dimensional structure based on the amino acid sequence.
  • Example 8 Evaluation of soil bacteria
  • various specific organisms inhabiting soil or seawater are detected and selected from gel capsules containing amplified polynucleotide using the technology disclosed in the present disclosure. It is possible to identify the area and indicate the abundance. Further, by analyzing the obtained genetic information in detail, it is possible to evaluate cultivars, livestock or aquaculture varieties suitable for the soil or seawater at the genome level. Furthermore, it is expected to be used for pesticide-free manufacturing methods using soil bacteria or marine bacteria.
  • DPBS phosphate buffered saline
  • the supernatant was filtered using a filter having a diameter of 5 ⁇ m (SMWP04700, Sigma-Aldrich) and then centrifuged at 10,000 ⁇ g for 5 minutes (75004263, Thermo Fisher Scientific) to remove the supernatant.
  • the pellets are resuspended in 10 mL PBS, dispensed in 1 mL each in a 1.5 mL tube (MCT-150-C, Axygen), and then centrifuged at 10,000 xg for 5 minutes to collect soil bacteria.
  • Bacteria Bacterial pellets were combined into a single 1.5 mL tube, washed twice by centrifugation with PBS, and then suspended in PBS to obtain a cell suspension of soil bacteria.
  • the cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%.
  • -10G, Sigma-Aldrich was added to prepare a soil bacterial suspension used for gel capsule preparation (cell final concentration: 1.5 ⁇ 10 3 cells / ⁇ L). Subsequently, the analysis was advanced and evaluated in the same procedure as the method described in Example 1 or 2.
  • Example 9 Analysis of aquatic microbial composition
  • 4 L of seawater or fresh water was collected in a sterilized plastic tank, filtered using a 5 ⁇ m diameter filter (SMWP04700, Sigma-Aldrich), and then filtered through a 0.22 ⁇ m diameter filter (GSWP04700, Sigma-Aldrich). By doing so, the bacterial fraction was trapped on the filter.
  • DPBS Phosphate Buffered Saline
  • Bacteria derived from seawater or freshwater are collected by dispensing 1 mL of a 10 mL bacterial suspension into a 1.5 mL tube (MCT-150-C, Axygen) and centrifuging at 10,000 xg for 5 minutes. Bacteria.
  • Bacterial cell suspensions derived from seawater or freshwater are obtained by collecting the bacterial cell pellets in a single 1.5 mL tube, washing them by centrifugation twice with PBS, and then suspending them in PBS. did. The cell concentration in the prepared cell suspension was measured (microscope: CKX41, OLYMPUS, bacterial calculator A161,2-5679-01, AS ONE), and ultra-low melting point agarose (A5030) was adjusted to a final concentration of 1.5%.
  • This disclosure is available in fields such as biological research, medicine, environment, and healthcare.
  • SEQ ID NO: 1 Forward primer
  • SEQ ID NO: 2 Reverse primer

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  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'évaluation d'un nombre absolu d'une composition de flore microbienne de manière peu coûteuse, simple et/ou très précise. Le procédé d'analyse d'une composition de flore microbienne comprend une étape d'évaluation de la composition de flore microbienne à partir d'un échantillon comprenant des acides nucléiques amplifiés dérivés de cellules respectives présentes dans la flore microbienne. Selon ce procédé, dans une exécution avant le séquençage du génome entier, le nombre absolu de la flore microbienne peut être évalué de manière peu coûteuse et simple, et après séquençage du génome entier, il devient possible de le comparer avec une séquence de gènes longs par la réduction, à partir d'informations de séquence numérique, des informations à analyser, ce qui permet d'augmenter le degré de précision.
PCT/JP2020/017792 2019-04-26 2020-04-24 Analyse numérique de la flore microbienne WO2020218553A1 (fr)

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US20160068899A1 (en) * 2013-03-14 2016-03-10 The Broad Institute Inc. Methods for quantitating dna using digital multiple displacment amplification
US20180371525A1 (en) * 2017-06-20 2018-12-27 Bio-Rad Laboratories, Inc. Mda using bead oligonucleotide
WO2019216271A1 (fr) * 2018-05-07 2019-11-14 ｂｉｔＢｉｏｍｅ株式会社 Procédé de réalisation d'analyse de cellules uniques et dispositif associé

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