WO2022131150A1 - Genome analysis method - Google Patents

Genome analysis method Download PDF

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WO2022131150A1
WO2022131150A1 PCT/JP2021/045487 JP2021045487W WO2022131150A1 WO 2022131150 A1 WO2022131150 A1 WO 2022131150A1 JP 2021045487 W JP2021045487 W JP 2021045487W WO 2022131150 A1 WO2022131150 A1 WO 2022131150A1
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base sequence
sample
genome analysis
cell
analysis method
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PCT/JP2021/045487
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French (fr)
Japanese (ja)
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章玄 岡本
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国立研究開発法人物質・材料研究機構
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Priority to JP2022569943A priority Critical patent/JP7473251B2/en
Publication of WO2022131150A1 publication Critical patent/WO2022131150A1/en

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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a genome analysis method.
  • Patent Document 1 describes "a step of using a sample containing two or more cells or cell-like structures, and encapsulating the cells or cell-like structures in the droplets one cell or structure unit at a time, and the droplets.
  • a first sample containing the cells by separating the cells and the membrane vesicles from a suspension containing the cells and the membrane vesicles produced by the cells and released outside the cells.
  • To prepare a second sample containing the membrane vesicles to determine the first base sequence, which is a base sequence derived from the cells contained in the first sample, and to use the second sample.
  • a genome analysis method comprising determining a second base sequence, which is a base sequence derived from the included membrane vesicles, and complementing the first base sequence using the second base sequence. .. [2] The genome analysis method according to [1], wherein the determination of the first base sequence is performed by single cell analysis of the first sample.
  • the polynucleotide is brought into contact with an amplification reagent to amplify the polynucleotide in the gel capsule, and the second base sequence, which is the base sequence of the polynucleotide derived from the membrane vesicle from the amplified polynucleotide.
  • the genomic analysis method according to any one of [1] to [3], which comprises. [5] The genome analysis method according to [4], wherein determining the second base sequence further comprises treating the second sample with a nucleolytic enzyme. [6] The genome analysis method according to [5], wherein the determination of the second base sequence is performed using each of the second sample that has undergone the treatment and the second sample that has not undergone the treatment. ..
  • the genome analysis method according to any one of [1] to [6], wherein the complement is to use the second base sequence as a fragment of the first base sequence.
  • the suspension is at least one selected from the group consisting of feces, saliva, sputum, surgical lavage fluid, blood, and a wiping solution for skin or body mucous membranes collected from humans or animals, and a swab.
  • [10] The method for genome analysis according to any one of [1] to [9], wherein the cell is a bacterium.
  • the above-mentioned bacteria are Porphyromonas, Prevotella, Beyonera, Fusobacterium, Parvimonas, Aggregatibacter, Actinobacillus, Actinobacillus, Bacteroides, Tannerela, Treponema, Capnocytophaga.
  • the genome analysis method according to [10] which is at least one bacterium selected from the group consisting of the genus Eikenella and the genus Capnocytophaga.
  • the present invention will be described in detail.
  • the description of the constituent elements described below may be based on a representative embodiment of the present invention, but the present invention is not limited to such embodiments.
  • the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.
  • polynucleotide refers to the polymer form of a nucleotide of any length, either a ribonucleotide or a deoxyribonucleotide. This term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, and RNA.
  • single cell analysis is synonymous with “single cell analysis”, and one cell is separated from a sample containing a plurality of types and / or a plurality of cells. It means a method for genome analysis.
  • Single cell analysis is, for example, a method in which one cell is separated from each of a large number of microwells arranged on a plane, and genome analysis is performed on each of the cells.
  • the apparatus described in Patent Document 1 the apparatus described in International Publication No. 2017/094101, the apparatus described in International Publication No. 2016/038670, and the like are known. And / or those using the method of.
  • single particle analysis means a method of separating cell-derived particles (excluding the cells themselves) and performing genome analysis for each particle.
  • the same method as the above-mentioned “single cell analysis” can be used.
  • membrane vesicles include “exosomes” produced and released by the endocytic pathway by outer membrane vesicles produced by bacteria and the like, animal cells and the like, and “absorption” caused by apoptosis.
  • Small bodies are also included, and among them, outer membrane vesicles (OMV) are preferable as the membrane vesicles.
  • Membrane vesicles produced by bacteria are produced so that part of the bacterial cell membrane is constricted outside the cells.
  • the microvesicle “outer membrane vesicle (OMV)” is mentioned, and its size is not particularly limited, but is often 10 to 1000 nm, and 10 to 300 nm for Gram-negative bacteria. Often, it is 50-150 nm for Gram-positive bacteria.
  • the genome analysis method of the present invention uses a cell and a suspension containing membrane vesicles produced in the cell and released outside the cell, and the "cell" is a multicellular organism. It includes both those of single-celled organisms and those of single-celled organisms.
  • the case where the cell is a bacterium will be described as an example. The following method can be similarly applied to animal cells and the like.
  • FIG. 1 is a flowchart of a genome analysis method (hereinafter, also referred to as “the present method”) according to an embodiment of the present invention.
  • the bacterium and the membrane vesicle are separated from the suspension containing the bacterium and the membrane vesicle produced by the bacterium and released outside the bacterium, and the first sample containing the bacterium is used.
  • sample preparation step, step S101 To prepare a second sample containing the above-mentioned membrane vesicles (sample preparation step, step S101).
  • first base sequence determination step S102 Determining the first base sequence, which is a base sequence derived from the above-mentioned bacteria contained in the first sample (first base sequence determination step, step S102), Determining the second base sequence, which is the base sequence derived from the membrane vesicle contained in the second sample (second base sequence determination step, S103), It is a genome analysis method including complementing the first base sequence using the second base sequence (complementation step, S104).
  • Step S101 is a step of separating the bacterium and the membrane vesicle from the suspension containing the bacterium and the membrane vesicle, and preparing a first sample containing the bacterium and a second sample containing the membrane vesicle. be.
  • the suspension used in this step contains bacteria and membrane vesicles. That is, the suspension contains membrane vesicles and the bacteria that produced the membrane vesicles.
  • This bacterium may be a single cultivated bacterial strain or a plurality of species (one or more of them may be a refractory bacterium).
  • the suspension may be, for example, a sample collected from humans or animals, such as feces, saliva, sputum, surgical cleaning solution, blood, skin / body mucosa wiping solution, and swab.
  • the collected sample may contain a group of bacteria and membrane vesicles produced by the bacteria contained therein. Bacterial groups and membrane vesicles may be separated from such collected samples and used as a first sample and a second sample, respectively.
  • Bacteria contained in the suspension are not particularly limited, and examples thereof include eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, racen, gram-negative bacteria, gram-positive bacteria, paleobacteria, and fungi.
  • the bacteria are preferably periodontal disease bacteria (the causative bacteria of periodontal disease).
  • periodontal disease bacteria include Capnocytophaga, Prevotella, Eikenella, Fusobacterium, Parvimonas, Aggregatibacter, Aggregatibacter, and Aggregatibacter.
  • the genus (Capnocytophaga) and the like can be mentioned.
  • Porphyromonas gingivalis or Bacteriorides gingivalis Prevotella intermedia, Prevotella intermedia, Eikenella parvula valula, vulura parbula, vayl ⁇ Porphyromonas micra, Aggregatibacter actinomycetemcomitans or Actinovacils actinomycetemcomitens Sensis (Tannella forsythesis), Treponema denticola, Campylobacter rectus, Eikenella corodence (Eikenella corrodens, etc.), and Capnocera corrodens.
  • the bacteria are called "Red Complex", Porphyromonas gingivalis, Treponema denticola, and Aggregatibacter actinomycetemcommitans (Aggregatibacter actinometry). At least one bacterium selected from the group is preferred.
  • the method for separating the bacteria and the membrane vesicles in the suspension is not particularly limited, and a known method can be used.
  • a known method can be used.
  • cells are removed from the culture medium by centrifugation, and this is used as the first sample, and the supernatant is used. May be used as the second sample.
  • the colonies can be suspended in buffer and separated in the same manner.
  • the centrifugal supernatant is filtered using a membrane filter (for example, pore size 0.1 to 0.9 ⁇ m), the cells remaining in the centrifugal supernatant can be easily separated. Since the centrifugation supernatant may contain contaminants such as proteins in addition to cells, for example, after concentrating with a 100-200 kDa cutoff filter, membrane vesicles are precipitated by ultracentrifugation and collected. You can also use the method of
  • the precipitate thus obtained may contain bacterial-derived structures (eg, sucrose, flagella, etc.), and in order to remove these, for example, A method of further purification may be used by using a density gradient centrifugation method using sucrose or the like.
  • bacterial-derived structures eg, sucrose, flagella, etc.
  • a method for preparing a sample including the above treatment will be described more specifically.
  • the culture is placed in a centrifuge tube with a volume of 1-100 mL (eg 50 mL) and 3000-9000 rpm (eg 7000 rpm) 1-20 ° C (eg 4 ° C). Then, centrifuge for 1 to 30 minutes (for example, 10 minutes).
  • the precipitate contains bacterial cells, so the cells are separated (as the first sample), and the supernatant is 0.1 to 0.9 ⁇ m (for example, 0.22 ⁇ m). ), Placed in another centrifuge tube, and stored at 1-10 ° C (eg, 4 ° C).
  • the supernatant is added to an ultracentrifugal tube, for example, with a centrifugal force of 150,000 to 300,000 ⁇ g (for example, 210,000 ⁇ g) and 1 to 10 ° C. (for example, 4 ° C.) from 1 to 1.
  • Ultracentrifuge for 3 hours (eg 2 hours). After ultracentrifugation, discard the supernatant and resuspend the precipitated adventitial vesicles (OMV) in buffer (eg, pH 7.4 PBS buffer) and store at 1-10 ° C (eg, 4 ° C). .. Further, this ultracentrifugation step may be repeated a plurality of times.
  • the obtained liquid may be diluted in order to adjust the number (concentration) of the membrane vesicles contained in the unit amount of the second sample. By doing so, it becomes easy to separate and acquire the membrane vesicles of each individual in the single particle analysis described later.
  • the method of dilution is not particularly limited, and examples thereof include a method of diluting the concentration of membrane vesicles to an appropriate concentration while observing the concentration of membrane vesicles in the liquid by a dynamic light scattering method or the like.
  • a method of observing the concentration of membrane vesicles the particles are irradiated with a laser, the Brownian motion of each particle is tracked from the scattered light (tracking method), and the diameter of the particles is determined from the diffusion rate based on the Stokes-Einstein equation. And the number are preferred.
  • the first sample contains one or more kinds of bacteria, and this step is a step of determining a first base sequence which is a genomic sequence derived from these (or others).
  • the method for determining the first base sequence include 16S ribosomal RNA analysis, metagenomic analysis, single cell analysis, and the like, and known techniques can be applied without particular limitation. Among them, single cell analysis is preferable because it is easy to obtain a more accurate genomic sequence of each bacterium.
  • the method is not particularly limited, but single cell analysis including each of the following steps is preferable.
  • FIG. 2 is a flow of single cell analysis including each of the above steps.
  • the "first nucleotide sequence determination step" represented by step S102 in FIG. 1 preferably includes the single cell analysis of FIG.
  • Step S201 is a step of encapsulating each individual bacterium in the droplet.
  • the first sample used in the encapsulation step contains bacteria.
  • the bacterium contained in the sample may be one kind or two or more kinds. At least one of the bacteria in the first sample produced membrane vesicles contained in the suspension and released them out of the cells.
  • the method of encapsulating each individual bacterium contained in the first sample in the droplet using the first sample is not particularly limited, and for example, a known method described in Patent Document 1 or the like can be used.
  • the droplets can be produced, for example, using a microchannel.
  • a microchannel By flowing the suspension described above into a microchannel and shearing the flow of the suspension, droplets containing individual bacteria can be produced. Shearing can be done at regular intervals.
  • oil may be used as a method of shearing the suspension. Examples of the oil include mineral oil, vegetable oil, silicone oil, fluorinated oil and the like.
  • the diameter of the droplet is preferably 1 to 250 ⁇ m, more preferably 10 to 200 ⁇ m.
  • FIG. 3 is a schematic cross-sectional view of a microchannel that can be used in this step.
  • the microchannel 301 is a device capable of producing a droplet and enclosing each individual bacterium in the droplet.
  • the micro flow path 301 is composed of a cross-shaped flow path formed by two flow paths that are substantially orthogonal to each other.
  • the first sample 302 containing the bacterium 303 flows from the top to the bottom of the drawing in the flow path extending from the top to the bottom in the drawing, and the flow path in the direction substantially orthogonal to this flows from the left and right to the center of the drawing. Oil 304 is flowing toward it.
  • the diameter of this flow path is not particularly limited and may be appropriately selected depending on the intended purpose, but is generally preferably 10 to 60 ⁇ m.
  • the first sample 302 containing the bacterium 303 is sheared by the oil 304 flowing from the left and right to the center of the drawing, and becomes a droplet 305 containing the bacterium 303.
  • Step S202 is a step of gelling the droplet to form a gel capsule.
  • the gelation method is not particularly limited.
  • the 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 the first sample.
  • the diameter of the gel capsule is preferably 1 to 250 ⁇ m, more preferably 10 to 200 ⁇ 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 gelation temperature is not particularly limited, but is preferably 4 to 10 ° C.
  • the material of the gel capsule may contain agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
  • a photocurable resin for example, PEG-DA
  • the gel capsule may be a hydrogel capsule.
  • hydrogel is meant that the solvent or dispersion medium retained by the network structure of the polymeric or colloidal particles is water.
  • Step S203 is a step of contacting the gel capsule with a lysing reagent to lyse the bacteria, elute the polynucleotide, and retain it in the gel capsule.
  • a lysing reagent to lyse the bacteria, elute the polynucleotide, and retain it in the gel capsule.
  • the polynucleotide in the bacterium can be eluted into the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed.
  • the dissolving reagent enzymes, surfactants, other denaturing agents, reducing agents, pH adjusting agents, combinations thereof and the like can be used.
  • Dissolving reagents include lysoteam, 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, dithiotreitol, "TCEP-HCl", sodium dodecyl, sodium deoxycholate, "Triton X-100", “Triton X-114", “NP-40”, “Brij-35” , “Brij-58”, “Tween 20", “Tween 80", octyl glucoside, octyl thioglucoside, "CHAPS", “CHASPO", dodecyl- ⁇ -D-malto
  • the method of contacting the gel capsule with the dissolving reagent is not particularly limited, and for example, the dissolving reagent is discharged to the gel capsule held in the container (for example, the wells of the microtube and the microplate) with an automatic microsyringer or the like. do it.
  • the automatic microsyringe may typically consist of a flow path through which the dissolving reagent flows, a pump, a valve, or the like for liquid flow.
  • Step S204 is a step of removing contaminants in the gel capsule.
  • the purified genetic substance for example, DNA
  • the possibility of contamination of molecules from the outside is eliminated. can do.
  • the step of centrifuging the test tube containing the gelled droplets, removing the supernatant and replacing it with a cleaning solution can be performed.
  • it can be performed by filtering the gelled droplets with a filter, removing the supernatant, passing the washing liquid through the filter, and finally collecting the gel capsules.
  • the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations such as amplification reactions can be smoothly performed.
  • the reaction such as DNA amplification is not easily inhibited.
  • the gel capsules hold the genetic material to be analyzed or amplified, so that the lysing reagent can be removed even in the analysis per cell with a small amount of genetic material. It is possible to use a strong lysing reagent or a combination of lysing reagents. And, using a strong lysing reagent or a combination of lysing reagents may enable more reliable amplification of nucleic acid and analysis of base sequence.
  • Step S205 is a step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule. After immersing in the amplification reagent, the temperature of the gel capsule may be adjusted if necessary.
  • the heating is preferably 60 ° C. or lower from the viewpoint that the gel (for example, agarose gel) is difficult to dissolve. Heating within this range is preferable in that it further promotes the amplification of DNA.
  • the enzyme used for amplification include fi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase. In this method, it is preferable to use a random primer in order to amplify the total DNA in the gel.
  • Step S206 is a step of determining the first base sequence from the amplified polynucleotide.
  • a method for determining the base sequence from the amplified polynucleotide commercially available reagent kits, devices, application software and the like can be used without particular limitation for any of library preparation, sequence, assembly and the like.
  • step S103 ⁇ Second base sequence determination step (step S103)> Returning to FIG. 1, the second base sequence determination step (step S103) will be described. This step is a step of determining the second base sequence using the second sample prepared in step S101.
  • the sample used in this step may be the second sample prepared in the sample preparation step (step S101), but may be the treated second sample obtained by pretreating the above sample.
  • the method for determining the second base sequence is not particularly limited, and may be metagenomic analysis or single particle analysis, but single particle analysis is preferable from the viewpoint that the amount of membrane vesicles required is smaller.
  • the method for single particle analysis is not particularly limited, and the same method as described above can be used as a method for single cell analysis of the first sample in the first base sequence determination step of step S102.
  • nucleic acid components are often attached to the surface of membrane vesicles. It is presumed that this nucleic acid component has various origins such as a fragment in which a bacterial cell has ruptured and a fragment derived from a coexisting bacterium.
  • the nucleic acid component attached to the surface of the membrane vesicle is likely to be derived from the bacterium that produced the membrane vesicle (hereinafter, also referred to as “host”).
  • the nucleic acid components contained in the sample are comprehensively analyzed.
  • a specific membrane small size is used among the nucleic acid components to be comprehensively analyzed. The probability of obtaining a sequence derived from the vesicle-producing bacterium is very low.
  • the nucleic acid component attached to the surface of the membrane vesicle is, for example, a component attached in the process of producing the membrane vesicle, and is highly likely to be derived from the host. That is, it is unlikely that a fragment (polynucleotide) derived from a host of a specific membrane vesicle can be obtained from a fragment obtained by comprehensively analyzing the first sample, but the nucleic acid component contained in the second sample is the host. It can be said that it is more likely that it is a fragment derived from.
  • Single cell analysis of such a second sample yields a second base sequence containing both a sequence derived from membrane vesicles and a sequence likely to be derived from the host. With this sequence, it is likely that the first base sequence can be complemented with a longer base length than when only the membrane vesicle sequence is used.
  • the method for single particle analysis of the second sample is not particularly limited, but the same method as the single cell analysis described with reference to FIG. 2, that is, the following steps can be performed in that a higher quality second base sequence can be obtained.
  • Single particle analysis including this order is preferable. -Encapsulating each individual membrane vesicle in the droplet using the second sample-Gelizing the droplet to form a gel capsule-Making the gel capsule in contact with a lysis reagent to contact the membrane vesicle Dissolve and elute the polynucleotide and retain it in the gel capsule ⁇ Remove contaminants in the gel capsule ⁇ Contact the polynucleotide with the amplification reagent to amplify it in the gel capsule ⁇ Amplified poly Determining the second base sequence from the nucleotides
  • Step S104 is a step of complementing the first base sequence by using the second base sequence. Complementation typically includes using the second base sequence as a fragment to improve the coverage of the first base sequence.
  • the second base sequence includes a base sequence derived from the membrane vesicle and a base sequence derived from the nucleic acid component attached to the surface of the membrane vesicle.
  • the second base sequence is considered to be a part of the first base sequence. That is, it is considered that the membrane vesicles having the second base sequence were produced by the bacteria having the first base sequence.
  • the step includes a step of amplifying the polynucleotide in a gel capsule. Therefore, nucleotides other than the polynucleotide derived from the bacterium that should be originally amplified may be amplified due to the binding of the primers to be used or the generation of a chimeric sequence. In addition, amplification may be insufficient for some reason, and as a result, the coverage by the obtained polynucleotide may not be sufficiently high (for example, about 40 to 80%). In such cases, this analysis method is more useful.
  • the whole genome is divided into fragments (reads) having a predetermined base length, each fragment is sequenced, and they are combined (assembly, binning). ..
  • the method for performing metagenomic analysis is not particularly limited, and a known method can be used. For example, an analysis method based on the whole genome shotgun method described in JP-A-2005-218421 can be used, and the method is known to those skilled in the art.
  • “Complementation” in this genome analysis method includes using a second base sequence as one of the above fragments (specific fragment).
  • the second base sequence contains a base sequence obtained from a membrane vesicle and is a part of the genomic sequence of the bacterium that produced the membrane vesicle.
  • the specific fragment is obtained by separating individual membrane vesicles and analyzing the base sequence, and thus is typically a metagenome. It has a longer base length and a more accurate sequence than the fragments used in the analysis.
  • using the specific fragment as a fragment of the whole genome means, for example, using the second base sequence as one of the fragments in the metagenomic analysis by the whole genome shotgun method. Since the specific fragment is a sequence derived from a single membrane vesicle generated and is a more accurate sequence, the obtained whole genome sequence tends to be of higher quality. In addition, the specific fragment typically has a longer base length than the other fragments, and thus often has a higher sequence coverage.
  • the method of using the specific fragment as a fragment of the whole genome may be a method other than the above.
  • the specific fragment may be mapped to the entire genome sequence.
  • “mapping" to the whole genome sequence means that the specific fragment coincides with a part of the region of the whole genome sequence.
  • Matching means that 90% or more of the sequences are identical, preferably 95% or more, more preferably 99% or more, and even more preferably completely identical.
  • mapping a specific fragment to the whole genome sequence the quality of the whole genome sequence can be checked. That is, using a specific fragment as a fragment of the whole genome may provide information for determining that the whole genome sequence of the region is accurate when mapped to the whole genome sequence.
  • the genome analysis method is a genome analysis method including the following steps. -A first sample containing bacteria and a second sample containing membrane vesicles are prepared from a suspension containing bacteria and membrane vesicles (step S401, sample preparation step). -The first base sequence is determined using the first sample (step S402, first base sequence determination step). -The second sample is treated with a nucleolytic enzyme to prepare a treated second sample (step S403, treatment step). -The second base sequence is determined using the treated second sample (step S404, second base sequence determination step). -The first base sequence is complemented using the second base sequence (step S405, complementing step).
  • FIG. 4 is a flow of the genome analysis method according to this embodiment.
  • This genome analysis method is the same as the genome analysis method shown in FIG. 1, except that it has a processing step. That is, step S401 is the same as step S101, step S402 is the same as step S102, step S404 is the same as step S103, step S405 is the same as step S104, and the public form is also The same is true. The description of these "similar parts" will be omitted, and the parts different from the genome analysis method shown in FIG. 1 will be mainly described.
  • This genome analysis method includes a step (treatment step, step S403) of treating the second sample with a nucleolytic enzyme to prepare a treated second sample.
  • the membrane vesicles contained in the second sample have a nucleic acid component that is likely to be derived from the host attached to the membrane vesicles.
  • the nucleic acid component is decomposed and removed. In the genome analysis method according to the present embodiment, it can be ensured that the second base sequence is a sequence derived from a membrane vesicle, so that the complementation of the first base sequence becomes easier.
  • the nucleic acid-degrading enzyme used in the treatment step is not particularly limited, and deoxyribonuclease and the like can be used. Specific examples thereof include a method in which 10 ⁇ L of the second sample is diluted 10-fold and 2 ⁇ L of DNase (2000U) is added thereto. This gives a treated second sample.
  • Step S404 is a step of determining the second base sequence using the treated second sample.
  • the single particle analysis described above is preferable in that the amount of the sample used is smaller.
  • this step preferably further comprises determining the second base sequence by single particle analysis using the untreated second sample.
  • the base sequence of each membrane vesicle is obtained from the treated second sample, and from the untreated second sample, in addition to the base sequence of each membrane vesicle, the surface of the membrane vesicle is obtained.
  • the base sequence of the attached nucleic acid component can be obtained.
  • the difference between the base sequence obtained from the second sample and the base sequence of the treated second sample includes the base sequence derived from the nucleic acid component attached to the surface of the membrane vesicle. ..
  • the above “difference” is likely to be derived from the host. By having this step, there is a possibility that the coverage range of the first base sequence by the obtained second base sequence becomes wider.
  • the "difference" it may be possible to determine whether the obtained sequence is derived from membrane vesicles or nucleic acid components attached to the surface, enabling more efficient genome analysis. become.
  • the method of using single cell analysis as the analysis method of the second sample is mainly described, but the genome analysis method according to the embodiment of the present invention is not limited to the above, and the second method may be used by another method.
  • the base sequence of the sample may be analyzed.
  • metagenomic analysis can be used as such a method.
  • MVs membrane vesicle samples
  • Bacteria bacterial cell samples
  • the specific procedure is as follows. First, saliva was centrifuged to roughly separate the bacterial cells and the membrane vesicles. Next, the separated centrifugal supernatant was filtered using a membrane filter, and the filtered centrifugal supernatant and the bacterial cells were separated. Since the centrifugation supernatant after filtration may contain contaminants such as proteins in addition to bacteria, concentrate with a 100-200 kDa cut-off filter and then precipitate membrane vesicles by ultracentrifugation. Collected. Since the precipitate thus obtained may contain structures such as bacterial groups (for example, flagella and flagella), sucrose is used to remove them. Further purification was performed using the density gradient centrifugation method used.
  • nucleic acid components not derived from the membrane vesicles may be attached to the surface of the membrane vesicles thus obtained, deoxyribonuclease is further acted on the separated and obtained membrane vesicles to these. Nucleic acid components were degraded.
  • the DNA sequences of the obtained membrane vesicle samples and bacterial cell samples were profiled for 192 particles of both samples by single particle analysis.
  • the fastq file obtained for each particle is assembled by software such as "SPADES", and the protein coding region is divided into the obtained assembly using "prokka” which is a software tool for genome annotation.
  • SPADES software
  • prokka a software tool for genome annotation.
  • a homology search was performed using the base sequence of the genome recorded in a public database or the like (for example, NCBI RefSeq) as a reference, and the mapped sequence region (as one form, the highest degree of agreement) was predicted and detected. It was determined that the cell having the most protein in the genome is the host cell of the particle.
  • FIG. 5 shows the percentage of particles in which Alphaproteobacteria bacterium 41-28 (Alphaproteobacteria 41-28) was determined to be a host cell in each sample, that is, particles determined to be derived from Alphaproteobacteria bacterium 41-28 with respect to 192 total particles. It is a figure which shows the ratio (Detected percentage). Further, FIG. 6 is a diagram showing the proportion of particles determined to be derived from TM7x in each sample, which was calculated in the same manner as above.
  • FIG. 7 maps the sequences of particles (52 particles) determined to be derived from Alphaproteobacteria 41-28 among the particles (MV) obtained from the membrane vesicle sample (Healthy) to the genomic DNA of Alphaproteobacteria 41-28. It is a figure which shows the result of this.
  • the horizontal axis represents the region of the genomic DNA of Alphaproteobacteria 41-28, and the region detected from each of the 52 particles is shown in black.
  • the results of 52 particles are shown side by side along the vertical axis.
  • mapping the genome sequence of the cell is determined by mapping the base sequence obtained from each particle to the genome of the cell determined to be the host cell using mapping software such as bowtie2 and BWA. We evaluated how much area was covered.
  • FIG. 8 is a diagram showing the results of mapping the sequences of the particles (86 particles) determined to be derived from TM7x among the particles (MV) obtained from the membrane vesicle sample (Patient) to the genomic DNA of TM7x. ..
  • the horizontal axis represents the region of the genomic DNA of TM7x, and the region detected from each of the 86 particles is shown in black.
  • the results of 86 particles are shown side by side along the vertical axis.
  • FIG. 9 is a diagram showing the ratio of genomic DNA length of each microorganism constructed by the sequence obtained from the membrane vesicle sample.
  • the horizontal axis shows the ratio of the region (cover region) detected by the DNA derived from the membrane vesicle to the entire genome sequence.
  • this genome analysis method by using the base sequence of a membrane vesicle for whole genome analysis including the cell from which it is produced, an accurate whole genome sequence can be obtained more quickly than the conventional method. Can be done.
  • This genome analysis method can grasp the whole picture of a complicated bacterial flora more accurately and quickly, and is useful for comprehensive data acquisition of the bacterial flora in the medical, environmental, and food industries. ..
  • Microchannel 302 First sample 303: Bacteria 304: Oil 305, 306: Droplets

Abstract

The present invention provides a genome analysis method with which it is possible to obtain a high-quality whole-genome analysis result in a simpler manner. This genome analysis method includes: separating, from a suspension that contains cells and membrane vesicles released outside the cells, the cells and the membrane vesicles from each other, and preparing a first sample that contains the cells and a second sample that contains the membrane vesicles; determining a first base sequence derived from the cells contained in the first sample; determining a second base sequence derived from the membrane vesicles contained in the second sample; and supplementing the first base sequence using the second base sequence.

Description

ゲノム解析方法Genome analysis method
　本発明は、ゲノム解析方法に関する。 The present invention relates to a genome analysis method.
　単一細胞解析を行う方法、及び、そのための装置が知られている。
　特許文献１には「２つ以上の細胞または細胞様構造物を含む試料を用い、該細胞または細胞様構造物を１細胞または構造物単位ずつ液滴中に封入する工程と、該液滴をゲル化してゲルカプセルを生成する工程と、該ゲルカプセルを１種以上の溶解用試薬に浸漬して前記細胞または細胞様構造物を溶解する工程であって、該細胞中のポリヌクレオチドが該ゲルカプセル内に溶出し該ポリヌクレオチドに結合する物質が除去された状態で前記ゲルカプセル内に保持される、工程と、該ポリヌクレオチドを増幅用試薬に接触させて該ポリヌクレオチドをゲルカプセル内で増幅する工程とを含む、細胞または細胞様構造物中のポリヌクレオチドを増幅する方法。」が記載されている。 Methods for performing single cell analysis and devices for that purpose are known.
Patent Document 1 describes "a step of using a sample containing two or more cells or cell-like structures, and encapsulating the cells or cell-like structures in the droplets one cell or structure unit at a time, and the droplets. A step of gelling to form a gel capsule and a step of immersing the gel capsule in one or more lysis reagents to lyse the cell or cell-like structure, wherein the polynucleotide in the cell is the gel. The step of retaining in the gel capsule with the substances eluted in the capsule and binding to the polynucleotide removed, and the step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. A method of amplifying a polynucleotide in a cell or cell-like structure, comprising the steps of. "
国際公開第２０１９／２１６２７１号International Publication No. 2019/216271
　多様な細胞を含む生物学的サンプルに難培養性細胞が含まれる場合、その難培養性細胞の機能解析のためには、その生物学的サンプルの全ゲノム解析が不可欠とされてきた。特許文献１に記載されるような単一細胞解析は、１細胞を分離し、その細胞中のポリヌクレオチドを増幅して読み取ることができるため、特に難培養性細胞の全ゲノム解析に大きく貢献している。また、個々の細胞に分離せずに行う、従来公知の全ゲノム（メタゲノム）解析も、依然として上記のような目的には有用である。 When a biological sample containing various cells contains refractory cells, whole-genome analysis of the biological sample has been indispensable for functional analysis of the refractory cells. Single cell analysis as described in Patent Document 1 can separate one cell and amplify and read the polynucleotide in the cell, which greatly contributes to the whole genome analysis of difficult-to-culture cells. ing. Also, conventionally known whole genome (metagenomic) analysis performed without separating into individual cells is still useful for the above-mentioned purpose.
　しかし、特許文献１に記載された単一細胞解析においては、各細胞由来のポリヌクレオチドを増幅してシークエンスするため、増幅過程に問題があると、配列カバー率が不十分になることがあった。また、メタゲノム解析においては、十分な品質のドラフトゲノムを得ようとすることは原理的に難しい場合が多い。 However, in the single cell analysis described in Patent Document 1, polynucleotides derived from each cell are amplified and sequenced. Therefore, if there is a problem in the amplification process, the sequence coverage may be insufficient. .. Moreover, in metagenomic analysis, it is often difficult in principle to obtain a draft genome of sufficient quality.
　そこで本発明は、より簡便に、高品質な全ゲノム解析結果を得ることができる、ゲノム解析方法を提供することを課題とする。 Therefore, it is an object of the present invention to provide a genome analysis method capable of obtaining a high-quality whole genome analysis result more easily.
　本発明者らは、上記課題を達成すべく鋭意検討した結果、以下の構成により上記課題を達成することができることを見出した。 As a result of diligent studies to achieve the above-mentioned problems, the present inventors have found that the above-mentioned problems can be achieved by the following configurations.
［１］　細胞と、上記細胞で産生され、上記細胞外に放出された膜小胞と、を含む懸濁液から、上記細胞と上記膜小胞とを分離し、上記細胞を含む第１試料と、上記膜小胞を含む第２試料とを調製することと、上記第１試料に含まれる上記細胞に由来する塩基配列である、第１塩基配列を決定することと、上記第２試料に含まれる上記膜小胞に由来する塩基配列である、第２塩基配列を決定することと、上記第２塩基配列を用いて、上記第１塩基配列を補完することと、を含む、ゲノム解析方法。
［２］　上記第１塩基配列の決定が、上記第１試料の単一細胞解析によって行われる、［１］に記載のゲノム解析方法。
［３］　上記第１塩基配列の決定が、上記第１試料のメタゲノム解析によって行われる、［１］に記載のゲノム解析方法。
［４］　上記第２塩基配列を決定することが、上記第２試料を用い、上記膜小胞の１個体ずつを液滴中に封入することと、上記液滴をゲル化してゲルカプセルを生成することと、上記ゲルカプセルを溶解試薬と接触させて、上記膜小胞を溶解させ、上記膜小胞中のポリヌクレオチドが上記ゲルカプセルに溶出し、上記ゲルカプセル内に保持させることと、上記ポリヌクレオチドを増幅試薬に接触させて、上記ポリヌクレオチドを上記ゲルカプセル内で増幅することと、上記増幅したポリヌクレオチドから、上記膜小胞に由来する上記ポリヌクレオチドの塩基配列である第２塩基配列を決定することと、を含む、［１］～［３］のいずれかに記載のゲノム解析方法。
［５］　上記第２塩基配列を決定することが、更に、上記第２試料を核酸分解酵素で処理することを含む、［４］に記載のゲノム解析方法。
［６］　上記第２塩基配列を決定することが、上記処理を経た上記第２試料と、上記処理を経ない上記第２試料のそれぞれを用いて行われる、［５］に記載のゲノム解析方法。
［７］　上記補完することが、上記第２塩基配列を上記第１塩基配列の断片として用いることである、［１］～［６］のいずれかに記載のゲノム解析方法。
［８］　上記補完することが、上記第１塩基配列のカバー率を向上させることである、［１］～［７］のいずれかに記載のゲノム解析方法。
［９］　上記懸濁液が、ヒト又は動物から採取した、糞便、唾液、喀痰、手術洗浄液、血液、並びに、皮膚又は身体粘膜の拭い液、及び、スワブからなる群より選択される少なくとも１種の採取試料である、［１］～［８］のいずれかに記載のゲノム解析方法。
［１０］　上記細胞が、細菌である、［１］～［９］のいずれかに記載のゲノム解析方法。
［１１］　上記細菌が、ポルフィロモナス属、プレボテラ属、ベイヨネラ属、フソバクテリウム属、パルビモナス属、アグリゲイティバクター属、アクチノマイセス属、アクチノバチルス属、バクテロイデス属、タンネレラ属、トレポネーマ属、カンピロバクター属、エイケネラ属、及び、カプノサイトファーガ属からなる群より選択される少なくとも１種の細菌である、［１０］に記載のゲノム解析方法。 [1] A first sample containing the cells by separating the cells and the membrane vesicles from a suspension containing the cells and the membrane vesicles produced by the cells and released outside the cells. To prepare a second sample containing the membrane vesicles, to determine the first base sequence, which is a base sequence derived from the cells contained in the first sample, and to use the second sample. A genome analysis method comprising determining a second base sequence, which is a base sequence derived from the included membrane vesicles, and complementing the first base sequence using the second base sequence. ..
[2] The genome analysis method according to [1], wherein the determination of the first base sequence is performed by single cell analysis of the first sample.
[3] The genome analysis method according to [1], wherein the determination of the first base sequence is performed by metagenomic analysis of the first sample.
[4] To determine the second base sequence, the second sample is used, one individual of the membrane vesicles is encapsulated in the droplet, and the droplet is gelled to form a gel capsule. In addition, the gel capsule is brought into contact with the lysis reagent to dissolve the membrane vesicle, and the polynucleotide in the membrane vesicle is eluted into the gel capsule and retained in the gel capsule. The polynucleotide is brought into contact with an amplification reagent to amplify the polynucleotide in the gel capsule, and the second base sequence, which is the base sequence of the polynucleotide derived from the membrane vesicle from the amplified polynucleotide. The genomic analysis method according to any one of [1] to [3], which comprises.
[5] The genome analysis method according to [4], wherein determining the second base sequence further comprises treating the second sample with a nucleolytic enzyme.
[6] The genome analysis method according to [5], wherein the determination of the second base sequence is performed using each of the second sample that has undergone the treatment and the second sample that has not undergone the treatment. ..
[7] The genome analysis method according to any one of [1] to [6], wherein the complement is to use the second base sequence as a fragment of the first base sequence.
[8] The genome analysis method according to any one of [1] to [7], wherein the complementation is to improve the coverage of the first base sequence.
[9] The suspension is at least one selected from the group consisting of feces, saliva, sputum, surgical lavage fluid, blood, and a wiping solution for skin or body mucous membranes collected from humans or animals, and a swab. The genome analysis method according to any one of [1] to [8], which is a sample collected from the above.
[10] The method for genome analysis according to any one of [1] to [9], wherein the cell is a bacterium.
[11] The above-mentioned bacteria are Porphyromonas, Prevotella, Beyonera, Fusobacterium, Parvimonas, Aggregatibacter, Actinobacillus, Actinobacillus, Bacteroides, Tannerela, Treponema, Capnocytophaga. The genome analysis method according to [10], which is at least one bacterium selected from the group consisting of the genus Eikenella and the genus Capnocytophaga.
　本発明によれば、より簡便に、高品質な全ゲノム解析結果を得ることができる、ゲノム解析方法が提供できる。 According to the present invention, it is possible to provide a genome analysis method capable of obtaining a high-quality whole genome analysis result more easily.
本発明の実施形態に係るゲノム解析方法のフロー図である。It is a flow chart of the genome analysis method which concerns on embodiment of this invention. 単一細胞解析による第１塩基配列の決定方法のフロー図である。It is a flow chart of the method of determining the first base sequence by single cell analysis. 単一細胞解析に用いることができるフローセルの模式図である。It is a schematic diagram of a flow cell which can be used for single cell analysis. 本発明の他の実施形態に係るゲノム解析方法のフロー図である。It is a flow chart of the genome analysis method which concerns on other embodiment of this invention. 唾液から採取した、膜小胞サンプル、及び、細菌サンプルのそれぞれにおけるＡｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　ｂａｃｔｅｒｉｕｍ　４１－２８（Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８）由来と判断された粒子の割合（Ｄｅｔｅｃｔｅｄ　ｐｅｒｃｅｎｔａｇｅ）を表す図である。It is a figure which shows the ratio (Detected percentage) of the particle determined to be derived from Alphaproteobacteria bacterium 41-28 (Alphaproteobacteria 41-28) in each of a membrane vesicle sample and a bacterial sample collected from saliva. 上記各サンプルにおけるＴＭ７ｘ由来と判断された粒子の割合を表す図である。It is a figure which shows the ratio of the particle which was determined to be derived from TM7x in each of the said samples. 膜小胞サンプル（Ｈｅａｌｔｈｙ）から得られた粒子（ＭＶ）のうち、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８由来と判断された粒子（５２粒子分）の配列を、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８のゲノムＤＮＡにマップした結果を表す図である。It shows the result of mapping the sequence of the particles (52 particles) determined to be derived from Alphaproteobacteria 41-28 among the particles (MV) obtained from the membrane vesicle sample (Healthy) to the genomic DNA of Alphaproteobacteria 41-28. It is a figure. 膜小胞サンプル（Ｐａｔｉｅｎｔ）から得られた粒子（ＭＶ）のうち、ＴＭ７ｘ由来と判断された粒子（８６粒子）の配列を、ＴＭ７ｘのゲノムＤＮＡにマップした結果を表す図である。It is a figure which shows the result of mapping the sequence of the particle (86 particle) determined to be derived from TM7x among the particle (MV) obtained from the membrane vesicle sample (Patient) to the genomic DNA of TM7x. 膜小胞サンプルから得られた配列によって構築された各微生物のゲノムＤＮＡ長の割合を表す図である。It is a figure which shows the ratio of the genomic DNA length of each microorganism constructed by the sequence obtained from the membrane vesicle sample.
　以下、本発明について詳細に説明する。
　以下に記載する構成要件の説明は、本発明の代表的な実施形態に基づいてなされることがあるが、本発明はそのような実施形態に制限されるものではない。
　なお、本明細書において、「～」を用いて表される数値範囲は、「～」の前後に記載される数値を下限値、及び、上限値として含む範囲を意味する。 Hereinafter, the present invention will be described in detail.
The description of the constituent elements described below may be based on a representative embodiment of the present invention, but the present invention is not limited to such embodiments.
In the present specification, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value.
（用語の定義）
　本明細書において、「ポリヌクレオチド」は、リボヌクレオチド、又は、デオキシリボヌクレオチドのいずれかの、任意の長さのヌクレオチドのポリマー形態を指す。この用語は上記分子の一次構造のみを指す。したがって、この用語は二本鎖と一本鎖のＤＮＡ、及び、ＲＮＡを含む。 (Definition of terms)
As used herein, "polynucleotide" refers to the polymer form of a nucleotide of any length, either a ribonucleotide or a deoxyribonucleotide. This term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, and RNA.
　本明細書において、「単一細胞解析」とは、「シングルセル解析」と同義であり、複数種類、及び／又は、複数個の細胞が含まれる検体（試料）から、一細胞を分取しゲノム解析する方法を意味する。
　単一細胞解析は、例えば、平面上に配列した多数のマイクロウェルのそれぞれに細胞を一つずつ分取し、その細胞の個々について、ゲノム解析を行う方法である。単一細胞解析には、例えば、特許文献１に記載された装置、国際公開第２０１７／０９４１０１号に記載された装置、及び、国際公開第２０１６／０３８６７０号に記載された装置等のような公知の装置、及び／又は、方法を使用するものが挙げられる。 In the present specification, "single cell analysis" is synonymous with "single cell analysis", and one cell is separated from a sample containing a plurality of types and / or a plurality of cells. It means a method for genome analysis.
Single cell analysis is, for example, a method in which one cell is separated from each of a large number of microwells arranged on a plane, and genome analysis is performed on each of the cells. For single cell analysis, for example, the apparatus described in Patent Document 1, the apparatus described in International Publication No. 2017/094101, the apparatus described in International Publication No. 2016/038670, and the like are known. And / or those using the method of.
　また、本明細書において、「単一粒子解析」とは、細胞由来の粒子（細胞自体を除く）を分取し、一粒子毎にゲノム解析する方法を意味する。単一粒子解析の方法としては、上記「単一細胞解析」と同様の方法が使用できる。 Further, in the present specification, "single particle analysis" means a method of separating cell-derived particles (excluding the cells themselves) and performing genome analysis for each particle. As a method for single particle analysis, the same method as the above-mentioned "single cell analysis" can be used.
　本明細書における、「膜小胞」には、細菌等により産生される外膜小胞、動物細胞等により、エンドサイトーシス経路により産生、放出される「エクソソーム」、及び、アポトーシスによって生ずる「アポトーシス小体」も含まれるものとし、なかでも、膜小胞としては、外膜小胞（ＯＭＶ）が好ましい。 In the present specification, "membrane vesicles" include "exosomes" produced and released by the endocytic pathway by outer membrane vesicles produced by bacteria and the like, animal cells and the like, and "absorption" caused by apoptosis. "Small bodies" are also included, and among them, outer membrane vesicles (OMV) are preferable as the membrane vesicles.
　細菌により産生される（「細菌に由来する」、又は、「細菌の」ということがある）膜小胞としては、細菌の細胞膜の一部が菌体外にくびり取られるようにして産生されたマイクロベシクルである「外膜小胞（ＯＭＶ；ｏｕｔｅｒ　ｍｅｍｂｒａｎｅ　ｖｅｓｉｃｌｅ）」が挙げられ、その大きさは、特に制限されないが、１０～１０００ｎｍである場合が多く、グラム陰性細菌では１０～３００ｎｍであることが多く、グラム陽性細菌では５０～１５０ｎｍであることが多い。 Membrane vesicles produced by bacteria (sometimes "derived from bacteria" or "bacterial") are produced so that part of the bacterial cell membrane is constricted outside the cells. The microvesicle "outer membrane vesicle (OMV)" is mentioned, and its size is not particularly limited, but is often 10 to 1000 nm, and 10 to 300 nm for Gram-negative bacteria. Often, it is 50-150 nm for Gram-positive bacteria.
［ゲノム解析方法］
　以下では、本発明のゲノム解析方法について詳述する。なお、本発明のゲノム解析方法は、細胞と、上記細胞で産生され、上記細胞外に放出された膜小胞を含む懸濁液を用いるものであり、上記「細胞」は、多細胞生物のもの、及び、単細胞生物のもののいずれも含まれる。以下の説明では、そのうち、細胞が細菌である場合を例に説明する。なお、以下の方法は、動物細胞等にも同様に適用可能である。 [Genome analysis method]
Hereinafter, the genome analysis method of the present invention will be described in detail. The genome analysis method of the present invention uses a cell and a suspension containing membrane vesicles produced in the cell and released outside the cell, and the "cell" is a multicellular organism. It includes both those of single-celled organisms and those of single-celled organisms. In the following description, the case where the cell is a bacterium will be described as an example. The following method can be similarly applied to animal cells and the like.
　図１は、本発明の実施形態に係るゲノム解析方法（以下「本方法」ともいう。）のフローチャートである。
　本方法は、細菌と、上記細菌で産生され、上記細菌外に放出された膜小胞を含む懸濁液から、上記細菌と上記膜小胞とを分離し、上記細菌を含む第１試料と、上記膜小胞を含む第２試料とを調製すること（試料調製工程、ステップＳ１０１）と、
　第１試料に含まれる上記細菌に由来する塩基配列である、第１塩基配列を決定すること（第１塩基配列決定工程、ステップＳ１０２）と、
　第２試料に含まれる上記膜小胞に由来する塩基配列である、第２塩基配列を決定すること（第２塩基配列決定工程、Ｓ１０３）と、
　第２塩基配列を用いて、第１塩基配列を補完すること（補完工程、Ｓ１０４）と、を含むゲノム解析方法である。 FIG. 1 is a flowchart of a genome analysis method (hereinafter, also referred to as “the present method”) according to an embodiment of the present invention.
In this method, the bacterium and the membrane vesicle are separated from the suspension containing the bacterium and the membrane vesicle produced by the bacterium and released outside the bacterium, and the first sample containing the bacterium is used. To prepare a second sample containing the above-mentioned membrane vesicles (sample preparation step, step S101).
Determining the first base sequence, which is a base sequence derived from the above-mentioned bacteria contained in the first sample (first base sequence determination step, step S102),
Determining the second base sequence, which is the base sequence derived from the membrane vesicle contained in the second sample (second base sequence determination step, S103),
It is a genome analysis method including complementing the first base sequence using the second base sequence (complementation step, S104).
・試料調製工程（ステップＳ１０１）
　ステップＳ１０１は、細菌と膜小胞とを含む懸濁液から、細菌と膜小胞とを分離し、細菌を含む第１試料と、膜小胞を含む第２試料と、を調製する工程である。 -Sample preparation step (step S101)
Step S101 is a step of separating the bacterium and the membrane vesicle from the suspension containing the bacterium and the membrane vesicle, and preparing a first sample containing the bacterium and a second sample containing the membrane vesicle. be.
　本工程で使用される懸濁液は、細菌と膜小胞とを含む。すなわち、懸濁液には、膜小胞と、その膜小胞を産生した細菌とが含まれている。この細菌は培養された単一の細菌株であってもよいし、複数種の細菌であってもよい（このうちの１種以上が難培養性細菌であってもよい）。 The suspension used in this step contains bacteria and membrane vesicles. That is, the suspension contains membrane vesicles and the bacteria that produced the membrane vesicles. This bacterium may be a single cultivated bacterial strain or a plurality of species (one or more of them may be a refractory bacterium).
　懸濁液としては、例えば、ヒト又は動物から採取した、糞便、唾液、喀痰、手術洗浄液、血液、皮膚・身体粘膜の拭い液、及び、スワブ等の採取試料であってもよい。一般に、採取試料中には、細菌群と、それに含まれる細菌により産生された膜小胞とが含まれる場合がある。
　このような採取試料から細菌群と膜小胞とを分離して、ぞれぞれを、第１試料と第２試料とにすればよい。 The suspension may be, for example, a sample collected from humans or animals, such as feces, saliva, sputum, surgical cleaning solution, blood, skin / body mucosa wiping solution, and swab. In general, the collected sample may contain a group of bacteria and membrane vesicles produced by the bacteria contained therein.
Bacterial groups and membrane vesicles may be separated from such collected samples and used as a first sample and a second sample, respectively.
　懸濁液に含まれる細菌としては特に制限されないが、真正細菌、大腸菌、枯草菌、藍色細菌、球菌、桿菌、ラセン菌、グラム陰性菌、グラム陽性菌、古細菌、及び、真菌等が挙げられる。細菌としては、例えば、Ｎｅｇｉｂａｃｔｅｒｉａ、Ｅｏｂａｃｔｅｒｉａ、Ｄｅｉｎｏｃｏｃｃｉ、Ｄｅｉｎｏｃｏｃｃｉ、Ｄｅｉｎｏｃｏｃｃａｌｅｓ、Ｔｈｅｒｍａｌｅｓ、Ｃｈｌｏｒｏｆｌｅｘｉ、Ａｎａｅｒｏｌｉｎｅａｅ、Ａｎａｅｒｏｌｉｎｅａｌｅｓ、Ｃａｌｄｉｌｉｎｅａｅ、Ｃｈｌｏｒｏｆｌｅｘａｌｅｓ、Ｈｅｒｐｅｔｏｓｉｐｈｏｎａｌｅｓ、Ｔｈｅｒｍｏｍｉｃｒｏｂｉａ、Ｔｈｅｒｍｏｍｉｃｒｏｂｉａｌｅｓ、Ｓｐｈａｅｒｏｂａｃｔｅｒａｌｅｓ、Ｋｔｅｄｏｎｏｂａｃｔｅｒｉａ、Ｋｔｅｄｏｎｏｂａｃｔｅｒａｌｅｓ、Ｔｈｅｒｍｏｇｅｍｍａｔｉｓｐｏｒａｌｅｓ、Ｇｌｙｃｏｂａｃｔｅｒｉａ、Ｃｙａｎｏｂａｃｔｅｒｉａ、Ｇｌｏｅｏｂａｃｔｅｒｏｐｈｙｃｅａｅ、Ｇｌｏｅｏｂａｃｔｅｒａｌｅｓ、Ｎｏｓｔｏｃｏｐｈｙｃｅａｅ、Ｓｙｎｅｃｈｏｃｏｃｃｏｐｈｙｃｉｄａｅ、Ｓｙｎｅｃｈｏｃｏｃｃａｌｅｓ、Ｎｏｓｔｏｃｏｐｈｙｃｉｄａｅ、Ｃｈｒｏｏｃｏｃｃａｌｅｓ、Ｏｓｃｉｌｌａｔｏｒｉａｌｅｓ、Ｎｏｓｔｏｃａｌｅｓ、Ｐｓｅｕｄａｎａｂａｅｎａｌｅｓ、Ｓｐｉｒｏｃｈａｅｔｅｓ、Ｓｐｉｒｏｃｈａｅｔｅｓ、Ｓｐｉｒｏｃｈａｅｔａｌｅｓ、Ｆｉｂｒｏｂａｃｔｅｒｅｓ、Ｆｉｂｒｏｂａｃｔｅｒｉａ、Ｇｅｍｍａｔｉｍｏｎａｄｅｔｅｓ、Ｇｅｍｍａｔｉｍｏｎａｄｅｔｅｓ、Ｇｅｍｍａｔｉｍｏｎａｄａｌｅｓ、Ｃｈｌｏｒｏｂｉ、Ｃｈｌｏｒｏｂｅａ、Ｃｈｌｏｒｏｂｉａｌｅｓ、Ｉｇｎａｖｉｂａｃｔｅｒｉａ、Ｉｇｎａｖｉｂａｃｔｅｒｉａｌｅｓ、Ｂａｃｔｅｒｏｉｄｅｔｅｓ、Ｂａｃｔｅｒｏｉｄｉａ、Ｂａｃｔｅｒｏｉｄａｌｅｓ、Ｆｌａｖｏｂａｃｔｅｒｉｉａ、Ｆｌａｖｏｂａｃｔｅｒｉａｌｅｓ、Ｓｐｈｉｎｇｏｂａｃｔｅｒｉｉａ、Ｓｐｈｉｎｇｏｂａｃｔｅｒｉａｌｅｓ、Ｃｙｔｏｐｈａｇｉａ、Ｃｙｔｏｐｈａｇａｌｅｓ、Ｐｌａｎｃｔｏｍｙｃｅｔｅｓ、Ｐｌａｎｃｔｏｍｙｃｅａ、Ｐｌａｎｃｔｏｍｙｃｅｔａｌｅｓ、Ｐｈｙｃｉｓｐｈａｅｒａｅ、Ｐｈｙｃｉｓｐｈａｅｒａｌｅｓ、Ｃｈｌａｍｙｄｉａｅ、Ｃｈｌａｍｙｄｉａｅ、Ｃｈｌａｍｙｄｉａｌｅｓ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａｅ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａｌｅｓ、Ｏｐｉｔｕｔａｅ、Ｏｐｉｔｕｔａｌｅｓ、Ｐｕｎｉｃｅｉｃｏｃｃａｌｅｓ、Ｓｐａｒｔｏｂａｃｔｅｒｉａ、Ｃｈｔｈｏｎｉｏｂａｃｔｅｒａｌｅｓ、Ｌｅｎｔｉｓｐｈａｅｒａｅ、Ｌｅｎｔｉｓｐｈａｅｒｉａ、Ｌｅｎｔｉｓｐｈａｅｒａｌｅｓ、Ｖｉｃｔｉｖａｌｌａｌｅｓ、Ｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｒｈｏｄｏｓｐｉｒｉｌｌａｌｅｓ、Ｒｉｃｋｅｔｔｓｉａｌｅｓ、Ｒｈｏｄｏｂａｃｔｅｒａｌｅｓ、Ｓｐｈｉｎｇｏｍｏｎａｄａｌｅｓ、Ｃａｕｌｏｂａｃｔｅｒａｌｅｓ、Ｒｈｉｚｏｂｉａｌｅｓ、Ｐａｒｖｕｌａｒｃｕｌａｌｅｓ、Ｋｏｒｄｉｉｍｏｎａｄａｌｅｓ、Ｓｎｅａｔｈｉｅｌｌａｌｅｓ、Ｋｉｌｏｎｉｅｌｌａｌｅｓ、Ｂｅｔａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｂｕｒｋｈｏｌｄｅｒｉａｌｅｓ、Ｈｙｄｒｏｇｅｎｏｐｈｉｌａｌｅｓ、Ｍｅｔｈｙｌｏｐｈｉｌａｌｅｓ、Ｎｅｉｓｓｅｒｉａｌｅｓ、Ｎｉｔｒｏｓｏｍｏｎａｄａｌｅｓ、Ｒｈｏｄｏｃｙｃｌａｌｅｓ、Ｐｒｏｃａｂａｃｔｅｒｉａｌｅｓ、Ｇａｍｍａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｃｈｒｏｍａｔｉａｌｅｓ、Ａｃｉｄｉｔｈｉｏｂａｃｉｌｌａｌｅｓ、Ｘａｎｔｈｏｍｏｎａｄａｌｅｓ、Ｃａｒｄｉｏｂａｃｔｅｒｉａｌｅｓ、Ｔｈｉｏｔｒｉｃｈａｌｅｓ、Ｌｅｇｉｏｎｅｌｌａｌｅｓ、Ｍｅｔｈｙｌｏｃｏｃｃａｌｅｓ、Ｏｃｅａｎｏｓｐｉｒｉｌｌａｌｅｓ、Ｐｓｅｕｄｏｍｏｎａｄａｌｅｓ、Ａｌｔｅｒｏｍｏｎａｄａｌｅｓ、Ｖｉｂｒｉｏｎａｌｅｓ、Ａｅｒｏｍｏｎａｄａｌｅｓ、Ｅｎｔｅｒｏｂａｃｔｅｒｉａｌｅｓ、Ｐａｓｔｅｕｒｅｌｌａｌｅｓ、Ｄｅｌｔａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｄｅｓｕｌｆｕｒｅｌｌａｌｅｓ、Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ、Ｄｅｓｕｌｆｏｂａｃｔｅｒａｌｅｓ、Ｄｅｓｕｌｆａｒｃｕｌａｌｅｓ、Ｄｅｓｕｌｆｕｒｏｍｏｎａｄａｌｅｓ、Ｓｙｎｔｒｏｐｈｏｂａｃｔｅｒａｌｅｓ、Ｂｄｅｌｌｏｖｉｂｒｉｏｎａｌｅｓ、Ｍｙｘｏｃｏｃｃａｌｅｓ、Ｅｐｓｉｌｏｎｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｃａｍｐｙｌｏｂａｃｔｅｒａｌｅｓ、Ｎａｕｔｉｌｉａｌｅｓ、Ａｃｉｄｏｂａｃｔｅｒｉａ、Ａｃｉｄｏｂａｃｔｅｒｉａ、Ａｃｉｄｏｂａｃｔｅｒｉａｌｅｓ、Ｈｏｌｏｐｈａｇａｅ、Ｈｏｌｏｐｈａｇａｌｅｓ、Ａｃａｎｔｈｏｐｌｅｕｒｉｂａｃｔｅｒａｌｅｓ、Ａｑｕｉｆｉｃａｅ、Ａｑｕｉｆｉｃａｅ、Ａｑｕｉｆｉｃａｌｅｓ、Ｄｅｆｅｒｒｉｂａｃｔｅｒｅｓ、Ｄｅｆｅｒｒｉｂａｃｔｅｒｅｓ、Ｇｅｏｖｉｂｒｉａｌｅｓ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａｌｅｓ、Ｎｉｔｒｏｓｐｉｒａｅ、Ｎｉｔｒｏｓｐｉｒａ、Ｎｉｔｒｏｓｐｉｒａｌｅｓ、Ｆｕｓｏｂａｃｔｅｒｉａ、Ｆｕｓｏｂａｃｔｅｒｉｉａ、Ｆｕｓｏｂａｃｔｅｒｉａｌｅｓ、Ｓｙｎｅｒｇｉｓｔｅｔｅｓ、Ｓｙｎｅｒｇｉｓｔｉａ、Ｓｙｎｅｒｇｉｓｔａｌｅｓ、Ｃａｌｄｉｓｅｒｉｃａ、Ｃａｌｄｉｓｅｒｉｃｉａ、Ｃａｌｄｉｓｅｒｉｃａｌｅｓ、Ｅｌｕｓｉｍｉｃｒｏｂｉａ、Ｅｌｕｓｉｍｉｃｒｏｂｉａ、Ｅｌｕｓｉｍｉｃｒｏｂｉａｌｅｓ、Ａｒｍａｔｉｍｏｎａｄｅｔｅｓ、Ａｒｍａｔｉｍｏｎａｄｉａ、Ａｒｍａｔｉｍｏｎａｄａｌｅｓ、Ｃｈｔｈｏｎｏｍｏｎａｄｅｔｅｓ、Ｃｈｔｈｏｎｏｍｏｎａｄａｌｅｓ、Ｆｉｍｂｒｉｉｍｏｎａｄｉａ、Ｆｉｍｂｒｉｉｍｏｎａｄａｌｅｓ、Ｐｏｓｉｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｔｏｇａｅ、Ｔｈｅｒｍｏｔｏｇａｅ、Ｔｈｅｒｍｏｔａｇａｌｅｓ、Ｆｉｒｍｉｃｕｔｅｓ、Ｂａｃｉｌｌｉ、Ｂａｃｉｌｌａｌｅｓ、Ｌａｃｔｏｂａｃｉｌｌａｌｅｓ、Ｃｌｏｓｔｒｉｄｉａ、Ｃｌｏｓｔｒｉｄｉａｌｅｓ、Ｈａｌａｎａｅｒｏｂｉａｌｅｓ、Ｔｈｅｒｍｏａｎａｅｒｏｂａｃｔｅｒａｌｅｓ、Ｎａｔｒａｎａｅｒｏｂｉａｌｅｓ、Ｎｅｇａｔｉｖｉｃｕｔｅｓ、Ｓｅｌｅｎｏｍｏｎａｄａｌｅｓ、Ｅｒｙｓｉｐｅｌｏｔｒｉｃｈｉａ、Ｅｒｙｓｉｐｅｌｏｔｒｉｃｈａｌｅｓ、Ｔｈｅｒｍｏｌｉｔｈｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｌｉｔｈｏｂａｃｔｅｒａｌｅｓ、Ｔｅｎｅｒｉｃｕｔｅｓ、Ｍｏｌｌｉｃｕｔｅｓ、Ｍｙｃｏｐｌａｓｍａｔａｌｅｓ、Ｅｎｔｏｍｏｐｌａｓｍａｔａｌｅｓ、Ａｃｈｏｌｅｐｌａｓｍａｔａｌｅｓ、Ａｎａｅｒｏｐｌａｓｍａｔａｌｅｓ、Ａｃｔｉｎｏｂａｃｔｅｒｉａ、Ａｃｔｉｎｏｂａｃｔｅｒｉａ、Ａｃｔｉｎｏｍｙｃｅｔａｌｅｓ、Ａｃｔｉｎｏｐｏｌｙｓｐｏｒａｌｅｓ、Ｂｉｆｉｄｏｂａｃｔｅｒｉａｌｅｓ、Ｃａｔｅｎｕｌｉｓｐｏｒａｌｅｓ、Ｃｏｒｙｎｅｂａｃｔｅｒｉａｌｅｓ、Ｆｒａｎｋｉａｌｅｓ、Ｇｌｙｃｏｍｙｃｅｔａｌｅｓ、Ｊｉａｎｇｅｌｌａｌｅｓ、Ｋｉｎｅｏｓｐｏｒｉａｌｅｓ、Ｍｉｃｒｏｃｏｃｃａｌｅｓ、Ｍｉｃｒｏｍｏｎｏｓｐｏｒａｌｅｓ、Ｐｒｏｐｉｏｎｉｂａｃｔｅｒｉａｌｅｓ、Ｐｓｅｕｄｏｎｏｃａｒｄｉａｌｅｓ、Ｓｔｒｅｐｔｏｍｙｃｅｔａｌｅｓ、Ｓｔｒｅｐｔｏｓｐｏｒａｎｇｉａｌｅｓ、Ｄｉｃｔｙｏｇｌｏｍｉ、Ｄｉｃｔｙｏｇｌｏｍｉａ、Ｄｉｃｔｙｏｇｌｏｍａｌｅｓ、Ｃｈｒｙｓｉｏｇｅｎｅｔｅｓ、Ｃｈｒｙｓｉｏｇｅｎｅｔｅｓ、Ｃｈｒｙｓｉｏｇｅｎａｌｅｓ、Ｈａｌｏｐｌａｓｍａｔａｌｅｓ等が挙げられる。 Bacteria contained in the suspension are not particularly limited, and examples thereof include eubacteria, Escherichia coli, bacilli, indigo bacteria, cocci, bacilli, racen, gram-negative bacteria, gram-positive bacteria, paleobacteria, and fungi. Be done.細菌としては、例えば、Ｎｅｇｉｂａｃｔｅｒｉａ、Ｅｏｂａｃｔｅｒｉａ、Ｄｅｉｎｏｃｏｃｃｉ、Ｄｅｉｎｏｃｏｃｃｉ、Ｄｅｉｎｏｃｏｃｃａｌｅｓ、Ｔｈｅｒｍａｌｅｓ、Ｃｈｌｏｒｏｆｌｅｘｉ、Ａｎａｅｒｏｌｉｎｅａｅ、Ａｎａｅｒｏｌｉｎｅａｌｅｓ、Ｃａｌｄｉｌｉｎｅａｅ、Ｃｈｌｏｒｏｆｌｅｘａｌｅｓ、Ｈｅｒｐｅｔｏｓｉｐｈｏｎａｌｅｓ、Ｔｈｅｒｍｏｍｉｃｒｏｂｉａ、Ｔｈｅｒｍｏｍｉｃｒｏｂｉａｌｅｓ、Ｓｐｈａｅｒｏｂａｃｔｅｒａｌｅｓ、Ｋｔｅｄｏｎｏｂａｃｔｅｒｉａ、Ｋｔｅｄｏｎｏｂａｃｔｅｒａｌｅｓ、Ｔｈｅｒｍｏｇｅｍｍａｔｉｓｐｏｒａｌｅｓ、Ｇｌｙｃｏｂａｃｔｅｒｉａ、Ｃｙａｎｏｂａｃｔｅｒｉａ、Ｇｌｏｅｏｂａｃｔｅｒｏｐｈｙｃｅａｅ、Ｇｌｏｅｏｂａｃｔｅｒａｌｅｓ、 Ｎｏｓｔｏｃｏｐｈｙｃｅａｅ、Ｓｙｎｅｃｈｏｃｏｃｃｏｐｈｙｃｉｄａｅ、Ｓｙｎｅｃｈｏｃｏｃｃａｌｅｓ、Ｎｏｓｔｏｃｏｐｈｙｃｉｄａｅ、Ｃｈｒｏｏｃｏｃｃａｌｅｓ、Ｏｓｃｉｌｌａｔｏｒｉａｌｅｓ、Ｎｏｓｔｏｃａｌｅｓ、Ｐｓｅｕｄａｎａｂａｅｎａｌｅｓ、Ｓｐｉｒｏｃｈａｅｔｅｓ、Ｓｐｉｒｏｃｈａｅｔｅｓ、Ｓｐｉｒｏｃｈａｅｔａｌｅｓ、Ｆｉｂｒｏｂａｃｔｅｒｅｓ、Ｆｉｂｒｏｂａｃｔｅｒｉａ、Ｇｅｍｍａｔｉｍｏｎａｄｅｔｅｓ、Ｇｅｍｍａｔｉｍｏｎａｄｅｔｅｓ、Ｇｅｍｍａｔｉｍｏｎａｄａｌｅｓ、Ｃｈｌｏｒｏｂｉ、Ｃｈｌｏｒｏｂｅａ、Ｃｈｌｏｒｏｂｉａｌｅｓ、Ｉｇｎａｖｉｂａｃｔｅｒｉａ、Ｉｇｎａｖｉｂａｃｔｅｒｉａｌｅｓ、Ｂａｃｔｅｒｏｉｄｅｔｅｓ、Ｂａｃｔｅｒｏｉｄｉａ、Ｂａｃｔｅｒｏｉｄａｌｅｓ、Ｆｌａｖｏｂａｃｔｅｒｉｉａ、 Ｆｌａｖｏｂａｃｔｅｒｉａｌｅｓ、Ｓｐｈｉｎｇｏｂａｃｔｅｒｉｉａ、Ｓｐｈｉｎｇｏｂａｃｔｅｒｉａｌｅｓ、Ｃｙｔｏｐｈａｇｉａ、Ｃｙｔｏｐｈａｇａｌｅｓ、Ｐｌａｎｃｔｏｍｙｃｅｔｅｓ、Ｐｌａｎｃｔｏｍｙｃｅａ、Ｐｌａｎｃｔｏｍｙｃｅｔａｌｅｓ、Ｐｈｙｃｉｓｐｈａｅｒａｅ、Ｐｈｙｃｉｓｐｈａｅｒａｌｅｓ、Ｃｈｌａｍｙｄｉａｅ、Ｃｈｌａｍｙｄｉａｅ、Ｃｈｌａｍｙｄｉａｌｅｓ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａｅ、Ｖｅｒｒｕｃｏｍｉｃｒｏｂｉａｌｅｓ、Ｏｐｉｔｕｔａｅ、Ｏｐｉｔｕｔａｌｅｓ、Ｐｕｎｉｃｅｉｃｏｃｃａｌｅｓ、Ｓｐａｒｔｏｂａｃｔｅｒｉａ、Ｃｈｔｈｏｎｉ ｏｂａｃｔｅｒａｌｅｓ、Ｌｅｎｔｉｓｐｈａｅｒａｅ、Ｌｅｎｔｉｓｐｈａｅｒｉａ、Ｌｅｎｔｉｓｐｈａｅｒａｌｅｓ、Ｖｉｃｔｉｖａｌｌａｌｅｓ、Ｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｒｈｏｄｏｓｐｉｒｉｌｌａｌｅｓ、Ｒｉｃｋｅｔｔｓｉａｌｅｓ、Ｒｈｏｄｏｂａｃｔｅｒａｌｅｓ、Ｓｐｈｉｎｇｏｍｏｎａｄａｌｅｓ、Ｃａｕｌｏｂａｃｔｅｒａｌｅｓ、Ｒｈｉｚｏｂｉａｌｅｓ、Ｐａｒｖｕｌａｒｃｕｌａｌｅｓ、Ｋｏｒｄｉｉｍｏｎａｄａｌｅｓ、Ｓｎｅａｔｈｉｅｌｌａｌｅｓ、Ｋｉｌｏｎｉｅｌｌａｌｅｓ、Ｂｅｔａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｂｕｒｋｈｏｌｄｅｒｉａｌｅｓ、Ｈｙｄｒｏｇｅｎｏｐｈｉｌａｌｅｓ、Ｍｅｔｈｙｌｏｐｈｉｌａｌｅｓ、Ｎｅｉｓｓｅｒｉａｌｅｓ、Ｎｉｔｒｏｓｏｍｏｎａｄａｌｅｓ、Ｒｈｏｄｏｃｙｃｌａｌｅｓ、Ｐｒｏｃａｂａｃｔｅｒｉａｌｅｓ、 Ｇａｍｍａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｃｈｒｏｍａｔｉａｌｅｓ、Ａｃｉｄｉｔｈｉｏｂａｃｉｌｌａｌｅｓ、Ｘａｎｔｈｏｍｏｎａｄａｌｅｓ、Ｃａｒｄｉｏｂａｃｔｅｒｉａｌｅｓ、Ｔｈｉｏｔｒｉｃｈａｌｅｓ、Ｌｅｇｉｏｎｅｌｌａｌｅｓ、Ｍｅｔｈｙｌｏｃｏｃｃａｌｅｓ、Ｏｃｅａｎｏｓｐｉｒｉｌｌａｌｅｓ、Ｐｓｅｕｄｏｍｏｎａｄａｌｅｓ、Ａｌｔｅｒｏｍｏｎａｄａｌｅｓ、Ｖｉｂｒｉｏｎａｌｅｓ、Ａｅｒｏｍｏｎａｄａｌｅｓ、Ｅｎｔｅｒｏｂａｃｔｅｒｉａｌｅｓ、Ｐａｓｔｅｕｒｅｌｌａｌｅｓ、Ｄｅｌｔａｐｒｏｔｅｏｂａｃｔｅｒｉａ、Ｄｅｓｕｌｆｕｒｅｌｌａｌｅｓ、Ｄｅｓｕｌｆｏｖｉｂｒｉｏｎａｌｅｓ、Ｄｅｓｕｌｆｏｂａｃｔｅｒａｌｅｓ、Ｄｅｓｕｌｆａｒｃｕｌａｌｅｓ、Ｄｅｓｕｌｆｕｒｏｍｏｎａｄａｌｅｓ、Ｓｙｎｔｒｏｐｈｏｂａｃｔｅｒａｌｅｓ、Ｂｄｅｌｌｏｖｉｂｒｉｏｎａｌｅｓ、Ｍｙｘｏｃｏｃｃａｌｅｓ、Ｅｐｓｉｌｏｎｐｒｏｔｅｏｂａｃｔｅｒｉａ、 Campylobacterales, Naturiales, Acidobacteria, Acidobacteria, Acidobacteriales, Holophagae, Holophagaceae, Canthopleuribacterales, Aquificota, Aquificota, Aquificota, Aquificota ｉｂｒｉａｌｅｓ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｄｅｓｕｌｆｏｂａｃｔｅｒｉａｌｅｓ、Ｎｉｔｒｏｓｐｉｒａｅ、Ｎｉｔｒｏｓｐｉｒａ、Ｎｉｔｒｏｓｐｉｒａｌｅｓ、Ｆｕｓｏｂａｃｔｅｒｉａ、Ｆｕｓｏｂａｃｔｅｒｉｉａ、Ｆｕｓｏｂａｃｔｅｒｉａｌｅｓ、Ｓｙｎｅｒｇｉｓｔｅｔｅｓ、Ｓｙｎｅｒｇｉｓｔｉａ、Ｓｙｎｅｒｇｉｓｔａｌｅｓ、Ｃａｌｄｉｓｅｒｉｃａ、Ｃａｌｄｉｓｅｒｉｃｉａ、Ｃａｌｄｉｓｅｒｉｃａｌｅｓ、Ｅｌｕｓｉｍｉｃｒｏｂｉａ、Ｅｌｕｓｉｍｉｃｒｏｂｉａ、Ｅｌｕｓｉｍｉｃｒｏｂｉａｌｅｓ、Ａｒｍａｔｉｍｏｎａｄｅｔｅｓ、Ａｒｍａｔｉｍｏｎａｄｉａ、Ａｒｍａｔｉｍｏｎａｄａｌｅｓ、Ｃｈｔｈｏｎｏｍｏｎａｄｅｔｅｓ、Ｃｈｔｈｏｎｏｍｏｎａｄａｌｅｓ、Ｆｉｍｂｒｉｉｍｏｎａｄｉａ、 Ｆｉｍｂｒｉｉｍｏｎａｄａｌｅｓ、Ｐｏｓｉｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｔｏｇａｅ、Ｔｈｅｒｍｏｔｏｇａｅ、Ｔｈｅｒｍｏｔａｇａｌｅｓ、Ｆｉｒｍｉｃｕｔｅｓ、Ｂａｃｉｌｌｉ、Ｂａｃｉｌｌａｌｅｓ、Ｌａｃｔｏｂａｃｉｌｌａｌｅｓ、Ｃｌｏｓｔｒｉｄｉａ、Ｃｌｏｓｔｒｉｄｉａｌｅｓ、Ｈａｌａｎａｅｒｏｂｉａｌｅｓ、Ｔｈｅｒｍｏａｎａｅｒｏｂａｃｔｅｒａｌｅｓ、Ｎａｔｒａｎａｅｒｏｂｉａｌｅｓ、Ｎｅｇａｔｉｖｉｃｕｔｅｓ、Ｓｅｌｅｎｏｍｏｎａｄａｌｅｓ、Ｅｒｙｓｉｐｅｌｏｔｒｉｃｈｉａ、Ｅｒｙｓｉｐｅｌｏｔｒｉｃｈａｌｅｓ、Ｔｈｅｒｍｏｌｉｔｈｏｂａｃｔｅｒｉａ、Ｔｈｅｒｍｏｌｉｔｈｏｂａｃｔｅｒａｌｅｓ、Ｔｅｎｅｒｉｃｕｔｅｓ、Ｍｏｌｌｉｃｕｔｅｓ、Ｍｙｃｏｐｌａｓｍａｔａｌｅｓ、Ｅｎｔｏｍｏｐｌａｓｍａｔａｌｅｓ、Ａｃｈｏｌｅｐｌａｓｍａｔａｌｅｓ、 Ａｎａｅｒｏｐｌａｓｍａｔａｌｅｓ、Ａｃｔｉｎｏｂａｃｔｅｒｉａ、Ａｃｔｉｎｏｂａｃｔｅｒｉａ、Ａｃｔｉｎｏｍｙｃｅｔａｌｅｓ、Ａｃｔｉｎｏｐｏｌｙｓｐｏｒａｌｅｓ、Ｂｉｆｉｄｏｂａｃｔｅｒｉａｌｅｓ、Ｃａｔｅｎｕｌｉｓｐｏｒａｌｅｓ、Ｃｏｒｙｎｅｂａｃｔｅｒｉａｌｅｓ、Ｆｒａｎｋｉａｌｅｓ、Ｇｌｙｃｏｍｙｃｅｔａｌｅｓ、Ｊｉａｎｇｅｌｌａｌｅｓ、Ｋｉｎｅｏｓｐｏｒｉａｌｅｓ、Ｍｉｃｒｏｃｏｃｃａｌｅｓ、Ｍｉｃｒｏｍｏｎｏｓｐｏｒａｌｅｓ、Ｐｒｏｐｉｏｎｉｂａｃｔｅｒｉａｌｅｓ、Ｐｓ Eudonocardiales, Streptosporangiales, Streptosporangiales, Dictyoglomus, Dictyoglomus, Dictyoglomus, Chrysiogenetes, Chrysiogenetes, Chrysiogenetes, Clysiogenes, etc.
　また、細菌は歯周病菌（歯周病の原因菌）が好ましい。歯周病菌としては、例えばポルフィロモナス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ）属、プレボテラ（Ｐｒｅｖｏｔｅｌｌａ）属、ベイヨネラ（Ｖｅｉｌｌｏｎｅｌｌａ）属、フソバクテリウム（Ｆｕｓｏｂａｃｔｅｒｉｕｍ）属、パルビモナス（Ｐａｒｖｉｍｏｎａｓ）属、アグリゲイティバクター（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ）属、アクチノマイセス（Ａｃｔｉｎｏｍｙｃｅｓ）属、アクチノバチルス（Ａｃｔｉｎｏｂａｃｉｌｌｕｓ）属、バクテロイデス（Ｂａｃｔｅｒｉｏｒｉｄｅｓ）属、タンネレラ（Ｔａｎｎｅｒｅｌｌａ）属、トレポネーマ（Ｔｒｅｐｏｎｅｍａ）属、カンピロバクター（Ｃａｍｐｙｌｏｂａｃｔａｒ）属、エイケネラ（Ｅｉｋｅｎｅｌｌａ）属、及び、カプノサイトファーガ（Ｃａｐｎｏｃｙｔｏｐｈａｇａ）属等が挙げられる。 In addition, the bacteria are preferably periodontal disease bacteria (the causative bacteria of periodontal disease). Examples of periodontal disease bacteria include Capnocytophaga, Prevotella, Eikenella, Fusobacterium, Parvimonas, Aggregatibacter, Aggregatibacter, and Aggregatibacter. The genus Actinomyces, the genus Actinobacillus, the genus Bacteriorides, the genus Tannerella, the genus Treponema, the genus Treponema, the genus Capnocytophaga, the genus Capnocytophaga, the genus Capnocytophaga, the genus Capnocytophaga. The genus (Capnocytophaga) and the like can be mentioned.
　より具体的には、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ　ｇｉｎｇｉｖａｌｉｓ）又はバクテロイデス・ジンジバリス（Ｂａｃｔｅｒｉｏｒｉｄｅｓ　ｇｉｎｇｉｖａｌｉｓ）、プレボテラ・インターメディア（Ｐｒｅｖｏｔｅｌｌａ　ｉｎｔｅｒｍｅｄｉａ）、ベイヨネラ・パルブーラ（Ｖｅｉｌｌｏｎｅｌｌａ　ｐａｒｖｕｌａ）、フソバクテリウム・ヌクレアタム（Ｆｕｓｏｂａｃｔｅｒｉｕｍ　ｎｕｃｌｅａｔｕｍ）、パルビモナス・ミクラ（Ｐａｒｖｉｍｏｎａｓ　ｍｉｃｒａ）、アグリゲイティバクター・アクチノミセテムコミタンス（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ　ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）又はアクチノバチルス・アクチノミセテムコミタンス（Ａｃｔｉｎｏｂａｃｉｌｌｕｓ　ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）、アクチノマイセス・ネスランディ（Ａｃｔｉｎｏｍｙｃｅｓ　ｎａｅｓｌｕｎｄｉｉ）、タンネレラ・フォーサイセンシス（Ｔａｎｎｅｒｅｌｌａ　ｆｏｒｓｙｔｈｅｎｓｉｓ）、トレポネーマ・デンティコラ（Ｔｒｅｐｏｎｅｍａ　ｄｅｎｔｉｃｏｌａ）、カンピロバクター・レクタス（Ｃａｍｐｙｌｏｂａｃｔａｒ　ｒｅｃｔｕｓ）、エイケネラ・コローデンス（Ｅｉｋｅｎｅｌｌａ　ｃｏｒｒｏｄｅｎｓ）、及び、カプノサイトファーガ・オクラセア（Ｃａｐｎｏｃｙｔｏｐｈａｇａ　ｏｃｈｒａｃｅａ）等が挙げられる。 More specifically, Porphyromonas gingivalis or Bacteriorides gingivalis, Prevotella intermedia, Prevotella intermedia, Eikenella parvula valula, vulura parbula, vayl・ Porphyromonas micra, Aggregatibacter actinomycetemcomitans or Actinovacils actinomycetemcomitens Sensis (Tannella forsythesis), Treponema denticola, Campylobacter rectus, Eikenella corodence (Eikenella corrodens, etc.), and Capnocera corrodens.
　なかでも、細菌としては、「レッドコンプレックス」と呼ばれる、ポルフィロモナス・ジンジバリス（Ｐｏｒｐｈｙｒｏｍｏｎａｓ　ｇｉｎｇｉｖａｌｉｓ）、トレポネーマ・デンティコラ（Ｔｒｅｐｏｎｅｍａ　ｄｅｎｔｉｃｏｌａ）、及び、アグリゲイティバクター・アクチノミセテムコミタンス（Ａｇｇｒｅｇａｔｉｂａｃｔｅｒ　ａｃｔｉｎｏｍｙｃｅｔｅｍｃｏｍｉｔａｎｓ）からなる群より選択される少なくとも１種の細菌が好ましい。 Among them, the bacteria are called "Red Complex", Porphyromonas gingivalis, Treponema denticola, and Aggregatibacter actinomycetemcommitans (Aggregatibacter actinometry). At least one bacterium selected from the group is preferred.
　懸濁液中の細菌と膜小胞とを分離する方法としては特に制限されず、公知の方法が使用できる。例えば、単一の細胞株の液体培養液であれば、膜小胞は培養上清に含まれているため、遠心分離によって、培養液から菌体を取り除き、これを第１試料とし、上清を第２試料とすればよい。細菌株を固体培養した場合には、コロニーを緩衝液中に懸濁して、同様の方法で分離できる。 The method for separating the bacteria and the membrane vesicles in the suspension is not particularly limited, and a known method can be used. For example, in the case of a liquid culture medium of a single cell line, since membrane vesicles are contained in the culture supernatant, cells are removed from the culture medium by centrifugation, and this is used as the first sample, and the supernatant is used. May be used as the second sample. When the bacterial strain is solid-cultured, the colonies can be suspended in buffer and separated in the same manner.
　分離した遠心上清をメンブランフィルター（例えば、孔径０．１～０．９μｍ）を用いてろ過すれば、遠心上清に残留する菌体を容易に分離することができる。
　遠心上清には、菌体以外にもタンパク質等の夾雑物質が含まれている場合があるので、例えば、１００～２００ｋＤａカットオフフィルタで濃縮した後、超遠心によって膜小胞を沈殿させて回収する方法も使用できる。 If the separated centrifugal supernatant is filtered using a membrane filter (for example, pore size 0.1 to 0.9 μm), the cells remaining in the centrifugal supernatant can be easily separated.
Since the centrifugation supernatant may contain contaminants such as proteins in addition to cells, for example, after concentrating with a 100-200 kDa cutoff filter, membrane vesicles are precipitated by ultracentrifugation and collected. You can also use the method of
　また、このようにして得られた沈殿物には、細菌由来の構造体（例えば、せん毛、及び、べん毛等）が含まれていることがあり、これらを除去するために、例えば、スクロース等を用いた密度勾配遠心法を用いて、更に精製する方法を用いてもよい。 In addition, the precipitate thus obtained may contain bacterial-derived structures (eg, sucrose, flagella, etc.), and in order to remove these, for example, A method of further purification may be used by using a density gradient centrifugation method using sucrose or the like.
　上記処理を含む試料の調製方法をより具体的に説明する。
　まず、所定の条件で所望の増殖相が達成されたら、培養液を容量が１～１００ｍＬ（例えば５０ｍＬ）の遠心管に入れ、３０００～９０００ｒｐｍ（例えば７０００ｒｐｍ）、１～２０℃（例えば４℃）で、１～３０分間（例えば、１０分間）遠心する。 A method for preparing a sample including the above treatment will be described more specifically.
First, when the desired growth phase is achieved under the given conditions, the culture is placed in a centrifuge tube with a volume of 1-100 mL (eg 50 mL) and 3000-9000 rpm (eg 7000 rpm) 1-20 ° C (eg 4 ° C). Then, centrifuge for 1 to 30 minutes (for example, 10 minutes).
　上清と沈殿物とに分離できたら沈殿物には菌体が含まれているため、これを分離し（第１試料とし）、上清を０．１～０．９μｍ（例えば、０．２２μｍ）のフィルターに通し、別の遠心管に収容し、１～１０℃（例えば、４℃）で保存する。 If the supernatant and the precipitate can be separated, the precipitate contains bacterial cells, so the cells are separated (as the first sample), and the supernatant is 0.1 to 0.9 μm (for example, 0.22 μm). ), Placed in another centrifuge tube, and stored at 1-10 ° C (eg, 4 ° C).
　次に、上清を超遠心チューブに加え、例えば、遠心力として１５０，０００～３００，０００×ｇ（例えば、２１０，０００×ｇ）で、１～１０℃（例えば、４℃）で１～３時間（例えば、２時間）超遠心する。超遠心後、上清を破棄し、沈殿した外膜小胞（ＯＭＶ）を緩衝液（例えば、ｐＨ７．４のＰＢＳバッファー）に再懸濁し、１～１０℃（例えば、４℃）で保存する。更に、この超遠心の工程を複数回繰り返してもよい。 Next, the supernatant is added to an ultracentrifugal tube, for example, with a centrifugal force of 150,000 to 300,000 × g (for example, 210,000 × g) and 1 to 10 ° C. (for example, 4 ° C.) from 1 to 1. Ultracentrifuge for 3 hours (eg 2 hours). After ultracentrifugation, discard the supernatant and resuspend the precipitated adventitial vesicles (OMV) in buffer (eg, pH 7.4 PBS buffer) and store at 1-10 ° C (eg, 4 ° C). .. Further, this ultracentrifugation step may be repeated a plurality of times.
　なお、第２試料の単位量あたりに含まれる膜小胞の数（濃度）を調整するために、得られた液を希釈してもよい。このようにすると、後述する単一粒子解析において、１個体ずつの膜小胞を分離取得しやすくなる。 The obtained liquid may be diluted in order to adjust the number (concentration) of the membrane vesicles contained in the unit amount of the second sample. By doing so, it becomes easy to separate and acquire the membrane vesicles of each individual in the single particle analysis described later.
　希釈の方法としては特に制限されないが、例えば、膜小胞の濃度を動的光散乱法等により液中の膜小胞の濃度を観察しながら、適当な濃度に希釈する方法が挙げられる。
　膜小胞の濃度の観察方法としては、粒子にレーザを照射し、その散乱光から各粒子のブラウン運動を追跡し（トラッキング法）、その拡散速度から、Ｓｔｏｋｅｓ－Ｅｉｎｓｔｅｉｎの式に基づき粒子の径と個数とを計算する方法が好ましい。 The method of dilution is not particularly limited, and examples thereof include a method of diluting the concentration of membrane vesicles to an appropriate concentration while observing the concentration of membrane vesicles in the liquid by a dynamic light scattering method or the like.
As a method of observing the concentration of membrane vesicles, the particles are irradiated with a laser, the Brownian motion of each particle is tracked from the scattered light (tracking method), and the diameter of the particles is determined from the diffusion rate based on the Stokes-Einstein equation. And the number are preferred.
＜第１塩基配列決定工程（ステップＳ１０２）＞
　第１試料には、１種又は複数種の細菌が含まれており、本工程は、これ（ら）に由来するゲノム配列である第１塩基配列を決定する工程である。
　第１塩基配列の決定方法としては、例えば、１６ＳリボソームＲＮＡ解析、メタゲノム解析、及び、単一細胞解析等が挙げられ、いずれも公知の技術が特に制限なく適用可能である。なかでも、個々の細菌のより正確なゲノム配列が得られやすい点で、単一細胞解析が好ましい。 <First base sequence determination step (step S102)>
The first sample contains one or more kinds of bacteria, and this step is a step of determining a first base sequence which is a genomic sequence derived from these (or others).
Examples of the method for determining the first base sequence include 16S ribosomal RNA analysis, metagenomic analysis, single cell analysis, and the like, and known techniques can be applied without particular limitation. Among them, single cell analysis is preferable because it is easy to obtain a more accurate genomic sequence of each bacterium.
　本工程における第１塩基配列の決定を単一細胞解析で行う場合、その方法としては特に制限されないが、以下の各工程を含む単一細胞解析が好ましい。 When the determination of the first base sequence in this step is performed by single cell analysis, the method is not particularly limited, but single cell analysis including each of the following steps is preferable.
・第１試料に含まれる細菌の１個体ずつを液滴中に封入すること（封入工程）
・液滴をゲル化してゲルカプセルを生成すること（ゲル化工程）
・ゲルカプセルを溶解試薬と接触させて、細菌を溶解させ、ポリヌクレオチドを溶出させ、ゲルカプセル内に保持すること（溶解工程）
・ゲルカプセル内の夾雑物質を除去すること（精製工程）
・ポリヌクレオチドを増幅試薬に接触させてポリヌクレオチドをゲルカプセル内で増幅させること（増幅工程）
・増幅したポリヌクレオチドから、第１塩基配列を決定すること（シークエンス工程） -Encapsulating each individual bacterium contained in the first sample in a droplet (encapsulation step)
・ Gelling droplets to form gel capsules (gelling process)
-Contacting the gel capsule with a lysis reagent to lyse the bacteria, elute the polynucleotide, and retain it in the gel capsule (dissolution step).
-Removing contaminants in gel capsules (purification process)
-Amplifying the polynucleotide in a gel capsule by contacting the polynucleotide with an amplification reagent (amplification step)
-Determining the first base sequence from the amplified polynucleotide (sequence step)
　図２は、上記各工程を含む単一細胞解析のフローである。図１のステップＳ１０２で表される「第１塩基配列決定工程」は、図２の単一細胞解析を含むことが好ましい。 FIG. 2 is a flow of single cell analysis including each of the above steps. The "first nucleotide sequence determination step" represented by step S102 in FIG. 1 preferably includes the single cell analysis of FIG.
・封入工程（ステップＳ２０１）
　ステップＳ２０１は、細菌の１個体ずつを液滴中に封入する工程である。封入工程で用いられる第１試料は、細菌を含む。試料に含まれる細菌は、１種でも２種以上でもよい。第１試料の細菌のうち少なくともいずれかは、懸濁液に含まれる膜小胞を産生し、菌体外に放出したものである。 Encapsulation step (step S201)
Step S201 is a step of encapsulating each individual bacterium in the droplet. The first sample used in the encapsulation step contains bacteria. The bacterium contained in the sample may be one kind or two or more kinds. At least one of the bacteria in the first sample produced membrane vesicles contained in the suspension and released them out of the cells.
　第１試料を用いて、第１試料に含まれる細菌の１個体ずつを液滴中に封入する方法としては特に制限されず、例えば、特許文献１等に記載された公知の方法が使用できる。 The method of encapsulating each individual bacterium contained in the first sample in the droplet using the first sample is not particularly limited, and for example, a known method described in Patent Document 1 or the like can be used.
　液滴の作製は、例えば、マイクロ流路を用いて行うことができる。上述した懸濁液をマイクロ流路中に流動させ、懸濁液の流れをせん断することにより、１個体ずつの細菌を封入した液滴を作製できる。せん断は、一定間隔で行うことができる。懸濁液のせん断の方法としては、例えば、オイルを用いればよい。オイルとしては、例えば、鉱物油、植物油、シリコーンオイル、及び、フッ素化オイル等が挙げられる。懸濁液中の細菌の量（濃度）、流路中の流速、せん断の間隔を調整し、当業者は、液滴あたり１個体の個体が封入されるように液滴作製を行うことが可能である。
　液滴の直径は、１～２５０μｍが好ましく、１０～２００μｍがより好ましい。 The droplets can be produced, for example, using a microchannel. By flowing the suspension described above into a microchannel and shearing the flow of the suspension, droplets containing individual bacteria can be produced. Shearing can be done at regular intervals. As a method of shearing the suspension, for example, oil may be used. Examples of the oil include mineral oil, vegetable oil, silicone oil, fluorinated oil and the like. By adjusting the amount (concentration) of bacteria in the suspension, the flow velocity in the flow path, and the shear interval, those skilled in the art can prepare droplets so that one individual is enclosed in each droplet. Is.
The diameter of the droplet is preferably 1 to 250 μm, more preferably 10 to 200 μm.
　図３は、本工程で使用できるマイクロ流路の模式的な断面図である。マイクロ流路３０１は、液滴を作製し、液滴中に細菌の１個体ずつを封入することができるデバイスである。
　マイクロ流路３０１は、２本の流路が略直交して形成された十字型の流路から構成されている。図面の、上から下方向に延びる流路には、細菌３０３を含む第１試料３０２が図面上から下に流れており、これと略直交する方向の流路には、図面の左右から中央に向けてオイル３０４が流れている。 FIG. 3 is a schematic cross-sectional view of a microchannel that can be used in this step. The microchannel 301 is a device capable of producing a droplet and enclosing each individual bacterium in the droplet.
The micro flow path 301 is composed of a cross-shaped flow path formed by two flow paths that are substantially orthogonal to each other. The first sample 302 containing the bacterium 303 flows from the top to the bottom of the drawing in the flow path extending from the top to the bottom in the drawing, and the flow path in the direction substantially orthogonal to this flows from the left and right to the center of the drawing. Oil 304 is flowing toward it.
　この流路の直径としては特に制限されず、目的に応じて適宜選択されればよいが、一般に、１０～６０μｍが好ましい。
　細菌３０３を含む第１試料３０２は、図面の左右から中央に流れるオイル３０４により剪断を受け、細菌３０３を含む液滴３０５となる。
　第１試料３０２中の細菌３０３の含有量を調整することで、液滴３０５に細菌３０３の１個体が封入されるよう、調整することができ、この場合、細菌３０３が封入されていない液滴３０６も生成される場合があってもよい。 The diameter of this flow path is not particularly limited and may be appropriately selected depending on the intended purpose, but is generally preferably 10 to 60 μm.
The first sample 302 containing the bacterium 303 is sheared by the oil 304 flowing from the left and right to the center of the drawing, and becomes a droplet 305 containing the bacterium 303.
By adjusting the content of the bacterium 303 in the first sample 302, it is possible to adjust so that one individual of the bacterium 303 is encapsulated in the droplet 305. In this case, the droplet in which the bacterium 303 is not encapsulated. 306 may also be produced.
・ゲル化工程（ステップＳ２０２）
　ステップＳ２０２は、液滴をゲル化してゲルカプセルを生成する工程である。ゲル化方法としては特に制限されない。液滴のゲル化は、液滴にゲルカプセルの材料が含まれるように構成し、作製した液滴を冷却することによって行うことができる。あるいは、液滴に対して光等の刺激を与えることによってゲル化を行うこともできる。液滴にゲルカプセルの材料が含まれるようにするには、例えば、第１試料にゲルカプセルの材料を含めておくことによって行うことができる。 -Gelification step (step S202)
Step S202 is a step of gelling the droplet to form a gel capsule. The gelation method is not particularly limited. The 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 the first sample.
　ゲルカプセルの直径としては、１～２５０μｍが好ましく、１０～２００μｍがより好ましい。ゲルカプセルの直径は、作製する液滴と同じであってもよいが、ゲル化に際して直径が変化してもよい。ゲル化の温度としては特に制限されないが、４～１０℃が好ましい。 The diameter of the gel capsule is preferably 1 to 250 μm, more preferably 10 to 200 μm. The diameter of the gel capsule may be the same as that of the droplet to be produced, but the diameter may change during gelation. The gelation temperature is not particularly limited, but is preferably 4 to 10 ° C.
　ゲルカプセルの材料は、アガロース、アクリルアミド、光硬化性樹脂（例えば、ＰＥＧ-ＤＡ）、ＰＥＧ、ゼラチン、アルギン酸ナトリウム、マトリゲル、及び、コラーゲン等を含んでもよい。 The material of the gel capsule may contain agarose, acrylamide, a photocurable resin (for example, PEG-DA), PEG, gelatin, sodium alginate, matrigel, collagen and the like.
　ゲルカプセルは、ヒドロゲルカプセルであってよい。「ヒドロゲル」とは、高分子物質またはコロイド粒子の網目構造によって保持されている溶媒あるいは分散媒が水であるものを意味する。 The gel capsule may be a hydrogel capsule. By "hydrogel" is meant that the solvent or dispersion medium retained by the network structure of the polymeric or colloidal particles is water.
・溶解工程（ステップＳ２０３）
　ステップＳ２０３は、ゲルカプセルを溶解試薬と接触させて、細菌を溶解させ、ポリヌクレオチドを溶出させ、ゲルカプセル内に保持する工程である。細菌を溶解することで、細菌中のポリヌクレオチドがゲルカプセル内に溶出し、ポリヌクレオチドに結合する物質が除去された状態でゲルカプセル内に保持され得る。溶解試薬としては、酵素、界面活性剤、その他変性剤、還元剤、及び、ｐＨ調製剤、並びに、これらの組合せ等が使用できる。 -Dissolution step (step S203)
Step S203 is a step of contacting the gel capsule with a lysing reagent to lyse the bacteria, elute the polynucleotide, and retain it in the gel capsule. By dissolving the bacterium, the polynucleotide in the bacterium can be eluted into the gel capsule and retained in the gel capsule with the substance binding to the polynucleotide removed. As the dissolving reagent, enzymes, surfactants, other denaturing agents, reducing agents, pH adjusting agents, combinations thereof and the like can be used.
　溶解試薬は、リゾチーム、ラビアーゼ、ヤタラーゼ、アクロモペプチダーゼ、プロテアーゼ、ヌクレアーゼ、ザイモリアーゼ、キチナーゼ、リソスタフィン、ムタノライシン、ドデシル硫酸ナトリウム、ラウリル硫酸ナトリウム、水酸化カリウム、水酸化ナトリウム、フェノール、クロロホルム、グアニジン塩酸塩、尿素、２－メルカプトエタノール、ジチオトレイトール、「ＴＣＥＰ－ＨＣｌ」、コール酸ナトリウム、デオキシコール酸ナトリウム、「Ｔｒｉｔｏｎ　Ｘ－１００」、「Ｔｒｉｔｏｎ　Ｘ－１１４」、「ＮＰ－４０」、「Ｂｒｉｊ－３５」、「Ｂｒｉｊ－５８」、「Ｔｗｅｅｎ　２０」、「Ｔｗｅｅｎ　８０」、オクチルグルコシド、オクチルチオグルコシド、「ＣＨＡＰＳ」、「ＣＨＡＰＳＯ」、ドデシル－β－Ｄ－マルトシド、「Ｎｏｎｉｄｅｔ　Ｐ－４０」、及び、「Ｚｗｉｔｔｅｒｇｅｎｔ　３－１２」等が挙げられる。
　また、溶解試薬は、リゾチーム、アクロモペプチダーゼ、プロテアーゼ、ドデシル硫酸ナトリウム、及び、水酸化カリウム等を含むことが好ましい。 Dissolving reagents include lysoteam, 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, dithiotreitol, "TCEP-HCl", sodium dodecyl, sodium deoxycholate, "Triton X-100", "Triton X-114", "NP-40", "Brij-35" , "Brij-58", "Tween 20", "Tween 80", octyl glucoside, octyl thioglucoside, "CHAPS", "CHASPO", dodecyl-β-D-maltoside, "Nonidet P-40", and "Zwittergent 3-12" and the like can be mentioned.
The dissolving reagent preferably contains lysozyme, achromopeptidase, protease, sodium dodecyl sulfate, potassium hydroxide and the like.
　ゲルカプセルを溶解試薬と接触させる方法は特に制限されないが、例えば、容器（例えば、マイクロチューブ、及び、マイクロプレートの各ウェル）に保持されたゲルカプセルに対して溶解試薬を自動マイクロシリンジ等で吐出すればよい。自動マイクロシリンジは、典型的には、溶解試薬を流通する流路、及び、液体流通のためのポンプ、弁等から構成されていてよい。 The method of contacting the gel capsule with the dissolving reagent is not particularly limited, and for example, the dissolving reagent is discharged to the gel capsule held in the container (for example, the wells of the microtube and the microplate) with an automatic microsyringer or the like. do it. The automatic microsyringe may typically consist of a flow path through which the dissolving reagent flows, a pump, a valve, or the like for liquid flow.
・精製工程（ステップＳ２０４）
　ステップＳ２０４は、ゲルカプセル内の夾雑物質を除去する工程である。本方法では、細菌の１個体を封入したゲルカプセルを用いるため、精製した遺伝物質（例えば、ＤＮＡ）をゲルカプセル内に保持することができ、また、外部からの分子の夾雑の可能性を排除することができる。 -Purification step (step S204)
Step S204 is a step of removing contaminants in the gel capsule. In this method, since a gel capsule containing one individual bacterium is used, the purified genetic substance (for example, DNA) can be retained in the gel capsule, and the possibility of contamination of molecules from the outside is eliminated. can do.
　また、操作面でも非常に簡単な操作で、１単位ごとの大量の細菌を並列処理することができる。ゲル化した液滴を含む試験管を遠心し、上清を除去し、洗浄液に置換するというステップを行うことができる。あるいは、ゲル化した液滴をフィルターでろ過し、上清を除去したのち、洗浄液を通液させ、最後にゲルカプセルを回収するというステップでも行うことができる。ゲルカプセルを用いることにより、遺伝物質を保持したまま、残留試薬を希薄化することができる。このステップは繰り返すことも可能である。阻害が出ないレベルにまで試薬を希薄化することで、下流の操作、例えば、増幅反応をスムーズに行うことができる。 In addition, it is possible to process a large amount of bacteria in parallel for each unit with a very simple operation. The step of centrifuging the test tube containing the gelled droplets, removing the supernatant and replacing it with a cleaning solution can be performed. Alternatively, it can be performed by filtering the gelled droplets with a filter, removing the supernatant, passing the washing liquid through the filter, and finally collecting the gel capsules. By using a gel capsule, the residual reagent can be diluted while retaining the genetic material. This step can be repeated. By diluting the reagent to a level that does not cause inhibition, downstream operations such as amplification reactions can be smoothly performed.
　溶解試薬が下流の反応の前に十分に除去されると、ＤＮＡ増幅等の反応が阻害されにくい点で好ましい。ゲルカプセルを用いる場合、ゲルカプセルによって解析又は増幅の対象となる遺伝物質が保持されるため、遺伝物質が少量である細胞１つあたりの解析においても溶解試薬の除去を行うことができ、そのため、強力な溶解試薬または溶解試薬の組合せを用いることが可能である。
　そして、強力な溶解試薬または溶解試薬の組合せを用いることは、より確実な核酸の増幅、及び、塩基配列の解析を可能にし得る。 When the lysing reagent is sufficiently removed before the downstream reaction, it is preferable that the reaction such as DNA amplification is not easily inhibited. When using gel capsules, the gel capsules hold the genetic material to be analyzed or amplified, so that the lysing reagent can be removed even in the analysis per cell with a small amount of genetic material. It is possible to use a strong lysing reagent or a combination of lysing reagents.
And, using a strong lysing reagent or a combination of lysing reagents may enable more reliable amplification of nucleic acid and analysis of base sequence.
・増幅工程（ステップＳ２０５）
　ステップＳ２０５は、ポリヌクレオチドを増幅試薬に接触させてポリヌクレオチドをゲルカプセル内で増幅させる工程である。増幅試薬に浸漬した後、必要に応じて、ゲルカプセルの温度を調整してもよい。 Amplification step (step S205)
Step S205 is a step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in a gel capsule. After immersing in the amplification reagent, the temperature of the gel capsule may be adjusted if necessary.
　加熱は、ゲル（例えば、アガロースゲル）が溶解しにくい観点で、６０℃以下が好ましい。この範囲内での加熱は、ＤＮＡの増幅をより促進する点で好ましい。
　増幅に用いる酵素としては、例えば、ｐｈｉ２９ポリメラーゼ、Ｂｓｔポリメラーゼ、Ａａｃポリメラーゼ、及び、リコンビナーゼポリメラーゼが挙げられる。
　本方法では、ゲル内の全ＤＮＡの増幅を行うために、ランダムプライマーを用いることが好ましい。 The heating is preferably 60 ° C. or lower from the viewpoint that the gel (for example, agarose gel) is difficult to dissolve. Heating within this range is preferable in that it further promotes the amplification of DNA.
Examples of the enzyme used for amplification include fi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase.
In this method, it is preferable to use a random primer in order to amplify the total DNA in the gel.
・シークエンス工程（ステップＳ２０６）
　ステップＳ２０６は、増幅したポリヌクレオチドから、第１塩基配列を決定する工程である。増幅したポリヌクレオチドからその塩基配列を決定する方法としては、ライブラリー調製、シークエンス、及び、アセンブリ等のいずれも市販の試薬キット、装置、及び、アプリケーションソフトウェア等を特に制限なく使用できる。 -Sequencing step (step S206)
Step S206 is a step of determining the first base sequence from the amplified polynucleotide. As a method for determining the base sequence from the amplified polynucleotide, commercially available reagent kits, devices, application software and the like can be used without particular limitation for any of library preparation, sequence, assembly and the like.
＜第２塩基配列決定工程（ステップＳ１０３）＞
　図１に戻り、第２塩基配列決定工程（ステップＳ１０３）について説明する。本工程は、ステップＳ１０１で調製した第２試料を用いて、第２塩基配列を決定する工程である。 <Second base sequence determination step (step S103)>
Returning to FIG. 1, the second base sequence determination step (step S103) will be described. This step is a step of determining the second base sequence using the second sample prepared in step S101.
　本工程で使用する試料は、試料調製工程（ステップＳ１０１）で調製された第２試料であればよいが、上記試料を前処理して得られる処理済みの第２試料であってもよい。 The sample used in this step may be the second sample prepared in the sample preparation step (step S101), but may be the treated second sample obtained by pretreating the above sample.
　第２塩基配列の決定方法としては特に制限されず、メタゲノム解析でも、単一粒子解析でもよいが、必要な膜小胞の量がより少なくて済む観点では、単一粒子解析が好ましい。単一粒子解析の方法としては特に制限されず、ステップＳ１０２の第１塩基配列決定工程において第１試料を単一細胞解析する方法としてすでに説明したのと同様の方法が使用できる。 The method for determining the second base sequence is not particularly limited, and may be metagenomic analysis or single particle analysis, but single particle analysis is preferable from the viewpoint that the amount of membrane vesicles required is smaller. The method for single particle analysis is not particularly limited, and the same method as described above can be used as a method for single cell analysis of the first sample in the first base sequence determination step of step S102.
　ここで、本発明者は、膜小胞の表面には、核酸成分が付着している場合が多いことを知見している。この核酸成分は、例えば菌体が破裂した断片や共存する細菌由来等様々な由来を持つと推測される。 Here, the present inventor has found that nucleic acid components are often attached to the surface of membrane vesicles. It is presumed that this nucleic acid component has various origins such as a fragment in which a bacterial cell has ruptured and a fragment derived from a coexisting bacterium.
　本発明者は、膜小胞の表面に付着している核酸成分は、膜小胞を産生した細菌（以下、「宿主」ともいう。）に由来する可能性が高いことを知見している。
　例えば、全ゲノムショットガン方式等、一般のメタゲノム解析では、試料に含まれる核酸成分を網羅的に分析することになるが、その場合、網羅的に分析される核酸成分のうち、特定の膜小胞を産生した細菌に由来する配列を得られる確率は非常に低い。 The present inventor has found that the nucleic acid component attached to the surface of the membrane vesicle is likely to be derived from the bacterium that produced the membrane vesicle (hereinafter, also referred to as “host”).
For example, in general metagenomic analysis such as the whole genome shotgun method, the nucleic acid components contained in the sample are comprehensively analyzed. In that case, among the nucleic acid components to be comprehensively analyzed, a specific membrane small size is used. The probability of obtaining a sequence derived from the vesicle-producing bacterium is very low.
　一方で、膜小胞の表面に付着している核酸成分は、例えば、その膜小胞の産生過程で付着した成分等であったりして、宿主に由来する可能性が高い。
　すなわち、第１試料を網羅解析して得られる断片から、特定の膜小胞の宿主に由来する断片（ポリヌクレオチド）を得られる可能性は低いが、第２試料に含まれる核酸成分は、宿主に由来する断片である可能性がより高いと言える。 On the other hand, the nucleic acid component attached to the surface of the membrane vesicle is, for example, a component attached in the process of producing the membrane vesicle, and is highly likely to be derived from the host.
That is, it is unlikely that a fragment (polynucleotide) derived from a host of a specific membrane vesicle can be obtained from a fragment obtained by comprehensively analyzing the first sample, but the nucleic acid component contained in the second sample is the host. It can be said that it is more likely that it is a fragment derived from.
　このような第２試料を単一細胞解析すると、膜小胞に由来する配列と、宿主に由来する可能性の高い配列の両方を含む第２塩基配列が得られる。この配列を用いれば、膜小胞の配列だけを用いた場合によりもより長い塩基長で、第１塩基配列を補完することができる可能性が高い。 Single cell analysis of such a second sample yields a second base sequence containing both a sequence derived from membrane vesicles and a sequence likely to be derived from the host. With this sequence, it is likely that the first base sequence can be complemented with a longer base length than when only the membrane vesicle sequence is used.
　第２試料を単一粒子解析する方法としては特に制限されないが、より高品質な第２塩基配列が得られる点で、図２で説明した単一細胞解析と同様の方法、すなわち、下記工程をこの順に含む単一粒子解析が好ましい。
・第２試料を用い、膜小胞の１個体ずつを液滴中に封入すること
・液滴をゲル化して、ゲルカプセルを生成すること
・ゲルカプセルを溶解試薬と接触させて、膜小胞を溶解させ、ポリヌクレオチドを溶出させ、ゲルカプセル内に保持すること
・ゲルカプセル内の夾雑物質を除去すること
・ポリヌクレオチドを増幅試薬と接触させて、ゲルカプセル内で増幅させること
・増幅したポリヌクレオチドから、第２塩基配列を決定すること The method for single particle analysis of the second sample is not particularly limited, but the same method as the single cell analysis described with reference to FIG. 2, that is, the following steps can be performed in that a higher quality second base sequence can be obtained. Single particle analysis including this order is preferable.
-Encapsulating each individual membrane vesicle in the droplet using the second sample-Gelizing the droplet to form a gel capsule-Making the gel capsule in contact with a lysis reagent to contact the membrane vesicle Dissolve and elute the polynucleotide and retain it in the gel capsule ・ Remove contaminants in the gel capsule ・ Contact the polynucleotide with the amplification reagent to amplify it in the gel capsule ・ Amplified poly Determining the second base sequence from the nucleotides
＜補完工程（ステップＳ１０４）＞
　ステップＳ１０４は、第２塩基配列を用いて、第１塩基配列を補完する工程である。補完とは、典型的には、第２塩基配列を断片として用いて、上記第１塩基配列のカバー率を向上させることが挙げられる。 <Complementary step (step S104)>
Step S104 is a step of complementing the first base sequence by using the second base sequence. Complementation typically includes using the second base sequence as a fragment to improve the coverage of the first base sequence.
　第２塩基配列は、膜小胞に由来する塩基配列と、膜小胞の表面に付着していた核酸成分に由来する塩基配列とを含んでいる。
　第２塩基配列の一部が第１塩基配列にマップする場合、その第２塩基配列は、第１塩基配列の一部と考えられる。すなわち、第２塩基配列の膜小胞は、第１塩基配列の細菌により産生されたと考えられる。 The second base sequence includes a base sequence derived from the membrane vesicle and a base sequence derived from the nucleic acid component attached to the surface of the membrane vesicle.
When a part of the second base sequence maps to the first base sequence, the second base sequence is considered to be a part of the first base sequence. That is, it is considered that the membrane vesicles having the second base sequence were produced by the bacteria having the first base sequence.
　特に、第１塩基配列を図２に示すような単一細胞解析によって得た場合、その工程中には、ゲルカプセル内でポリヌクレオチドを増幅する工程が含まれている。従って、使用するプライマー同士が結合したり、キメラ配列が生成したりして本来増幅されるべき細菌に由来するポリヌクレオチド以外のヌクレオチドが増幅される場合がある。また、何らかの原因で増幅が不十分となり、結果として、得られるポリヌクレオチドによるカバー率が十分高まらない（例えば、４０～８０％程度になる）場合がある。このような場合には、本解析方法はより有用である。 In particular, when the first base sequence is obtained by single cell analysis as shown in FIG. 2, the step includes a step of amplifying the polynucleotide in a gel capsule. Therefore, nucleotides other than the polynucleotide derived from the bacterium that should be originally amplified may be amplified due to the binding of the primers to be used or the generation of a chimeric sequence. In addition, amplification may be insufficient for some reason, and as a result, the coverage by the obtained polynucleotide may not be sufficiently high (for example, about 40 to 80%). In such cases, this analysis method is more useful.
　また、補完としては、例えば、第１塩基配列の取得をメタゲノム解析で実施した場合に得られるドラフトゲノムの品質を向上するために第２塩基配列を使用する方法も挙げられる。 Further, as a complement, for example, a method of using the second base sequence in order to improve the quality of the draft genome obtained when the acquisition of the first base sequence is carried out by metagenomic analysis can be mentioned.
　典型的な全ゲノムショットガン方式のメタゲノム解析では、全ゲノムを所定の塩基長を有する断片（リード）に分割し、それぞれの断片をシークエンスして、それらを組み合わせること（アセンブリ、ビニング）で行われる。なお、メタゲノム解析を行う方法としては特に制限されず、公知の方法が使用できる。例えば、特開２００５－２１８４２１号公報等に記載の全ゲノムショットガン方式による解析方法を使用でき、その方法は当業者にとって公知である。 In a typical whole-genome shotgun metagenomic analysis, the whole genome is divided into fragments (reads) having a predetermined base length, each fragment is sequenced, and they are combined (assembly, binning). .. The method for performing metagenomic analysis is not particularly limited, and a known method can be used. For example, an analysis method based on the whole genome shotgun method described in JP-A-2005-218421 can be used, and the method is known to those skilled in the art.
　本ゲノム解析方法における「補完」には上記断片の一つ（特定断片）として、第２塩基配列を用いることが含まれる。第２塩基配列は、膜小胞から得られた塩基配列を含み、その膜小胞を産生した細菌のゲノム配列の一部である。特に、第２塩基配列が単一細胞解析により得られたものである場合、特定断片は、個々の膜小胞を分離し、その塩基配列を解析したものであるため、典型的には、メタゲノム解析で用いられる断片よりも塩基長がより長く、更に配列もより正確である。 "Complementation" in this genome analysis method includes using a second base sequence as one of the above fragments (specific fragment). The second base sequence contains a base sequence obtained from a membrane vesicle and is a part of the genomic sequence of the bacterium that produced the membrane vesicle. In particular, when the second base sequence is obtained by single cell analysis, the specific fragment is obtained by separating individual membrane vesicles and analyzing the base sequence, and thus is typically a metagenome. It has a longer base length and a more accurate sequence than the fragments used in the analysis.
　ここで、特定断片を全ゲノムの断片として用いる、とは、例えば、第２塩基配列を、全ゲノムショットガン方式によるメタゲノム解析における断片の一つとして用いることが挙げられる。特定断片は、生成した単一の膜小胞に由来する配列であり、より正確性の高い配列であるため、得られる全ゲノム配列がより品質の高いものになりやすい。
　また、特定断片は、典型的には他の断片よりも塩基長が長いため、配列カバー率をより高めることが多い。 Here, using the specific fragment as a fragment of the whole genome means, for example, using the second base sequence as one of the fragments in the metagenomic analysis by the whole genome shotgun method. Since the specific fragment is a sequence derived from a single membrane vesicle generated and is a more accurate sequence, the obtained whole genome sequence tends to be of higher quality.
In addition, the specific fragment typically has a longer base length than the other fragments, and thus often has a higher sequence coverage.
　また、特定断片を全ゲノムの断片として用いる方法は、上記以外の方法であってもよい。例えば、特定断片を全ゲノム配列にマップすることであってもよい。ここで、全ゲノム配列に「マップする」とは、特定断片が、全ゲノム配列の一部の領域と一致することを意味する。一致とは、配列の９０％以上が同一であることを意味し、９５％以上が好ましく、９９％以上がより好ましく、完全に同一であることが更に好ましい。 Further, the method of using the specific fragment as a fragment of the whole genome may be a method other than the above. For example, the specific fragment may be mapped to the entire genome sequence. Here, "mapping" to the whole genome sequence means that the specific fragment coincides with a part of the region of the whole genome sequence. Matching means that 90% or more of the sequences are identical, preferably 95% or more, more preferably 99% or more, and even more preferably completely identical.
　特定断片を全ゲノム配列にマップすることにより、全ゲノム配列の品質をチェックすることができる。すなわち、特定断片を全ゲノムの断片として用いることは、全ゲノム配列にマップされる場合、その領域の全ゲノム配列が正確であると判断するための情報を提供することであってもよい。 By mapping a specific fragment to the whole genome sequence, the quality of the whole genome sequence can be checked. That is, using a specific fragment as a fragment of the whole genome may provide information for determining that the whole genome sequence of the region is accurate when mapped to the whole genome sequence.
＜本発明の他の実施形態＞
　本発明の他の実施形態に係るゲノム解析方法は、以下の工程を含むゲノム解析方法である。
・細菌と膜小胞とを含む懸濁液から、細菌を含む第１試料と、膜小胞を含む第２試料とを調製する（ステップＳ４０１、試料調製工程）。
・第１試料を用いて、第１塩基配列を決定する（ステップＳ４０２、第１塩基配列決定工程）。
・第２試料を核酸分解酵素で処理して、処理済み第２試料を調製する（ステップＳ４０３、処理工程）。
・処理済み第２試料を用いて、第２塩基配列を決定する（ステップＳ４０４、第２塩基配列決定工程）。
・第２塩基配列を用いて、第１塩基配列を補完する（ステップＳ４０５、補完工程）。 <Other Embodiments of the present invention>
The genome analysis method according to another embodiment of the present invention is a genome analysis method including the following steps.
-A first sample containing bacteria and a second sample containing membrane vesicles are prepared from a suspension containing bacteria and membrane vesicles (step S401, sample preparation step).
-The first base sequence is determined using the first sample (step S402, first base sequence determination step).
-The second sample is treated with a nucleolytic enzyme to prepare a treated second sample (step S403, treatment step).
-The second base sequence is determined using the treated second sample (step S404, second base sequence determination step).
-The first base sequence is complemented using the second base sequence (step S405, complementing step).
　図４は、本実施形態に係るゲノム解析方法のフローである。本ゲノム解析方法は、処理工程を有すること以外は、図１に示されたゲノム解析方法と同様である。すなわち、ステップＳ４０１は、ステップＳ１０１と同様であり、ステップＳ４０２は、ステップＳ１０２と同様であり、ステップＳ４０４は、ステップＳ１０３と同様であり、ステップＳ４０５は、ステップＳ１０４と同様であり、公的形態も同様である。これらの「同様である部分」の説明は省略し、図１に示されたゲノム解析方法と異なる部分を主に説明する。 FIG. 4 is a flow of the genome analysis method according to this embodiment. This genome analysis method is the same as the genome analysis method shown in FIG. 1, except that it has a processing step. That is, step S401 is the same as step S101, step S402 is the same as step S102, step S404 is the same as step S103, step S405 is the same as step S104, and the public form is also The same is true. The description of these "similar parts" will be omitted, and the parts different from the genome analysis method shown in FIG. 1 will be mainly described.
　本ゲノム解析方法は、第２試料を核酸分解酵素で処理して、処理済み第２試料を調製する工程（処理工程、ステップＳ４０３）を有する。 This genome analysis method includes a step (treatment step, step S403) of treating the second sample with a nucleolytic enzyme to prepare a treated second sample.
　すでに説明したとおり、第２試料に含まれる膜小胞には、その宿主に由来する可能性が高い核酸成分が付着している。本実施形態に係るゲノム解析方法では、その核酸成分を分解除去する点に特徴の一つがある。
　本実施形態に係るゲノム解析方法では、第２塩基配列が、確実に膜小胞に由来する配列であることを保証することができるため、第１塩基配列の補完がより容易になる。 As described above, the membrane vesicles contained in the second sample have a nucleic acid component that is likely to be derived from the host attached to the membrane vesicles. One of the features of the genome analysis method according to the present embodiment is that the nucleic acid component is decomposed and removed.
In the genome analysis method according to the present embodiment, it can be ensured that the second base sequence is a sequence derived from a membrane vesicle, so that the complementation of the first base sequence becomes easier.
　処理工程で使用する核酸分解酵素としては特に制限されず、デオキシリボヌクレアーゼ等が使用できる。具体的には、第２試料の１０μＬを１０倍希釈して、そこへ２μＬのＤＮａｓｅ（２０００Ｕ）を添加する方法等が挙げられる。これにより処理済み第２試料が得られる。 The nucleic acid-degrading enzyme used in the treatment step is not particularly limited, and deoxyribonuclease and the like can be used. Specific examples thereof include a method in which 10 μL of the second sample is diluted 10-fold and 2 μL of DNase (2000U) is added thereto. This gives a treated second sample.
　ステップＳ４０４は、処理済み第２試料を用いて、第２塩基配列を決定する工程である。本工程で使用する配列決定方法は、使用する試料の量がより少量である点で、すでに説明した単一粒子解析が好ましい。
　なお、本工程は、上記に加えて更に、未処理の第２試料を用いて、単一粒子解析で第２塩基配列を決定することを含むことが好ましい。 Step S404 is a step of determining the second base sequence using the treated second sample. As the sequencing method used in this step, the single particle analysis described above is preferable in that the amount of the sample used is smaller.
In addition to the above, this step preferably further comprises determining the second base sequence by single particle analysis using the untreated second sample.
　これにより、処理済み第２試料から、個々の膜小胞の塩基配列が得られて、未処理の第２試料からは、個々の膜小胞の塩基配列に加えて、膜小胞の表面に付着していた核酸成分の塩基配列が得られる。 As a result, the base sequence of each membrane vesicle is obtained from the treated second sample, and from the untreated second sample, in addition to the base sequence of each membrane vesicle, the surface of the membrane vesicle is obtained. The base sequence of the attached nucleic acid component can be obtained.
　すなわち、第２試料から得られる塩基配列と、処理済み第２試料の塩基配列との差分には、膜小胞の表面に付着していた核酸成分に由来する塩基配列が含まれる可能性が高い。すでに説明したとおり、上記「差分」は、宿主に由来する可能性が高い。本工程を有することで、得られる第２塩基配列による第１塩基配列のカバー範囲がより広くなる可能性がある。また、「差分」を利用すれば、得られた配列が膜小胞に由来するのか、表面に付着していた核酸成分に由来するのかを判断できる場合があり、より効率的にゲノム解析が可能になる。 That is, it is highly possible that the difference between the base sequence obtained from the second sample and the base sequence of the treated second sample includes the base sequence derived from the nucleic acid component attached to the surface of the membrane vesicle. .. As described above, the above "difference" is likely to be derived from the host. By having this step, there is a possibility that the coverage range of the first base sequence by the obtained second base sequence becomes wider. In addition, by using the "difference", it may be possible to determine whether the obtained sequence is derived from membrane vesicles or nucleic acid components attached to the surface, enabling more efficient genome analysis. become.
　なお、上記では、第２試料の解析方法として単一細胞解析を用いる方法を主に記載したが、本発明の実施形態に係るゲノム解析方法としては上記に制限されず、他の方法によって第２試料の塩基配列を解析してもよい。そのような方法としては例えば、メタゲノム解析が使用できる。 In the above, the method of using single cell analysis as the analysis method of the second sample is mainly described, but the genome analysis method according to the embodiment of the present invention is not limited to the above, and the second method may be used by another method. The base sequence of the sample may be analyzed. For example, metagenomic analysis can be used as such a method.
　以下、本発明を実施例により説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited thereto.
[実施例：膜小胞由来ＤＮＡを用いた希少微生物種のゲノム再構築]
　唾液採取キットを用いて、健常者（Ｈｅａｌｔｈｙ）、及び、歯周病患者（Ｐａｔｉｅｎｔｓ）の唾液を採取した。採取した唾液から、膜小胞と細菌とを分離して、それぞれ、膜小胞サンプル（ＭＶｓ）と細菌細胞のサンプル（Ｂａｃｔｅｒｉａ）とした。 [Example: Genome reconstruction of a rare microbial species using membrane vesicle-derived DNA]
Using the saliva collection kit, saliva of healthy subjects and patients with periodontal disease was collected. Membrane vesicles and bacteria were separated from the collected saliva and used as membrane vesicle samples (MVs) and bacterial cell samples (Bacteria), respectively.
　具体的な手順は以下のとおりである。まず、唾液を遠心分離して、菌体と、膜小胞とを粗分離した。次に、分離した遠心上清について、メンブランフィルターを用いてろ過し、ろ過後の遠心上清と、菌体とを分離した。
　ろ過後の遠心上清には、細菌体以外にもタンパク質等の夾雑物質が含まれている場合があるので、１００～２００ｋＤａカットオフフィルタで濃縮した後、超遠心によって膜小胞を沈殿させて回収した。このようにして得られた沈殿物には、細菌群等の構造体（例えば、せん毛、及び、べん毛等）が含まれていることがあるため、これらを除去するために、スクロースを用いた密度勾配遠心法を用いて、更に精製した。 The specific procedure is as follows. First, saliva was centrifuged to roughly separate the bacterial cells and the membrane vesicles. Next, the separated centrifugal supernatant was filtered using a membrane filter, and the filtered centrifugal supernatant and the bacterial cells were separated.
Since the centrifugation supernatant after filtration may contain contaminants such as proteins in addition to bacteria, concentrate with a 100-200 kDa cut-off filter and then precipitate membrane vesicles by ultracentrifugation. Collected. Since the precipitate thus obtained may contain structures such as bacterial groups (for example, flagella and flagella), sucrose is used to remove them. Further purification was performed using the density gradient centrifugation method used.
　このようにして得られた膜小胞の表面には、膜小胞に由来しない核酸成分が付着している場合があるため、分離取得された膜小胞に更にデオキシリボヌクレアーゼを作用させ、これらの核酸成分を分解した。 Since nucleic acid components not derived from the membrane vesicles may be attached to the surface of the membrane vesicles thus obtained, deoxyribonuclease is further acted on the separated and obtained membrane vesicles to these. Nucleic acid components were degraded.
　次に、得られた膜小胞サンプル、及び、細菌細胞のサンプルについて、単一粒子解析により、両サンプルの１９２個の粒子について、ＤＮＡ配列をプロファイリングした。プロファイリングにあたっては、各粒子ごとに得られたｆａｓｔｑファイルを「ＳＰＡＤＥＳ」等のソフトウェアによってアセンブリし、得られたアセンブリに対しゲノムアノテーションのためのソフトウェアツールである「ｐｒｏｋｋａ」等を用いてタンパク質コード領域を予測・検出し、各領域について、公共データベース等（例えばＮＣＢＩ　ＲｅｆＳｅｑ）に収録されたゲノムの塩基配列をリファレンスとして相同性検索を行い、マップされた（一形態として、一致度の最も高い）配列領域をゲノム中に最も多く有する細胞がその粒子の宿主細胞であると判断した。 Next, the DNA sequences of the obtained membrane vesicle samples and bacterial cell samples were profiled for 192 particles of both samples by single particle analysis. In profiling, the fastq file obtained for each particle is assembled by software such as "SPADES", and the protein coding region is divided into the obtained assembly using "prokka" which is a software tool for genome annotation. For each region, a homology search was performed using the base sequence of the genome recorded in a public database or the like (for example, NCBI RefSeq) as a reference, and the mapped sequence region (as one form, the highest degree of agreement) was predicted and detected. It was determined that the cell having the most protein in the genome is the host cell of the particle.
図５は、各サンプルにおけるＡｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　ｂａｃｔｅｒｉｕｍ　４１－２８（Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８）が宿主細胞と判定された粒子の割合、すなわち、全粒子１９２個に対して、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　ｂａｃｔｅｒｉｕｍ　４１－２８由来と判断された粒子の割合（Ｄｅｔｅｃｔｅｄ　ｐｅｒｃｅｎｔａｇｅ）を表す図である。
　また、図６は、上記と同様に計算された、各サンプルにおけるＴＭ７ｘ由来と判断された粒子の割合を表す図である。 FIG. 5 shows the percentage of particles in which Alphaproteobacteria bacterium 41-28 (Alphaproteobacteria 41-28) was determined to be a host cell in each sample, that is, particles determined to be derived from Alphaproteobacteria bacterium 41-28 with respect to 192 total particles. It is a figure which shows the ratio (Detected percentage).
Further, FIG. 6 is a diagram showing the proportion of particles determined to be derived from TM7x in each sample, which was calculated in the same manner as above.
　図７は、膜小胞サンプル（Ｈｅａｌｔｈｙ）から得られた粒子（ＭＶ）のうち、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８由来と判断された粒子（５２粒子分）の配列を、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８のゲノムＤＮＡにマップした結果を表す図である。
　横軸は、Ａｌｐｈａｐｒｏｔｅｏｂａｃｔｅｒｉａ　４１－２８のゲノムＤＮＡの領域を表しており、５２個の各粒子から検出された領域が黒く示されている。図７では、縦軸に沿って５２個の粒子の結果が並べて示されている。 FIG. 7 maps the sequences of particles (52 particles) determined to be derived from Alphaproteobacteria 41-28 among the particles (MV) obtained from the membrane vesicle sample (Healthy) to the genomic DNA of Alphaproteobacteria 41-28. It is a figure which shows the result of this.
The horizontal axis represents the region of the genomic DNA of Alphaproteobacteria 41-28, and the region detected from each of the 52 particles is shown in black. In FIG. 7, the results of 52 particles are shown side by side along the vertical axis.
　なお、マッピングにあたっては、宿主細胞であると判定された細胞のゲノムに対し、ｂｏｗｔｉｅ２、ＢＷＡ等のマッピングソフトウェアを用いて、各粒子から取得された塩基配列をマッピングすることで、その細胞のゲノム配列のどれだけの領域をカバーできているかを評価した。 In mapping, the genome sequence of the cell is determined by mapping the base sequence obtained from each particle to the genome of the cell determined to be the host cell using mapping software such as bowtie2 and BWA. We evaluated how much area was covered.
　図８は、膜小胞サンプル（Ｐａｔｉｅｎｔ）から得られた粒子（ＭＶ）のうち、ＴＭ７ｘ由来と判断された粒子（８６粒子）の配列を、ＴＭ７ｘのゲノムＤＮＡにマップした結果を表す図である。横軸は、ＴＭ７ｘのゲノムＤＮＡの領域を表しており、８６個の各粒子から検出された領域が黒く示されている。図８では、縦軸に沿って８６個の粒子の結果が並べて示されている。 FIG. 8 is a diagram showing the results of mapping the sequences of the particles (86 particles) determined to be derived from TM7x among the particles (MV) obtained from the membrane vesicle sample (Patient) to the genomic DNA of TM7x. .. The horizontal axis represents the region of the genomic DNA of TM7x, and the region detected from each of the 86 particles is shown in black. In FIG. 8, the results of 86 particles are shown side by side along the vertical axis.
　図９は、膜小胞サンプルから得られた配列によって構築された各微生物のゲノムＤＮＡ長の割合を表す図である。横軸は、全ゲノム配列に対して、膜小胞由来のＤＮＡで検出された領域（カバー領域）が占める割合を示している。 FIG. 9 is a diagram showing the ratio of genomic DNA length of each microorganism constructed by the sequence obtained from the membrane vesicle sample. The horizontal axis shows the ratio of the region (cover region) detected by the DNA derived from the membrane vesicle to the entire genome sequence.
　上記の結果から、細菌自体が希少で、ショットガンメタゲノム解析や細菌サンプルの１細胞ゲノム解析では、ゲノムＤＮＡを正しく得ることが難しい場合であっても、膜小胞を使うと、例えば、８０％以上の領域が検出（再構築）できる。ショットガンメタゲノム解析等で再構築された細菌のゲノムＤＮＡを、膜小胞のＤＮＡ解析結果により補完すれば、より正しく、簡便に、希少細菌のゲノムＤＮＡの解析が行える。 From the above results, even if the bacteria themselves are rare and it is difficult to obtain genomic DNA correctly by shotgun metagenome analysis or single-cell genome analysis of bacterial samples, using membrane vesicles, for example, 80%. The above areas can be detected (reconstructed). If the bacterial genomic DNA reconstructed by shotgun metagenome analysis or the like is supplemented with the results of membrane vesicle DNA analysis, the genomic DNA of rare bacteria can be analyzed more correctly and easily.
　本ゲノム解析方法によれば、膜小胞の塩基配列を、その産生元である細胞を含む全ゲノム解析に用いることで、従来の方法と比較してより迅速に正確な全ゲノム配列を得ることができる。本ゲノム解析方法は、複雑な細菌叢等の全体像をより正確により迅速に把握することができ、医療、環境、及び、食品産業等において、細菌叢の包括的なデータ取得に役立つものである。 According to this genome analysis method, by using the base sequence of a membrane vesicle for whole genome analysis including the cell from which it is produced, an accurate whole genome sequence can be obtained more quickly than the conventional method. Can be done. This genome analysis method can grasp the whole picture of a complicated bacterial flora more accurately and quickly, and is useful for comprehensive data acquisition of the bacterial flora in the medical, environmental, and food industries. ..
３０１　　：マイクロ流路
３０２　　：第１試料
３０３　　：細菌
３０４　　：オイル
３０５、３０６　　：液滴

  301: Microchannel 302: First sample 303: Bacteria 304: Oil 305, 306: Droplets

Claims (11)

  1. 　細胞と、前記細胞で産生され、前記細胞外に放出された膜小胞と、を含む懸濁液から、前記細胞と前記膜小胞とを分離し、前記細胞を含む第１試料と、前記膜小胞を含む第２試料とを調製することと、
    　前記第１試料に含まれる前記細胞に由来する塩基配列である、第１塩基配列を決定することと、
    　前記第２試料に含まれる前記膜小胞に由来する塩基配列である、第２塩基配列を決定することと、
    　前記第２塩基配列を用いて、前記第１塩基配列を補完することと、を含む、ゲノム解析方法。     The cell and the membrane vesicle are separated from the suspension containing the cell and the membrane vesicle produced by the cell and released to the outside of the cell, and the first sample containing the cell and the said. Preparing a second sample containing membrane vesicles and
    Determining the first base sequence, which is the base sequence derived from the cells contained in the first sample,
    Determining the second base sequence, which is the base sequence derived from the membrane vesicle contained in the second sample,
    A method for genome analysis, comprising complementing the first base sequence with the second base sequence.
  2. 　前記第１塩基配列の決定が、前記第１試料の単一細胞解析によって行われる、請求項１に記載のゲノム解析方法。 The genome analysis method according to claim 1, wherein the determination of the first base sequence is performed by single cell analysis of the first sample.
  3. 　前記第１塩基配列の決定が、前記第１試料のメタゲノム解析によって行われる、請求項１に記載のゲノム解析方法。 The genome analysis method according to claim 1, wherein the determination of the first base sequence is performed by metagenomic analysis of the first sample.
  4. 　前記第２塩基配列を決定することが、
    　前記第２試料を用い、前記膜小胞の１個体ずつを液滴中に封入することと、
    　前記液滴をゲル化してゲルカプセルを生成することと、
    　前記ゲルカプセルを溶解試薬と接触させて、前記膜小胞を溶解させ、前記膜小胞中のポリヌクレオチドが前記ゲルカプセルに溶出し、前記ゲルカプセル内に保持させることと、
    　前記ポリヌクレオチドを増幅試薬に接触させて、前記ポリヌクレオチドを前記ゲルカプセル内で増幅することと、
    　前記増幅したポリヌクレオチドから、前記膜小胞に由来する前記ポリヌクレオチドの塩基配列である第２塩基配列を決定することと、を含む、請求項１～３のいずれか１項に記載のゲノム解析方法。     Determining the second base sequence
    Using the second sample, each individual membrane vesicle is encapsulated in a droplet.
    Gelling the droplets to form gel capsules
    The gel capsule is brought into contact with a lysing reagent to dissolve the membrane vesicle, and the polynucleotide in the membrane vesicle is eluted into the gel capsule and retained in the gel capsule.
    Amplification of the polynucleotide in the gel capsule by contacting the polynucleotide with an amplification reagent
    The genomic analysis according to any one of claims 1 to 3, comprising determining a second base sequence, which is a base sequence of the polynucleotide derived from the membrane vesicle, from the amplified polynucleotide. Method.
  5. 　前記第２塩基配列を決定することが、
    　更に、前記第２試料を核酸分解酵素で処理することを含む、請求項４に記載のゲノム解析方法。     Determining the second base sequence
    The genome analysis method according to claim 4, further comprising treating the second sample with a nucleolytic enzyme.
  6. 　前記第２塩基配列を決定することが、
    　前記処理を経た前記第２試料と、前記処理を経ない前記第２試料のそれぞれを用いて行われる、請求項５に記載のゲノム解析方法。     Determining the second base sequence
    The genome analysis method according to claim 5, wherein each of the second sample that has undergone the treatment and the second sample that has not undergone the treatment is used.
  7. 　前記補完することが、前記第２塩基配列を前記第１塩基配列の断片として用いることである、請求項１～６のいずれか１項に記載のゲノム解析方法。 The genome analysis method according to any one of claims 1 to 6, wherein the complement is to use the second base sequence as a fragment of the first base sequence.
  8. 　前記補完することが、前記第１塩基配列のカバー率を向上させることである、請求項１～７のいずれか１項に記載のゲノム解析方法。 The genome analysis method according to any one of claims 1 to 7, wherein the complement is to improve the coverage of the first base sequence.
  9. 　前記懸濁液が、ヒト又は動物から採取した、糞便、唾液、喀痰、手術洗浄液、血液、並びに、皮膚又は身体粘膜の拭い液、及び、スワブからなる群より選択される少なくとも１種の採取試料である、請求項１～８のいずれか１項に記載のゲノム解析方法。 At least one sample selected from the group consisting of feces, saliva, sputum, surgical lavage fluid, blood, and skin or body mucosa wipes, and swabs, from which the suspension is collected from humans or animals. The genome analysis method according to any one of claims 1 to 8.
  10. 　前記細胞が、細菌である、請求項１～９のいずれか１項に記載のゲノム解析方法。 The genome analysis method according to any one of claims 1 to 9, wherein the cell is a bacterium.
  11. 　前記細菌が、ポルフィロモナス属、プレボテラ属、ベイヨネラ属、フソバクテリウム属、パルビモナス属、アグリゲイティバクター属、アクチノマイセス属、アクチノバチルス属、バクテロイデス属、タンネレラ属、トレポネーマ属、カンピロバクター属、エイケネラ属、及び、カプノサイトファーガ属からなる群より選択される少なくとも１種の細菌である、請求項１０に記載のゲノム解析方法。

          The bacteria are Porphyromonas, Prevotella, Beyonella, Fusobacterium, Parbimonas, Aggregatibacter, Actinobacillus, Actinobacillus, Bacteroides, Tannerela, Treponema, Capnocytophaga, Eikenella. The method for genome analysis according to claim 10, wherein the bacterium is at least one bacterium selected from the group consisting of the genus Capnocytophaga.
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