JP7473251B2 - Genome Analysis Methods - Google Patents

Description

The present invention relates to a genome analysis method.

Methods for performing single cell analysis and devices therefor are known.
Patent Document 1 describes a method for amplifying a polynucleotide in a cell or cell-like structure, comprising the steps of: using a sample containing two or more cells or cell-like structures, and encapsulating the cells or cell-like structures in liquid droplets one cell or structure at a time; gelling the droplets to produce gel capsules; immersing the gel capsules in one or more types of lysis reagents to lyse the cells or cell-like structures, wherein polynucleotides in the cells are dissolved into the gel capsule and are retained in the gel capsule in a state in which substances that bind to the polynucleotides have been removed; and contacting the polynucleotides with an amplification reagent to amplify the polynucleotides in the gel capsule.

International Publication No. 2019/216271

When a biological sample containing a variety of cells contains cells that are difficult to culture, whole genome analysis of the biological sample has been considered essential for functional analysis of the cells that are difficult to culture. Single cell analysis as described in Patent Document 1 can isolate a single cell and amplify and read the polynucleotides in that cell, and therefore has made a significant contribution to whole genome analysis, particularly of cells that are difficult to culture. In addition, conventional whole genome (metagenomic) analysis, which is performed without isolating individual cells, is still useful for the above-mentioned purpose.

However, in the single-cell analysis described in Patent Document 1, polynucleotides derived from each cell are amplified and sequenced, so if there is a problem in the amplification process, the sequence coverage rate may be insufficient. Also, in metagenomic analysis, it is often difficult in principle to obtain a draft genome of sufficient quality.

Therefore, the objective of the present invention is to provide a genome analysis method that can more easily obtain high-quality whole genome analysis results.

As a result of intensive research into achieving the above object, the inventors have discovered that the above object can be achieved by the following configuration.

[1] A genome analysis method comprising: separating cells and membrane vesicles produced by the cells and released outside the cells from a suspension containing the cells and the membrane vesicles, and preparing a first sample containing the cells and a second sample containing the membrane vesicles; determining a first base sequence that is a base sequence derived from the cells contained in the first sample; determining a second base sequence that is a base sequence derived from the membrane vesicles contained in the second sample; and complementing the first base sequence using the second base sequence.
[2] The genome analysis method according to [1], wherein the first base sequence is determined by single-cell analysis of the first sample.
[3] The genome analysis method according to [1], wherein the first base sequence is determined by metagenomic analysis of the first sample.
[4] The method of any one of [1] to [3], wherein determining the second base sequence includes using the second sample to encapsulate each of the membrane vesicles in a droplet, gelling the droplet to generate a gel capsule, contacting the gel capsule with a dissolution reagent to dissolve the membrane vesicles, and the polynucleotide in the membrane vesicle is eluted into the gel capsule and retained in the gel capsule, contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule, and determining a second base sequence, which is the base sequence of the polynucleotide derived from the membrane vesicle, from the amplified polynucleotide.
[5] The genome analysis method according to [4], wherein determining the second base sequence further comprises treating the second sample with a nuclease.
[6] The genome analysis method according to [5], wherein determining the second base sequence is performed using each of the second sample that has been subjected to the treatment and the second sample that has not been subjected to the treatment.
[7] The genome analysis method according to any one of [1] to [6], wherein the complementation is using 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 complementing is to improve the coverage of the first base sequence.
[9] The genome analysis method according to any one of [1] to [8], wherein the suspension is at least one sample selected from the group consisting of feces, saliva, sputum, surgical irrigation fluid, blood, and skin or body mucosa swabs collected from a human or animal.
[10] The genome analysis method according to any one of [1] to [9], wherein the cell is a bacterium.
[11] The genome analysis method according to [10], wherein the bacterium is at least one bacterium selected from the group consisting of the genera Porphyromonas, Prevotella, Veillonella, Fusobacterium, Parvimonas, Aggregatibacter, Actinomyces, Actinobacillus, Bacteroides, Tannerella, Treponema, Campylobacter, Eikenella, and Capnocytophaga.

The present invention provides a genome analysis method that can more easily obtain high-quality whole genome analysis results.

FIG. 1 is a flow diagram of a genome analysis method according to an embodiment of the present invention. FIG. 2 is a flow diagram of a method for determining a first base sequence by single-cell analysis. FIG. 1 is a schematic diagram of a flow cell that can be used for single cell analysis. FIG. 11 is a flow diagram of a genome analysis method according to another embodiment of the present invention. FIG. 1 shows the percentage of particles determined to be derived from Alphaproteobacteria bacterium 41-28 (Alphaproteobacteria 41-28) (Detected percentage) in each of a membrane vesicle sample and a bacterial sample collected from saliva. FIG. 13 shows the ratio of particles determined to be derived from TM7x in each of the above samples. FIG. 1 shows the results of mapping the sequences of particles (52 particles) determined to be derived from Alphaproteobacteria 41-28 among particles (MV) obtained from a membrane vesicle sample (Healthy) to the genomic DNA of Alphaproteobacteria 41-28. FIG. 1 shows the results of mapping the sequences of particles (86 particles) determined to be derived from TM7x among particles (MV) obtained from a membrane vesicle sample (Patient) to the genomic DNA of TM7x. FIG. 1 shows the percentage of genomic DNA length for each microorganism assembled from sequences obtained from membrane vesicle samples.

The present invention will be described in detail below.
The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.
In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower limit and upper limit.

(Definition of terms)
As used herein, "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, and RNA.

In this specification, "single cell analysis" is synonymous with "single cell analysis" and refers to a method of isolating and genomic analyzing a single cell from a specimen (sample) containing multiple types and/or multiple cells.
Single cell analysis is a method of, for example, sorting cells into each of a number of microwells arranged on a plane and performing genome analysis on each of the cells. Examples of single cell analysis include those using known devices and/or methods such as the device described in Patent Document 1, the device described in International Publication No. 2017/094101, and the device described in International Publication No. 2016/038670.

In addition, in this specification, "single particle analysis" refers to a method of separating cell-derived particles (excluding the cells themselves) and analyzing the genome of each particle. As a method for single particle analysis, the same method as the above-mentioned "single cell analysis" can be used.

In this specification, "membrane vesicles" include outer membrane vesicles produced by bacteria, etc., "exosomes" produced and released by animal cells, etc. via the endocytosis pathway, and "apoptotic bodies" generated by apoptosis. Of these, outer membrane vesicles (OMVs) are preferred as membrane vesicles.

Membrane vesicles produced by bacteria (sometimes referred to as "derived from bacteria" or "bacterial") include "outer membrane vesicles (OMVs)," which are microvesicles produced when part of the bacterial cell membrane is constricted outside the bacterial body; their size is not particularly limited, but is often 10-1000 nm, often 10-300 nm in gram-negative bacteria, and often 50-150 nm in gram-positive bacteria.

[Genome analysis method]
The genome analysis method of the present invention will be described in detail below. The genome analysis method of the present invention uses a suspension containing cells and membrane vesicles produced by the cells and released outside the cells, and the "cells" include both those of multicellular organisms and those of unicellular organisms. In the following explanation, the case where the cells are bacteria will be described as an example. The following method can also be applied to animal cells, etc.

FIG. 1 is a flowchart of a genome analysis method according to an embodiment of the present invention (hereinafter also referred to as "the method").
This method includes separating the bacteria and the membrane vesicles from a suspension containing the bacteria and membrane vesicles produced by the bacteria and released outside the bacteria, and preparing a first sample containing the bacteria and a second sample containing the membrane vesicles (sample preparation step, step S101);
determining a first base sequence, which is a base sequence derived from the bacterium contained in the first sample (first base sequence determining step, step S102);
Determining a second base sequence, which is a base sequence derived from the membrane vesicles contained in a second sample (second base sequence determination step, S103);
and complementing the first base sequence with the second base sequence (complementing step, S104).

Sample preparation process (step S101)
Step S101 is a process of separating bacteria and membrane vesicles from a suspension containing bacteria and membrane vesicles, and preparing a first sample containing bacteria and a second sample containing membrane vesicles.

The suspension used in this process contains bacteria and membrane vesicles. That is, the suspension contains membrane vesicles and the bacteria that produced the membrane vesicles. The bacteria may be a cultured single bacterial strain, or multiple species of bacteria (one or more of which may be difficult-to-culture bacteria).

The suspension may be, for example, a sample collected from a human or animal, such as feces, saliva, sputum, surgical irrigation fluid, blood, skin or body mucous membrane wipes, swabs, etc. In general, the sample may contain bacteria and membrane vesicles produced by the bacteria contained therein.
The bacteria group and the membrane vesicles may be separated from such a collected sample to provide a first sample and a second sample, respectively.

The bacteria contained in the suspension are not particularly limited, but include eubacteria, Escherichia coli, Bacillus subtilis, cyanobacteria, cocci, bacilli, spiral bacteria, gram-negative bacteria, gram-positive bacteria, archaea, and fungi. Examples of bacteria include Neibacteria, Eobacteria, Deinococci, Deinococci, Deinococcales, Thermales, Chloroflexi, Anaerolineae, Anaerolineals, Caldilineae, Chloroflexales, Herpetosiphonales, Thermomicrobia, Thermomicrobiale. s, Sphaerobacteriales, Ktedonobacteria, Ktedonobacteriales, Thermogemmatisporales, Glycobacteria, Cyanobacteria, Gloeobacterophyceae, Gloeobacteriales, Nostoc Ophycidae, Chroococcales, Oscillatoryes, Nostocales, Pseudobaenales, Spirochaetes, Spirochaetes, Spirochaetes, Fibrobacteres, Fibrobacteria, Gemmatimonadetes, Gemmatimonadetes, Gemmatimonadales, Chlorobi, Chlorobea, Chlorobiales, Ignavibacteria, Ignavibacteriales, Bacteroidetes, Bacteroidia, Bacteroidales, Flavobacteria, Flavobacteriales, Sphingobacteria, Sphingobacteriales, Cytophagia, Cytophagales, Planctomyc etes, Planctomycea, Planctomycetales, Phycisphaerae, Phycisphaerales, Chlamydiae, Chlamydiae, Chlamydiales, Verrucomicrobia, Verrucomicrobiae, Verrucomicrobiales, Opitutae, Opitutales, Punicicoccales, Spartobac teria, Chthoniobacteriales, Lentisphaerae, Lentisphaeria, Lentisphaerales, Victivallales, Proteobacteria, Alphaproteobacteria, Rhodospirillales, Rickettsiales, Rhodobacteriales, Sphingomonadales, Caulobacteriales, Rhizobiales, Parvulares, Kordiimonadales, Sneathiellales, Kiloniellales, Betaproteobacteria, Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Rhodocyclales, Procabacter iiales, Gammaproteobacteria, Chromatiales, Acidithiobacillales, Xanthomonadales, Cardiobacteriales, Thiotrichales, Legionellales, Methylococcales, Oceanospirillales, Pseudomonadales, Alteromonadales, Vibrionales , Aeromonadales, Enterobacteriales, Pasteurellales, Deltaproteobacteria, Desulfurellales, Desulfovibrionales, Desulfobacteriales, Desulfarculares, Desulfuromonadales, Syntrophobacteriales, Bdellovibrionales, Myx Occoccales, Epsilonproteobacteria, Campylobacteriales, Nautileales, Acidobacteria, Acidobacteria, Acidobacteria, Holophagae, Holophagales, Acanthopleuribacteriales, Aquificae, Aquificae, Aquificales, Deferribacter eres, Deferribacteres, Geovibriales, Thermodesulfobacteria, Thermodesulfobacteria, Thermodesulfobacteria, Nitrospirae, Nitrospira, Nitrospirales, Fusobacteria, Fusobacteria, Fusobacteriales, Synergistetes, Synergistia, Synergistales, Caldiserica, Caldiserica, Caldisericales, Elusimicrobia, Elusimicrobia, Elusimicrobiales, Armatimonadetes, Armatimonadia, Armatimonadales, Chthonomonadetes, Chthonomonadales, Fimbrii monadia, Fimbriimonadales, Posibacteria, Thermotogae, Thermotogae, Thermotagales, Firmicutes, Bacilli, Bacillales, Lactobacillales, Clostridia, Clostridiales, Halanaerobiales, Thermoanaerobacteriales, Natranaerob iales, Negativicutes, Selenomonadales, Erysipelotrichia, Erysipelotrichales, Thermolithobacteria, Thermolithobacterales, Tenericutes, Mollicutes, Mycoplasmatales, Entomoplasmatales, Acholeplasmatales, Anaeropla smatales, Actinobacteria, Actinobacteria, Actinomycetales, Actinopolysporales, Bifidobacteriales, Catenulisporales, Corynebacteriales, Frankiales, Glycomycetales, Jiangellales, Kineosporiales, Micrococcal ... Examples of such species include romnosporales, Propionibacteriales, Pseudonocardiales, Streptomycetales, Streptosporangiales, Dictyoglomi, Dictyoglomia, Dictyoglomales, Chrysiogenetes, Chrysiogenetes, Chrysiogenenes, and Haloplasmatales.

In addition, the bacteria is preferably periodontal disease bacteria (bacteria that cause periodontal disease). Examples of periodontal disease bacteria include bacteria of the genus Porphyromonas, Prevotella, Veillonella, Fusobacterium, Parvimonas, Aggregatibacter, Actinomyces, etc. Examples of the genera include the genera Actinobacillus, Bacteroides, Tannerella, Treponema, Campylobacter, Eikenella, and Capnocytophaga.

More specifically, Porphyromonas gingivalis or Bacteroides gingivalis, Prevotella intermedia, Veillonella parvula, Fusobacterium nucleatum, Parvimonas micra, Aggregatibacter actinomycetemcomitans, actinomycetemcomitans or Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Tannerella forsythensis, Treponema denticola, Campylobacter rectus, Eikenella corrodens, and Capnocytophaga ochracea.

Among them, the bacteria is preferably at least one type selected from the group consisting of Porphyromonas gingivalis, Treponema denticola, and Aggregatibacter actinomycetemcomitans, which are known as the "red complex."

There are no particular limitations on the method for separating the bacteria and membrane vesicles in the suspension, and any known method can be used. For example, in the case of a liquid culture of a single cell line, the membrane vesicles are contained in the culture supernatant, so the bacterial cells can be removed from the culture liquid by centrifugation and used as the first sample, and the supernatant can be used as the second sample. When the bacterial strain is cultured on a solid state, the colonies can be suspended in a buffer solution and separated in a similar manner.

If the separated centrifugal supernatant is filtered using a membrane filter (for example, pore size 0.1 to 0.9 μm), the bacteria remaining in the centrifugal supernatant can be easily separated.
Since the centrifugal supernatant may contain impurities such as proteins in addition to the bacterial cells, for example, a method can be used in which the supernatant is concentrated using a 100 to 200 kDa cutoff filter, and then the membrane vesicles are precipitated and collected by ultracentrifugation.

In addition, the precipitate obtained in this manner may contain bacterial structures (e.g., ciliata and flagella, etc.), and in order to remove these, further purification may be performed using, for example, density gradient centrifugation using sucrose, etc.

The sample preparation method including the above treatment will now be described in more detail.
First, when a desired growth phase is achieved under predetermined conditions, the culture medium is placed in a centrifuge tube with a volume of 1 to 100 mL (e.g., 50 mL) and centrifuged at 3000 to 9000 rpm (e.g., 7000 rpm) at 1 to 20°C (e.g., 4°C) for 1 to 30 minutes (e.g., 10 minutes).

Once the supernatant and precipitate have been separated, the precipitate contains the bacterial cells, so separate this (the first sample), pass the supernatant through a 0.1 to 0.9 μm (e.g., 0.22 μm) filter, place in a separate centrifuge tube, and store at 1 to 10°C (e.g., 4°C).

Next, the supernatant is added to an ultracentrifuge tube and ultracentrifuged, for example, at a centrifugal force of 150,000 to 300,000 x g (e.g., 210,000 x g) at 1 to 10°C (e.g., 4°C) for 1 to 3 hours (e.g., 2 hours). After ultracentrifugation, the supernatant is discarded and the precipitated outer membrane vesicles (OMVs) are resuspended in a buffer solution (e.g., PBS buffer at pH 7.4) and stored at 1 to 10°C (e.g., 4°C). Furthermore, this ultracentrifugation process may be repeated multiple times.

The obtained liquid may be diluted to adjust the number (concentration) of membrane vesicles contained per unit amount of the second sample. This makes it easier to separate and obtain individual membrane vesicles in the single particle analysis described below.

The method of dilution is not particularly limited, and examples thereof include a method in which the concentration of membrane vesicles in a liquid is monitored by dynamic light scattering or the like while diluting the liquid to an appropriate concentration.
A preferred method for observing the concentration of membrane vesicles is to irradiate the particles with a laser, track the Brownian motion of each particle from the scattered light (tracking method), and calculate the particle diameter and number from the diffusion speed based on the Stokes-Einstein equation.

<First base sequence determination step (step S102)>
The first sample contains one or more types of bacteria, and this step is a step of determining a first base sequence, which is a genomic sequence derived from the bacteria(s).
Methods for determining the first base sequence include, for example, 16S ribosomal RNA analysis, metagenomics analysis, and single-cell analysis, and any known technique can be applied without particular limitation. Among these, single-cell analysis is preferred because it is easier to obtain a more accurate genome sequence of an individual bacterium.

When the first base sequence in this process is determined by single-cell analysis, the method is not particularly limited, but single-cell analysis including the following steps is preferred.

Encapsulating each individual bacterium contained in the first sample in a droplet (encapsulation process)
- Gelling the droplets to produce gel capsules (gelling process)
Contacting the gel capsule with a lysis reagent to lyse the bacteria and elute the polynucleotides, which are retained within the gel capsule (lysis step);
・Removing impurities from the gel capsule (purification process)
- Contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule (amplification step);
- Determining a first base sequence from the amplified polynucleotide (sequencing step)

Figure 2 is a flow chart of single-cell analysis including each of the above steps. It is preferable that the "first base sequence determination step" represented by step S102 in Figure 1 includes the single-cell analysis of Figure 2.

Encapsulation process (step S201)
Step S201 is a process of encapsulating bacteria one by one in a droplet. The first sample used in the encapsulation process contains bacteria. The sample may contain one type or two or more types of bacteria. At least one of the bacteria in the first sample produces membrane vesicles contained in the suspension and releases them outside the bacterial body.

There are no particular limitations on the method for encapsulating individual bacteria contained in the first sample into droplets using the first sample, and publicly known methods such as those described in Patent Document 1 can be used.

The droplets can be produced, for example, using a microchannel. The above-mentioned suspension is caused to flow in a microchannel, and the flow of the suspension is sheared to produce droplets in which individual bacteria are encapsulated. The shearing can be performed at regular intervals. For example, oil can be used to shear the suspension. Examples of oil include mineral oil, vegetable oil, silicone oil, and fluorinated oil. By adjusting the amount (concentration) of bacteria in the suspension, the flow rate in the channel, and the shearing interval, a person skilled in the art can produce droplets in which one individual bacteria is encapsulated per droplet.
The diameter of the droplets is preferably from 1 to 250 μm, more preferably from 10 to 200 μm.

3 is a schematic cross-sectional view of a microchannel that can be used in this process. The microchannel 301 is a device that can create droplets and encapsulate individual bacteria in the droplets.
The microchannel 301 is composed of a cross-shaped channel formed by two channels intersecting at approximately right angles. In the channel extending from top to bottom in the drawing, a first sample 302 containing bacteria 303 flows from top to bottom in the drawing, and in the channel extending in a direction approximately perpendicular to the first sample 302, oil 304 flows from the left and right to the center in the drawing.

The diameter of this flow path is not particularly limited and may be appropriately selected depending on the purpose, but generally, it is preferably 10 to 60 μm.
A first sample 302 containing bacteria 303 is sheared by oil 304 flowing from the left and right to the center of the drawing, and becomes droplets 305 containing bacteria 303 .
By adjusting the content of bacteria 303 in the first sample 302, it is possible to adjust so that a single bacterium 303 is encapsulated in the droplet 305, and in this case, it is also possible that a droplet 306 not encapsulating bacteria 303 may also be generated.

Gelation process (step S202)
Step S202 is a process of gelling the droplets to generate gel capsules. There are no particular limitations on the gelling method. The droplets can be gelled by configuring the droplets to contain the material of the gel capsules and cooling the droplets thus prepared. Alternatively, gelling can be performed by applying a stimulus such as light to the droplets. To make the droplets contain the material of the gel capsules, for example, the first sample can contain the material of the gel capsules.

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 droplets to be produced, but may change during gelation. The gelation temperature is not particularly limited, but is preferably 4 to 10°C.

Materials for the gel capsule may include agarose, acrylamide, photocurable resin (e.g., PEG-DA), PEG, gelatin, sodium alginate, Matrigel, and collagen, among others.

The gel capsule may be a hydrogel capsule. By "hydrogel" is meant a water-based solvent or dispersion medium held together by a network of polymeric or colloidal particles.

Dissolving step (step S203)
Step S203 is a step of contacting the gel capsule with a lysis reagent to lyse the bacteria, elute the polynucleotide, and retain it in the gel capsule. By lysing the bacteria, the polynucleotide in the bacteria is eluted into the gel capsule, and the polynucleotide can be retained in the gel capsule in a state in which substances that bind to the polynucleotide have been removed. As the lysis reagent, an enzyme, a surfactant, other denaturants, a reducing agent, a pH adjuster, or a combination thereof can be used.

Lysis reagents include lysozyme, labiase, yatalase, achromopeptidase, protease, nuclease, zymolyase, chitinase, lysostaphin, mutanolysin, sodium dodecyl sulfate, sodium lauryl sulfate, potassium hydroxide, sodium hydroxide, phenol, chloroform, guanidine hydrochloride, urea, 2-mercaptoethanol, dithiothreitol, "TCEP-HCl", sodium cholate, sodium deoxycholate, "Triton X-100", "Triton X-114", "NP-40", "Brij-35", "Brij-58", "Tween 20", "Tween 80", octyl glucoside, octyl thioglucoside, "CHAPS", "CHAPSO", dodecyl-β-D-maltoside, "Nonidet P-40", and "Zwittergent 3-12" and others.
The lysis reagent preferably contains lysozyme, achromopeptidase, protease, sodium dodecyl sulfate, potassium hydroxide, and the like.

The method for contacting the gel capsule with the dissolution reagent is not particularly limited, but for example, the dissolution reagent may be discharged into the gel capsule held in a container (e.g., a microtube or each well of a microplate) using an automatic microsyringe or the like. The automatic microsyringe may typically be composed of a flow path for distributing the dissolution reagent, and a pump, a valve, etc. for liquid distribution.

Purification step (step S204)
Step S204 is a process of removing contaminants from within the gel capsule. In this method, since a gel capsule containing a single bacterium is used, the purified genetic material (e.g., DNA) can be retained within the gel capsule, and the possibility of contaminating molecules from outside can be eliminated.

In terms of operation, it is also very simple to process large amounts of bacteria in parallel, one unit at a time. The steps can be performed by centrifuging the test tube containing the gelled droplets, removing the supernatant, and replacing it with a washing solution. Alternatively, the steps can be performed by filtering the gelled droplets, removing the supernatant, passing a washing solution through them, and finally recovering the gel capsules. By using gel capsules, the remaining reagents can be diluted while retaining the genetic material. This step can also be repeated. By diluting the reagents to a level where no inhibition occurs, downstream operations, such as amplification reactions, can be performed smoothly.

It is preferable that the lysis reagent is sufficiently removed before the downstream reaction, since it is less likely to inhibit reactions such as DNA amplification. When using a gel capsule, the gel capsule retains the genetic material to be analyzed or amplified, so that removal of the lysis reagent can be performed even in the analysis of a single cell, which contains only a small amount of genetic material, and therefore a strong lysis reagent or combination of lysis reagents can be used.
Furthermore, the use of a strong lysis reagent or combination of lysis reagents may enable more reliable nucleic acid amplification and base sequence analysis.

Amplification step (step S205)
Step S205 is a step of contacting the polynucleotide with an amplification reagent to amplify the polynucleotide in the gel capsule. After immersion in the amplification reagent, the temperature of the gel capsule may be adjusted as necessary.

Heating is preferably performed at 60° C. or less from the viewpoint of preventing the gel (e.g., agarose gel) from dissolving easily. Heating within this range is preferable from the viewpoint of further promoting DNA amplification.
Enzymes used for amplification include, for example, phi29 polymerase, Bst polymerase, Aac polymerase, and recombinase polymerase.
In this method, random primers are preferably used to achieve amplification of the total DNA in the gel.

Sequencing process (step S206)
Step S206 is a step of determining a first base sequence from the amplified polynucleotide. The method of determining the base sequence from the amplified polynucleotide can be any method, such as library preparation, sequencing, assembly, etc., using commercially available reagent kits, devices, application software, etc., without particular limitation.

<Second base sequence determination step (step S103)>
1, the second base sequence determination step (step S103) will be described. This step is a step of determining a second base sequence using the second sample prepared in step S101.

The sample used in this process may be the second sample prepared in the sample preparation process (step S101), but it may also be a 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 preferred from the viewpoint of requiring a smaller amount of membrane vesicles. The method for single particle analysis is not particularly limited, and the same method as that already described as the method for single-cell analysis of the first sample in the first base sequence determination step of step S102 can be used.

Here, the inventors have found that nucleic acid components are often attached to the surface of membrane vesicles. These nucleic acid components are presumed to have various origins, such as fragments of ruptured bacterial cells or from coexisting bacteria.

The present inventors have found that the nucleic acid components attached to the surface of membrane vesicles are highly likely to be derived from the bacteria (hereinafter also referred to as the "host") that produced the membrane vesicles.
For example, in general metagenomic analysis, such as the whole genome shotgun method, the nucleic acid components contained in a sample are comprehensively analyzed. In such cases, however, the probability of obtaining a sequence derived from the bacterium that produced a specific membrane vesicle among the nucleic acid components comprehensively analyzed is extremely low.

On the other hand, the nucleic acid components attached to the surface of the membrane vesicles are likely to be derived from the host, for example, as components attached during the production process of the membrane vesicles.
In other words, it is unlikely that fragments (polynucleotides) derived from a specific host of membrane vesicles will be obtained from fragments obtained by comprehensive analysis of the first sample, but it can be said that the nucleic acid components contained in the second sample are more likely to be fragments derived from the host.

Single-cell analysis of such a second sample will yield a second base sequence that contains both sequences derived from the membrane vesicles and sequences likely derived from the host. This sequence is likely to be able to complement the first base sequence with a longer base length than would be possible using only the membrane vesicle sequence.

The method for performing single particle analysis of the second sample is not particularly limited, but a method similar to the single cell analysis described in FIG. 2, i.e., single particle analysis including the following steps in this order, is preferred, in that it allows obtaining a second base sequence of higher quality.
Using a second sample, encapsulating each of the membrane vesicles in a droplet; gelling the droplet to produce a gel capsule; contacting the gel capsule with a dissolution reagent to dissolve the membrane vesicles and elute the polynucleotides and retain them in the gel capsule; removing contaminants in the gel capsule; contacting the polynucleotides with an amplification reagent to amplify them in the gel capsule; and determining a second base sequence from the amplified polynucleotides.

<Complementation process (step S104)>
Step S104 is a step of complementing the first base sequence with the second base sequence. The complementing typically includes using the second base sequence as a fragment to improve the coverage rate of the first base sequence.

The second base sequence contains a base sequence derived from the membrane vesicle and a base sequence derived from a nucleic acid component attached to the surface of the membrane vesicle.
If a portion of the second base sequence maps to the first base sequence, the second base sequence is considered to be a portion of the first base sequence, i.e., the membrane vesicle of the second base sequence is considered to have been produced by the bacterium of the first base sequence.

In particular, when the first base sequence is obtained by single-cell analysis as shown in Figure 2, the process includes a step of amplifying a polynucleotide in a gel capsule. Therefore, the primers used may bind to each other or a chimeric sequence may be generated, resulting in the amplification of nucleotides other than the polynucleotide derived from the bacterium that should be amplified. In addition, amplification may be insufficient for some reason, and as a result, the coverage rate of the obtained polynucleotides may not be sufficiently high (for example, about 40 to 80%). In such cases, the present analysis method is more useful.

Another example of complementation is a method in which a second base sequence is used to improve the quality of a draft genome obtained when a first base sequence is obtained by metagenomic analysis.

In a typical whole genome shotgun metagenomic analysis, the whole genome is divided into fragments (reads) of a predetermined base length, each fragment is sequenced, and the fragments are combined (assembly, binning). There are no particular limitations on the method for performing metagenomic analysis, and any known method can be used. For example, the whole genome shotgun analysis method described in JP 2005-218421 A and the like can be used, and this method is known to those skilled in the art.

In this genome analysis method, "complementation" includes using a second base sequence as one of the above fragments (specific fragment). The second base sequence includes a base sequence obtained from a membrane vesicle, and is part of the genome 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 isolating individual membrane vesicles and analyzing their base sequences, and therefore typically has a longer base length and a more accurate sequence than the fragments used in metagenomic analysis.

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 metagenomic analysis by the whole genome shotgun method. The specific fragment is a sequence derived from a single membrane vesicle produced, and is a more accurate sequence, so that the obtained whole genome sequence is likely to be of higher quality.
Furthermore, the specific fragment typically has a longer base length than the other fragments, and therefore often provides 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. For example, the specific fragment may be mapped to the whole genome sequence. Here, "mapping" to the whole genome sequence means that the specific fragment matches a partial region of the whole genome sequence. Matching means that 90% or more of the sequence is identical, preferably 95% or more, more preferably 99% or more, and even more preferably completely identical.

By mapping the specific fragment to the whole genome sequence, the quality of the whole genome sequence can be checked. That is, using the specific fragment as a fragment of the whole genome, when mapped to the whole genome sequence, may provide information to determine that the whole genome sequence of that region is accurate.

<Other embodiments of the present invention>
A 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).
A first base sequence is determined using the first sample (step S402, first base sequence determination step).
The second sample is treated with a nuclease to prepare a treated second sample (step S403, treatment step).
The treated second sample is used to determine a second base sequence (step S404, second base sequence determination step).
The first base sequence is complemented by using the second base sequence (step S405, complementation step).

Figure 4 is a flow chart of the genome analysis method according to this embodiment. This genome analysis method is similar to the genome analysis method shown in Figure 1, except that it has a processing step. That is, step S401 is similar to step S101, step S402 is similar to step S102, step S404 is similar to step S103, step S405 is similar to step S104, and the public form is also similar. An explanation of these "similar parts" will be omitted, and the parts that differ from the genome analysis method shown in Figure 1 will be mainly explained.

This genome analysis method includes a step of treating the second sample with a nuclease to prepare a treated second sample (treatment step, step S403).

As already explained, the membrane vesicles contained in the second sample have attached thereto nucleic acid components that are likely to be derived from the host. One of the features of the genome analysis method according to the present embodiment is that the nucleic acid components are decomposed and removed.
In the genome analysis method according to this embodiment, it is possible to ensure that the second base sequence is a sequence derived from a membrane vesicle, making it easier to complement the first base sequence.

There are no particular limitations on the nucleic acid decomposition enzyme used in the treatment step, and deoxyribonuclease, etc., can be used. Specifically, a method can be used in which 10 μL of the second sample is diluted 10 times and 2 μL of DNase (2000 U) is added thereto. This results in a treated second sample.

Step S404 is a step of determining a second base sequence using the treated second sample. The sequencing method used in this step is preferably the single particle analysis described above, since it requires a smaller amount of sample.
In addition to the above, this step preferably further includes determining a second base sequence by single particle analysis using an untreated second sample.

As a result, the base sequences of individual membrane vesicles are obtained from the treated second sample, and from the untreated second sample, in addition to the base sequences of individual membrane vesicles, the base sequences of nucleic acid components attached to the surface of the membrane vesicles are obtained.

In other words, the difference between the base sequence obtained from the second sample and the base sequence of the treated second sample is likely to include a base sequence derived from a nucleic acid component attached to the surface of the membrane vesicle. As already explained, the "difference" is likely to be derived from the host. By having this process, the coverage of the first base sequence by the obtained second base sequence may be wider. Furthermore, by utilizing the "difference", it may be possible to determine whether the obtained sequence is derived from the membrane vesicle or from a nucleic acid component attached to the surface, making genome analysis more efficient.

Although the above mainly describes a method of using single-cell analysis as a method of analyzing the second sample, the genome analysis method according to the embodiment of the present invention is not limited to the above, and the base sequence of the second sample may be analyzed by other methods. For example, metagenomic analysis can be used as such a method.

The present invention will be described below with reference to examples, but the present invention is not limited to these.

[Example: Genome reconstruction of rare microbial species using DNA derived from membrane vesicles]
Saliva was collected from healthy subjects (Health) and patients with periodontal disease (Patient) using a saliva collection kit. Membrane vesicles and bacteria were separated from the collected saliva to obtain membrane vesicle samples (MVs) and bacterial cell samples (Bacteria), respectively.

The specific procedure is as follows: First, the saliva was centrifuged to roughly separate the bacterial cells and the membrane vesicles. Next, the separated centrifugation supernatant was filtered using a membrane filter, and the filtered centrifugation supernatant was separated from the bacterial cells.
Since the centrifugal supernatant after filtration may contain contaminating substances such as proteins in addition to the bacterial bodies, it was concentrated using a 100-200 kDa cutoff filter, and then the membrane vesicles were precipitated and collected by ultracentrifugation. Since the precipitate thus obtained may contain structures of the bacterial groups and the like (e.g., ciliata and flagella, etc.), in order to remove these, it was further purified using density gradient centrifugation using sucrose.

Because the surface of the membrane vesicles obtained in this manner may contain nucleic acid components that are not derived from the membrane vesicles, the isolated membrane vesicles were further treated with deoxyribonuclease to decompose these nucleic acid components.

Next, the DNA sequences of 192 particles from the obtained membrane vesicle sample and the bacterial cell sample were profiled by single particle analysis. For profiling, the fastq files obtained for each particle were assembled using software such as "SPADES", and protein coding regions were predicted and detected for the obtained assemblies using software tools such as "prokka", which is a software tool for genome annotation. For each region, a homology search was performed using the genomic base sequences recorded in public databases (e.g., NCBI RefSeq) as references, and the cell with the most mapped sequence regions (as a form of highest degree of identity) in its genome was determined to be the host cell of that particle.

FIG. 5 is a diagram showing the percentage of particles in each sample in which Alphaproteobacteria bacterium 41-28 (Alphaproteobacteria 41-28) was determined to be a host cell, that is, the percentage of particles determined to be derived from Alphaproteobacteria bacterium 41-28 (Detected percentage) relative to a total of 192 particles.
FIG. 6 is a diagram showing the proportion of particles determined to be derived from TM7x in each sample, calculated in the same manner as above.

FIG. 7 shows the results of mapping the sequences of particles (52 particles) determined to be derived from Alphaproteobacteria 41-28 among particles (MV) obtained from a membrane vesicle sample (Healthy) to the genomic DNA of Alphaproteobacteria 41-28.
The horizontal axis represents the region of the genomic DNA of Alphaproteobacteria 41-28, and the regions detected from each of the 52 particles are shown in black. In Figure 7, the results of the 52 particles are arranged along the vertical axis.

In mapping, the base sequences obtained from each particle were mapped to the genome of cells determined to be host cells using mapping software such as Bowtie2 and BWA to evaluate how much of the cell's genome sequence had been covered.

Figure 8 shows the results of mapping the sequences of particles (86 particles) determined to be derived from TM7x among particles (MV) obtained from a 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 Figure 8, the results of the 86 particles are arranged along the vertical axis.

Figure 9 shows the proportion of genomic DNA length for each microorganism constructed from sequences obtained from membrane vesicle samples. The horizontal axis shows the proportion of the region detected in DNA derived from membrane vesicles (covered region) to the entire genome sequence.

From the above results, even if the bacteria themselves are rare and it is difficult to obtain genomic DNA correctly using shotgun metagenomic analysis or single-cell genomic analysis of bacterial samples, by using membrane vesicles, for example, it is possible to detect (reconstruct) more than 80% of the region. By complementing the bacterial genomic DNA reconstructed by shotgun metagenomic analysis or the like with the results of DNA analysis of membrane vesicles, it is possible to analyze the genomic DNA of rare bacteria more accurately and simply.

According to this genome analysis method, by using the base sequence of membrane vesicles in whole genome analysis including the cells from which they were produced, it is possible to obtain accurate whole genome sequences more quickly than with conventional methods. This genome analysis method makes it possible to grasp the overall picture of complex bacterial flora more accurately and quickly, and is useful for obtaining comprehensive data on bacterial flora in the medical, environmental, and food industries, etc.

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

Claims (11)

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, and preparing a first sample containing the cells and a second sample containing the membrane vesicles;
determining a first base sequence, the first base sequence being a base sequence derived from the cell contained in the first sample;
determining a second base sequence, the second base sequence being a base sequence derived from the membrane vesicles contained in the second sample;
and complementing the first base sequence using the second base sequence. The genome analysis method described in claim 1, wherein the determination of the first base sequence is performed by single-cell analysis of the first sample. The genome analysis method described in claim 1, wherein the first base sequence is determined by metagenomic analysis of the first sample. Determining the second base sequence
encapsulating the membrane vesicles one by one in droplets using the second sample;
gelling the droplets to form gel capsules;
contacting the gel capsule with a dissolution reagent to dissolve the membrane vesicles, and dissolving the polynucleotides in the membrane vesicles into the gel capsule and retaining them within the gel capsule;
contacting the polynucleotide with an amplification reagent to amplify the polynucleotide within the gel capsule;
The genome analysis method according to any one of claims 1 to 3, further comprising determining a second base sequence from the amplified polynucleotide, the second base sequence being the base sequence of the polynucleotide derived from the membrane vesicle. Determining the second base sequence
The genome analysis method according to claim 4 , further comprising treating the second sample with a nuclease. Determining the second base sequence
The genome analysis method according to claim 5 , wherein the method is performed using both the second sample that has been subjected to the treatment and the second sample that has not been subjected to the treatment. A genome analysis method according to any one of claims 1 to 6, wherein the complementing comprises using the second base sequence as a fragment of the first base sequence. A genome analysis method according to any one of claims 1 to 7, wherein the complementing is to improve the coverage rate of the first base sequence. The genome analysis method according to any one of claims 1 to 8, wherein the suspension is at least one type of sample selected from the group consisting of feces, saliva, sputum, surgical irrigation fluid, blood, and skin or body mucosa swabs and swabs collected from a human or animal. A genome analysis method according to any one of claims 1 to 9, wherein the cell is a bacterium. The genome analysis method according to claim 10, wherein the bacterium is at least one type of bacterium selected from the group consisting of the genera Porphyromonas, Prevotella, Veillonella, Fusobacterium, Parvimonas, Aggregatibacter, Actinomyces, Actinobacillus, Bacteroides, Tannerella, Treponema, Campylobacter, Eikenella, and Capnocytophaga.

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