CN105937053A - Establishment method of gene library of fecal flora based on high-throughput gene sequencing - Google Patents

Abstract

An establishment method of a gene library of fecal flora based on high-throughput gene sequencing employs a ''nested PCR'' method for enrichment amplification on 16S rDNA, so as to reduce the host and food residue genome pollution by the maximum; and V3 and V6 are combined for specific amplification and massive parallel sequencing, so as to obtain the a target gene sequence database of the flora. The library can increase the identification of the bacterial flora from Genus level to Species level. The method can maximally avoid the interference of host cell nucleic acid, and completely and efficiently detect 16SrDNA tag sequence of all bacteria varieties, so as to accurately determine the ecological structure of the intestinal flora.

Description

A kind of method setting up faecal microbiota gene library based on high flux gene sequencing

Technical field

The invention belongs to the molecular biology for detection technical field of antibacterial in biological technical field, particularly relate to one Plant the method setting up faecal microbiota gene library based on high flux gene sequencing.

Background technology

Enteric microorganism is more and more explained with diet and healthy relation in recent years, has benefited to a great extent The fast development of enteric microorganism investigative technique.The research of enteric microorganism substantially experienced by and cultivates the method relied on, non-cultivation The conventional molecular biological method relied on, 3 stages of high flux group method based on order-checking.

By comparing feature and the application of 3 stage research methoies, people may find that cultivating the method relied on is identifying Can obtain corresponding bacterial strain while strain, facilitate follow-up study, but culture method itself is wasted time and energy, enteric microorganism is with anaerobism Bacterium and facultative anaerobe are main, cultivate more difficult, and strain changes than regular meeting during cultivating so that There is bottleneck in its application.The non-conventional molecular biological method relied on of cultivating directly can extract intestinal without cultivating from sample Microbial genome, utilizes molecular biology method to carry out separating, identifies and quantitatively so that it is result can be reacted more accurately The composition of high abundance strain and actual proportions in enteric microorganism.

In page 1,122 1124 of periodical Journal of Clinical Microbiology, Kevin Jeng is open The quick detection of Salmonella, entitled Application of a 16S rRNA PCR High-Resolution Melt Analysis Assay for Rapid Detection of Salmonella Bacteremia.This invention is by for sand Door Salmonella reactivity serotype enteritis bacteremia patients is analyzed, to determine its infection conditions.

But conventional molecular biological method also exists the defect that flux is low, targeted approach such as real-time quantitative PCR is the most only Energy research one or a class enteric microorganism, rather than the gradient deformation gel electrophoresis (Denaturing that targeted approach is the most the most frequently used Gradient gel electrophoresis, DGGE) it is limited to sensitivity, often can only study abundant micro-life in intestinal Thing.Intestinal microflora composition complexity, every kind of antibacterial all forms the mutual relation network of complexity with other antibacterials, low-abundance The role that the same performer of strain is important, so conventional molecular biological result of study often has one-sidedness.

16SrDNA, because of its sequence great diversity between species, becomes " molecular clock " of systematic bacteriology research, has 9 Individual variable region and 9 spaced features in conserved region, variable region is different because of antibacterial, and the phylogeny of degree of variation and antibacterial Closely related.Therefore the sequence by analyzing variable region i.e. can get the taxonomy feature of each antibacterial.

In conventional molecular biological method, the most also the 16SrDNA of single culture is checked order and reflects by application Sanger method Fixed, but it is low to be limited to flux, and efficiency is low, it is impossible to meet scientific research, clinical needs well.

Summary of the invention

It is an object of the invention to for solve the technical problem that above, it is provided that a kind of acquisition covers whole microbiologic population The method of situation, thinks that realizing quick, convenient and accurate detection people's Intestinal flora composition sets up base with research bacterium colony ecologic structure The bacterium colony data base of plinth.

Concrete, in order to solve the problems referred to above, the invention provides structure and the merit of the whole strain of a kind of enteric microorganism The method for building up in the target gene sequence storehouse of energy, the most concrete, it is provided that one sets up feces based on high flux gene sequencing The method of flora gene library, the method uses the method for " nest-type PRC " that 16S rDNA is carried out Enrichment Amplification, subtracts to greatest extent Lack host and food debris genome pollution, V3 and V6 has been combined simultaneously, carry out specific amplified and carry out large-scale parallel survey Sequence, it is thus achieved that the target gene sequence storehouse of flora.Utilize this sequence library, it is possible to by Bacteria identification subordinate (Genus) rank, be promoted to Plant (Species) rank.

Concrete, according to said method, wherein the method for " nest-type PRC " includes carrying out 16S from total genomic dna RDNA total length Enrichment Amplification, the primer sets used by amplification is made up of 3 pairs of PCR primer, and these 3 pairs of primer sequences are as follows:

1st pair of PCR primer sequence is:

SEQ ID NO.1:5 '-AGAGTTTGATCCTGGCTCA-3 ' (16S-27F)

SEQ ID NO.2:5 '-GGTTACCTTGTTACGACT-3 ' (16S-1492R)

2nd pair of PCR primer sequence is:

SEQ ID NO.3:5 '-CTCCTACGGGAGGCAGCA-3 ' (V3-357F)

SEQ ID NO.4:5 '-ATTACCGCGGCTGCTGG-3 ' (v3-534R)

3rd pair of PCR primer sequence is:

SEQ ID NO.5:5 '-CAACGCGAAGAACCTTAC-3 ' (v6-967F)

SEQ ID NO.6:5 '-GGGTTGCGCTCGTTG-3 ' (v6-1100R)

In above-mentioned primer sets, the region, site 27/1492 of the 1st couple of PCR primer sequence amplification bacterial 16 S rDNA, amplification is produced The a length of 1465bps of thing, about 15kb.

In above-mentioned primer sets, the region, site 357/534 of the 2nd couple of PCR primer sequence amplification bacterial 16 S rDNA V3, expand Increase product length and be about 177bps；

In above-mentioned primer sets, the region, site 967/1100 of the 3rd couple of PCR primer sequence amplification bacterial 16 S rDNA V6, expand Increase product length and be about 133bps；

To this end, present invention also offers a kind of method setting up faecal microbiota gene library based on high flux gene sequencing, The method comprises the steps:

Step one, extraction faecal microbiota total genomic dna；

Step 2, with above-mentioned 1st pair of primer total genomic dna carried out 16S rDNA total length Enrichment Amplification, and use 16S The V3 district of rDNA and the 2nd pair, V6 district primer and the 3rd pair of primer carry out specific amplified respectively；

Step 3, carry out large-scale parallel order-checking, set up library.

In said method, the product of the specific amplified of step 2 is about 177bps and 133bps respectively.

In said method, the large-scale parallel order-checking of step 3 uses Ion Torrent individualized operation's gene order-checking Instrument carries out large-scale parallel order-checking.

In said method, step one is extracted faecal microbiota total genomic dna and is tried according to according to (TIANGEN company DP328) Condition described in agent box operating instruction is extracted faecal microbiota total genomic dna and is carried out, it is preferred that carry out condition according to intestinal Optimizing, the condition after optimization is as follows:

The cracking temperature of sample pretreatment being adjusted to 95 DEG C and hatches 10min, the Gram-positive that can make more difficult breaking cellular wall is thin Bacterium cracking breaking cellular wall so that extract product can cover whole microorganism, makes DNA structure integrity keep optimum state simultaneously；Sample After product cracking, on the basis of original one piece, then add one piece of inhibitor suction sheet InhibitEX, fecal specimens can be directed to Impurity removal PCR inhibitory action.

Preferably, in above-mentioned steps two, the method that total genomic dna carries out 16S rDNA total length Enrichment Amplification is " nest Formula PCR tri-one-step circulation method ".

Concrete, the operating procedure of nest-type PRC three one-step circulation method is:

First antibacterial total genomic dna being diluted to concentration is 1ng/ul, and taking 1 μ l is the template mould as amplification for the first time Plate, carries out PCR amplification with the 1st pair of primer, obtains the PCR primer of 16S rDNA；Second time amplification, with its amplification enrichment of 1ul It is template that the PCR primer of 16S rDNA dilutes 50 times, and primer is the 2nd pair of primer and the 3rd pair of primer, by identical dosing system with anti- Condition is answered to carry out secondary amplification.

Wherein the reaction system of nest-type PRC three one-step circulation method is: PCR is the 50 total systems of μ l, specifically 2 × Taq PCR StarMix with Loading Dye 25 μ l, Primer-F/Primer-R 2 μ l, thin to 1ng/ below μ l of concentration dilution Bacterium gDNA template 2ul, supplies 50 μ l systems with aquesterilisa.

Wherein the reaction condition of nest-type PRC three one-step circulation method is: 95 DEG C of denaturations 3min, then 95 DEG C of degeneration 30sec, 58 DEG C annealing 30sec, 72 DEG C extend 30sec, altogether circulation 28 times, last 72 DEG C re-extend 5min.

In said method, 16S rDNA and V3, V6 district PCR primer authentication method be: takes after 5 μ l have expanded for the first time V3, V6 district PCR primer of 16S rDNA product and second time amplification directly runs 1% agarose gel electrophoresis.

V3, V6 district PCR primer recovery method is: utilize Backman brand AgencourtAMPure magnetic bead by sample and magnetic Bead is amassed 1.5 times of volume purification and is reclaimed purpose fragment.

In said method, it is preferred that the operating procedure of step 3 is as follows:

(1) end-filling DNA, purification reclaim purpose fragment: utilize Backman brand AgencourtAMPure magnetic bead by Sample reclaims purpose fragment with 1.5 times of volumes of magnetic bead volume (75 μ L) purification；

(2) jointing, nick translation, purification recovery purpose fragment: utilize Backman brand AgencourtAMPure Magnetic bead is pressed sample and is reclaimed purpose fragment with 1.2 times of volumes of magnetic bead volume (120 μ L) purification；

(3) amplified library, purification reclaim purpose fragment: utilize Backman brand AgencourtAMPure magnetic bead by sample Purpose fragment is reclaimed with 1.2 times of volumes of magnetic bead volume (120 μ L) purification；

(4) library Quality Control and decision template dilution gfactor:

Detectable concentration: take the library after 2 μ l amplifications, library is diluted to the dilution gfactor of final concentration 100pM, passes through Qubit 2.0HS DNA luciferase assay reagent carries out Concentration Testing；

Detection fragment distribution: according to Qubit detectable concentration, testing sample is diluted to 1ng/ μ l, passes through High Sensitivity DNA Assay test kit chip detection；

Respectively V3, V6 equimolar concentration is mixed.Take 100ng as building storehouse initial amount.

(5) prepared by template: use Ion OneTouch 200Template Kit OT2 test kit and OneTouch instrument Carry out emulsion-based PCR；Use MyOne beads magnetic bead and Ion OneTouch ES instrument to carry out ISPs enrichment, finally give 3 ' ends Positive template library with magnetic bead；

(6) order-checking and sequencing result analysis: by the library for preparing at the Ion PGM of Life technologies company Checking order on sequencer high-flux sequence instrument, sample generation sequencing reading length is at 200bp, and depth requirements is at more than 100X, number According to amount at more than 0.05M reads；Through FastQC-3.4.1.1 analyze sequencing quality control, use QIIME software, by with data base Comparison, can obtain the ecological species composition of flora, ratio.By obtaining the ecological species composition of flora, ratio judges advantage Inferior position bacterium situation.

In said method, purification recovery product Quality Control in V3, V6 district is built storehouse initial amount with decision and is:

(1) detectable concentration: take 2 μ l V3, V6 district purification and reclaim product, by Qubit 2.0HS DNA luciferase assay reagent Carry out Concentration Testing；

(2) detection fragment distribution: according to Qubit detectable concentration, testing sample is diluted to 1ng/ μ l, passes through High Sensitivity DNA Assay test kit chip detection.

Present invention also offers a kind of primer carried out from total genomic dna used by 16S rDNA total length Enrichment Amplification Group, this primer sets is made up of 3 pairs of PCR primer, and these 3 pairs of primer sequences are as follows:

1st pair of PCR primer sequence is:

SEQ ID NO.1:5 '-AGAGTTTGATCCTGGCTCA-3 '

SEQ ID NO.2:5 '-GGTTACCTTGTTACGACT-3 '

2nd pair of PCR primer sequence is:

SEQ ID NO.3:5 '-CTCCTACGGGAGGCAGCA-3 '

SEQ ID NO.4:5 '-ATTACCGCGGCTGCTGG-3 '

3rd pair of PCR primer sequence is:

SEQ ID NO.5:5 '-CAACGCGAAGAACCTTAC-3 '

SEQ ID NO.6:5 '-GGGTTGCGCTCGTTG-3 '.

The present invention compared with prior art has the advantage that

(1) building storehouse speed fast, flux is big: detection method has been arranged in pairs or groups the fastest high-flux sequence platform life brand Ion Torrent PGM sequenator, up to a hundred sample parallel detections, upper machine order-checking the time be only 3 hours, library meets section Grind, clinical needs；

(2) library high specific, high sensitivity: V3 and V6 combines, and flora, from identifying " belonging to (Genus) " rank, promotes To the rank identifying " planting (Species) ".Meanwhile, the method for " nest-type PRC " is selected to carry out Enrichment Amplification to 16S rDNA, Decrease host and food debris genome pollution to greatest extent；

(3) library is set up and is used biopsy in situ: forms with the flora of fresh or frozen sample in-situ biopsy feces, is not required to Through screening and culturing, thus original flora ecology is kept to form.

(4) present invention is with high flux group as means, it is possible to quickly, convenient, synchronize, efficiently, high special, examine with sensitivity Measure the ecological composition of original flora, it is thus achieved that cover the library information of whole microbiologic population, be truly realized life micro-to intestinal The holistic approach of the 26S Proteasome Structure and Function of the whole strain of thing.

Accompanying drawing explanation

Fig. 1 is the testing result of embodiment faecal microbiota total genome extracting.

Fig. 2 is the testing result of embodiment faecal microbiota 16SrDNA amplification.

Fig. 3 is embodiment faecal microbiota V3, the testing result of V6 amplification.

Fig. 4 is 2100 testing results that embodiment faecal microbiota V3 amplification purification reclaims.

Fig. 5 is 2100 testing results that embodiment faecal microbiota V6 amplification purification reclaims.

Fig. 6 is 2100 testing results after embodiment faecal microbiota V3, V6 library construction.

Fig. 7 is the data volume result after embodiment faecal microbiota V3, the upper machine of V6 library order-checking.

Fig. 8 is embodiment faecal microbiota V3, the upper machine post-fragment distribution results of V6 library order-checking.

Fig. 9 is microorganism percentage ratio thermal map and the block diagram of embodiment faecal microbiota.

Figure 10 is the microorganism richness block diagram of embodiment faecal microbiota.

In accompanying drawing 1-8, the implication of foreign language is as follows:

M refers to maker 1kb, DNA molecular amount standard；

Bp, base pair number

16s rDNA, 16s rDNA；

Kb, 1,000 base pair numbers

V3, ribosome 16s DNA V3 district；

V6, ribosome 16s DNA V6 district；

FU, value of electrical signals；

Total Bases, total amount of data；

M, Mb, Mbytes；

Key Signal, key signal value；

ISP Loading, Ion Sphere Particles Loading, ion ball particle coverage；

ISP Density, Ion Sphere Particles Density, ion ball particle density；

Wells, nanometer hole count；

GZ-170-2015.09.08-16s v3v6-200, order-checking serial number and order-checking sample name；

Loading Density, Ion Sphere Particles Density average, ion ball ion concentration is put down Average；

(Avg-76%), this ion ball ion concentration measure meansigma methods be 76%, normal range be 65-85% it Between, belong to preferred values.

None bp Mean, without meansigma methods；

Median, intermediate value；

Mode, mode；

Read Length, sequencing sequence length；

Read length Histogram sequencing sequence length histogram；

Count, count value；

In accompanying drawing 9, the implication of foreign language is as follows:

Color Key and Histogram, color signal and block diagram；

Value, value；

Count, timing point；

AB1.1, carries the fecal specimens 1 that clostridium difficile AB toxin is positive

AB1.2, carries the fecal specimens 2 that clostridium difficile AB toxin is positive

NC, normal control, blank

Sequence number	English	Chinese translation
			1	Enhydrobacter	Aquatic Pseudomonas
2	Sphingomonas	Sphingol single-cell
			3	Clostridium	Clostridium；Fusobacterium
4	SMB53	SMB53 Pseudomonas
			5	Enterococcus	Enterobacter
6	Ruminococcus	Ruminococcus
			7	Parabacteroides	Purple zygosaccharomyces
8	Lactobacillus	Lactobacillus
			9	Dorea	Delhi Austria Pseudomonas
10	Anaerotruncus	Anaerotruncus Pseudomonas
			11	Kaistobacter	Sphingolipid Zymomonas mobilis
12	Oscillospira	Oscillospira
			13	Lachnospira	Lachnospira
14	Fusobacterium	Fusobacterium
			15	Arcobacter	Arch bar Pseudomonas
16	Roseburia	Bordetella
			17	Bacteroides	Bacteroides
18	Staphylococcus	Staphylococcus
			19	Acinetobacter	Acinetobacter
20	Chryseobacterium	Chryseobacterium
			21	Faecalibacterium	Faecalibacterium Pseudomonas
22	Nesterenkonia	Genus Nesterenkonia
			23	Deinococcus	Abnormal cocci belongs to
24	Allobaculum	Allobaculum Pseudomonas
			25	Phascolarctobacterium	Koala Bacillus
26	Sutterella	Saudi Bordetella
			27	Coprococcus	Coprecoccus
28	Propionibacterium	Propionibacterium
			29	Corynebacterium	Corynebacterium

In accompanying drawing 10, the implication of foreign language is as follows:

Sequence number	English	Chinese translation
			1	Arcobacter	Arch bar Pseudomonas
2	Chryseobacterium	Chryseobacterium
			3	Phascolarctobacterium	Koala Bacillus
4	Propionibacterium	Propionibacterium

5	Deinococcus	Abnormal cocci belongs to
			6	Roseburia	Bordetella
7	Nesterenkonia	Genus Nesterenkonia
			8	Coprococcus	Coprecoccus
9	Lactobacillus	Lactobacillus
			10	Anaerotruncus	Anaerotruncus Pseudomonas
11	Lachnospira	Lachnospira
			12	Allobaculum	Allobaculum Pseudomonas
13	Faecalibacterium	Faecalibacterium Pseudomonas
			14	Fusobacterium	Fusobacterium
15	SMB53	SMB53 Pseudomonas
			16	Dorea	Delhi Austria Pseudomonas
17	Oscillospira	Oscillospira
			18	Sphingomonas	Sphingol single-cell
19	Staphylococcus	Staphylococcus
			20	Parabacteroides	Purple zygosaccharomyces
21	Sutterella	Saudi Bordetella
			22	Kaistobacter	Sphingolipid Zymomonas mobilis
23	Acinetobacter	Acinetobacter
			24	Bacteroides	Bacteroides
25	Enterococcus	Enterobacter
			26	Enhydrobacter	Aquatic Pseudomonas
27	Clostridium	Clostridium；Fusobacterium
			28	Corynebacterium	Corynebacterium
29	Ruminococcus	Ruminococcus

AB1-1, carries the fecal specimens 1 that clostridium difficile AB toxin is positive

AB1-2, carries the fecal specimens 2 that clostridium difficile AB toxin is positive

NC, normal control, blank

Detailed description of the invention

Below by combining the drawings and specific embodiments, technical scheme is described in further details, but this Bright it is not limited to following example.There is provided for Guangzhou servicemen norm one group of material used in embodiment be diagnosed as carry difficult Patient's feces that difficult clostridium AB toxin is positive.

The experiment flow setting up library of the present invention comprises the following steps that.

1. extraction faecal microbiota total genomic dna:

Excrement is extracted in strict accordance with TIANamp Stool DNA Kit (TIANGEN company DP328) test kit operating instruction Just flora total genomic dna, and carried out condition optimizing for intestinal: the cracking temperature of sample pretreatment is adjusted to 95 DEG C Hatch 10min, the gram-positive bacterium cracking breaking cellular wall of more difficult breaking cellular wall can be made, it is ensured that extract product can cover whole micro-life Thing, makes DNA structure integrity keep optimum state simultaneously；After sample dissociation, then add one piece of inhibitor suction sheet InhibitEX, can be directed to fecal specimens Impurity removal PCR inhibitory action.Concrete operations are as follows:

1.1 extract faecal microbiota total genomic dna

(1) weigh in fecal sample 50mg to 1.5ml centrifuge tube, and pipe is placed on ice.

(if in liquid sample then transferase 12 00 μ l to centrifuge tube)

(2) adding 1.5ml buffer GSL in sample, intermittent oscillation 3min to sample mixes.

Hatch 10min for (3) 95 DEG C.

(4) vortex 15sec.12,000rpm is centrifuged 1min.Supernatant 1ml is to new 1.5ml centrifuge tube in transfer.

(5) adding one piece of inhibitor suction sheet InhibitEX, vibration is thoroughly opened resuspended to suction sheet.Incubated at room 5min, makes suction sheet fully to act on.

(6) 12,000rpm is centrifuged 3min.

(7) previous step gained supernatant is transferred to new 1.5ml centrifuge tube, repeats step 6.

(8) transfer gained supernatant 400 μ l is to new 1.5ml centrifuge tube, adds 20 μ l ProteinaseK.

(9) 400 μ l buffer GB, vortex 15sec are added.

Hatch 10min for (10) 70 DEG C.

(11) adding 400 μ l dehydrated alcohol, vortex mixes.

(12) previous step gained solution joins in an adsorption column 12, and 000rpm is centrifuged 30sec, outwells waste liquid, will Adsorption column is put in collecting pipe.

(13) adding 500 μ l buffer GD in adsorption column, 12,000rpm are centrifuged 30sec, outwell waste liquid, by adsorption column Put in collecting pipe.

(14) adding 600 μ l rinsing liquid PW in adsorption column CR2,12,000rpm are centrifuged 30sec, outwell waste liquid, adsorption column CR2 puts in collecting pipe.

(15) repetitive operation step 14.

(16) putting back in collecting pipe by adsorption column, 12,000rpm are centrifuged 2min, outwell waste liquid.Adsorption column is placed in room temperature Place several minutes, thoroughly to dry rinsing liquid remaining in adsorbing material.

(18) adsorption column is proceeded in a clean centrifuge tube, to the middle part of adsorbed film unsettled dropping 50 μ lpH value The seedless sour water of 7.5, is placed on 65 DEG C of baking oven 5min, and 12,000rpm are centrifuged 2min, are collected in centrifuge tube by solution.DNA produces Thing should be saved in-20 DEG C.

1.2 detection of flora total genome concentration and Quality Controls

To extracting after DNA fragmentation trace ultraviolet spectrophotometer and 1% agarose gel electrophoresis detectable concentration with pure Degree.DNA should have notable absworption peak at OD260, and OD260/OD280 ratio should be 1.8-2.0, and concentration is 500ng/ μ l.Electrophoresis In glue figure, at about 15kb, band is single clearly, illustrates that DNA is up-to-standard.Antibacterial total genome electrophoretogram is as shown in Figure 1: M: Marker；1-18: flora total genomic dna.

2. design PCR primer enrichment 16SrDNA and capture V3, V6 district

2.1 enrichment 16SrDNA and capture V3, V6 district

Design 3 carries out 16S rDNA total length Enrichment Amplification to the PCR primer total genomic dna to extracting, it is ensured that expand The product increased can be completely covered high Sudden change region；Then taking 1 μ lPCR product is template, with V3 district and the V6 district of 16S rDNA Special primer carries out special capture respectively.

One couple of PCR primers sequence is:

16S-27F:5’-AGAGTTTGATCCTGGCTCA-3’

16S-1492R:5’-GGTTACCTTGTTACGACT-3’

The region, site 27/1492 of amplification bacterial 16 S rDNA, a length of 1465bps of amplified production, about 15kb；

Second pair of PCR primer sequence is:

V3-357F:5’-CTCCTACGGGAGGCAGCA-3’

v3-534R:5’-ATTACCGCGGCTGCTGG-3’

The region, site 357/534 of amplification bacterial 16 S rDNA V3, amplified production length is about 177bps；

3rd pair of PCR primer sequence is:

v6-967F:5’-CAACGCGAAGAACCTTAC-3’

v6-1100R:5’-GGGTTGCGCTCGTTG-3’

The region, site 967/1100 of amplification bacterial 16 S rDNA V6, amplified production length is about 133bps；

Following reaction system: PCR is coordinated to be the 50 total systems of μ l with above-mentioned primer:

Composition	Volume (μ L)
		2×Taq PCR StarMix with Loading Dye	25
Primer-F/Primer-R	2
		DNA	1
Nuclease-Free Water	22
		Total	50

First antibacterial total genomic dna is diluted to concentration is 1ng/ μ l, and taking 1 μ l is template reaction condition for " nest-type PRC Three one-step circulation methods "

Second time amplification, with PCR primer of the 16SrDNA of its amplification enrichment of 1ul dilute 50 times as template, respectively with V3 and V6 district two, to primer, carries out secondary amplification by identical dosing system and reaction condition.The concentration dilution of purpose template is to 1ng/ μ l Hereinafter be conducive to improving the specificity of PCR.

2.2 16SrDNA and V3, V6 district PCR primer are identified

V3, V6 district PCR primer taking the 16SrDNA product after 5 μ l have expanded for the first time and second time amplification directly runs 1% Agarose gel electrophoresis, as shown in Figure 2 and Figure 3.Fig. 2, amplified production 16S rDNA are about 15kb；Fig. 3, V3, V6 district amplified production It is about 177bps and 154bps respectively.

2.3V3, V6 district PCR primer magnetic bead reclaims

Backman brand AgencourtAMPure magnetic bead is utilized to reclaim mesh by 1.5 times of volume purification of sample and magnetic bead volume Fragment.Concrete grammar is as follows:

(1) by sample moisturizing to 50 μ l, adding the magnetic bead (75 μ l) of 1.5 times of volumes, with pipettor piping and druming mixing, room temperature is incubated Educate 5 minutes；

(2) sample pipe is placed on magnetic frame, waits 2-3 minute until solution is clarified completely in pipe.

(3) careful Aspirate supernatant, removes and discards supernatant.

(4) add the 500 fresh configurations of μ L 70% alcoholic solution.Clean magnetic bead by rotary tube, rotate 180 °.Altogether Rotate 5 times thoroughly to clean.Note: rotate and magnetic can be utilized to break up pearl, remove substrate.

(5) ethanol of residual removed by placement sample pipe after removing supernatant, then rotating centrifugal after clarifying on magnetic frame Solution.

(6) magnetic bead is air-dried the most completely 5 minutes, until magnetic bead surfaces is dried, no liquid gloss.

(7) 25 μ L LowTE are added in sample pipe and shake 10 seconds.With rifle head upper and lower pressure-vaccum solution repeatedly to ensure to wash Take off and mix completely.

(8) sample pipe is placed on magnetic frame, after solution is clarified, the solution (supernatant) of eluting is transferred to one Preserve in individual new .5ml low absorption pipe.

2.4V3, V6 district purification reclaims product Quality Control and decision and builds storehouse initial amount

(1) Qubit detectable concentration

Take 2 μ l V3, V6 district purification and reclaim product, carry out concentration inspection by Qubit 2.0HS DNA luciferase assay reagent Survey.Testing result: V3 product be 53ng/ μ l, V6 product be 25ng/ μ l.From concentration, the yield phase that this magnetic bead reclaims is described For cutting glue absorption method more efficient, compensate for cutting glue method simultaneously and cannot remove the deficiency that large fragment is polluted.

(2) Agilent 2100 detects fragment distribution

According to Qubit detectable concentration, testing sample is diluted to 1ng/ μ l, by High Sensitivity DNA Assay test kit chip detection.Testing result: V3 district main peak is 179bp and 205bp size；V6 district main peak is 154bp size. From fragment distribution, V3, V6 district band all meets expection size, there are two main peaks for V3 district, and the main peak of 205bp may It it is the amplified production of the distinctive dominant bacteria of intestinal.Clip size, between 160-200, is conducive to building 200base and reads long survey Sequence DNA library.Agilent 2100 instrument the clip size in library is detected such as Fig. 4,5.

3. large-scale parallel order-checking

Library construction uses the DNA library of life brand to build test kit, according to the Standard Operating Procedure of 200bp amplicon Carry out building storehouse, and the segment characterizations for intestinal 16SrDNA is optimized.Library construction is before and after target sequence two End is plus the site from primer coupling and for identifying that different samples insert special signature's double-stranded adapters of about ten bases.This mark Sign joint, provide coupling site for amplified reaction below and order-checking and identify signal.

3.1 end-filling DNA

(1) 1.5ml in low absorption mixes in managing and mixes following agent formulations, is then placed within room temperature condition hatching 30 minutes.

Composition	Volume (μ L)
		DNA 100ng input	44
10×End-Repair Buffer	5
		End Repair Enzymes	1
Total	50

(2) utilize Backman brand Agencourt AMPure magnetic bead by sample and 1.5 times of volumes of magnetic bead volume (75 μ L) Purification reclaims purpose fragment, and this concentration of empirical tests can ensure that and retains target main peak 200base in Piece Selection to greatest extent, removes The dimeric pollution of joint.

3.2 jointings, nick translation

(1) mix in coupled reaction system and mix following agent formulations.

Composition	Volume (μ L)
		DNA	25
Ion P1Adapters	2
		Ion Xpress Barcode X	2

10×Ligase Buffer	10
		DNA ligase	2
Nuclease-free water	49
		dNTP Mix	2
Nick Repair Polymerase	8
		Total	100

(2) hatch according to following procedure in PCR instrument:

Temperature (DEG C)	Time
		25	25min
72	5min
		4	∞

(3) utilize Backman brand Agencourt AMPure magnetic bead by sample and 1.2 times of volume (120 μ of magnetic bead volume L) purification reclaims purpose fragment, and this concentration of empirical tests can ensure that and retains the target master after adding joint in Piece Selection to greatest extent Peak.

3.3 amplified library

(1) mix in PCR pipe and mix following agent formulations.

Composition	Volume (μ L)
		Platinum HiFi	70
Library Amplification Primer Mix	5
		Adaptor-ligated DNA	25
Total	100

(2) following procedure is set and carries out PCR reaction.

(3) utilize Backman brand Agencourt AMPure magnetic bead by sample and 1.2 times of volume (120 μ of magnetic bead volume L) purification reclaims purpose fragment, and this concentration of empirical tests can ensure that and retains target main peak 300 in Piece Selection to greatest extent, goes simultaneously Except joint dimer and primer pollute.

3.4 library Quality Controls and decision template dilution gfactor

(1) Qubit detectable concentration

Take the library after 2 μ l amplifications, carry out Concentration Testing by Qubit 2.0HS DNA luciferase assay reagent.Detection knot Really: V3 and V6 fragment mixing library is 1.68ng/ μ l.From concentration, in 1-10ng/ μ l scope, the most not excessive amplification and lose Very.(2) Agilent 2100 detects fragment distribution

According to Qubit detectable concentration, testing sample is diluted to 1ng/ μ l, by High Sensitivity DNA Assay test kit chip detection.Testing result: V3 and V6 fragment mixing library is 237bp, 263bp and 288bp size；This be Target sequence adds two end connector 85bp, and to be actually inserted into fragment be 152bp, 178bp and 203bp, and this adds before building storehouse V3 and V6 district fragment template is completely the same, and size meets order-checking length requirement, and this batch sample library construction quality is thus described Qualified.Library is diluted to the dilution of final concentration 100pM by the molar concentration of the 50-1000bp scope according to Agilent 2100 offer The factor.16SV3V6 library Agilent 2100 testing result is shown in Fig. 6.

Prepared by 3.5 templates

After library construction completes, by millions of times of sequence cluster amplification in water-in-oil emulsion PCR, and enter as template Machine order-checking on row.Empirical tests takes the 100pM library dilution gfactor of 6 μ l can reduce the polyclone phenomenon that main peak disperses to bring, and can obtain Obtain more sequenator data volume.Use Ion OneTouch 200Template Kit OT2 test kit and OneTouch instrument Carry out emulsion-based PCR；Use MyOne beads magnetic bead and Ion OneTouch ES instrument to carry out ISPs enrichment, finally give 3 ' ends Positive template library with magnetic bead.

3.6Ion Torrent PGM checks order

Before PGM sequenator is initialized, first press Ion PGM Sequencing 200kit v2 test kit and clean standard Sequenator pipeline priority chlorine water and 18M Ω deionized water are cleaned 15min by operating process.Then, according to installing W1, W2, W3 Reaction solution carries out initializing regulation pH value, and correction to pH is 7.5-7.6.After initialization, with 100% isopropanol and annealing buffer Liquid Annealing buffer cleans 314 sequence testing chips, is then put into reacting hole signal detection on PGM sequenator.Meanwhile, Xiang Fu The library collected adds sequencing primer, library internal reference Control Ion Spheres.Then 95 DEG C are put in PCR instrument, 2min； 37 DEG C, 2min anneals degeneration, adds order-checking polymerase, is loaded on sequence testing chip by library sample.Finally, chip is put into On PGM sequenator, arranging 500 flow order-checking circulations, react two hours, order-checking completes.

3.7 sequencing result analyses

Sample is checked order by upper machine, and the most original sequencing data obtained stores with the form of FASTQ form.

This sequencing data result is reported: the 314 actually measured effective coverages of gene chip reach 76%, and finally produce is original Sequencing data total amount is 45.1Mb；Sequencing reading length be 150bp, 179,205bp, all reflect the purpose fragment physical length of insertion. (it is the data volume result after embodiment faecal microbiota V3, the upper machine of V6 library order-checking see Fig. 7；Fig. 8 be embodiment faecal microbiota V3, The upper machine post-fragment distribution results of V6 library order-checking)

The FASTQ result that this 16S rRNA order-checking obtains, (does not has by using QIIME software to carry out operating unit classification Filter), there is the operating unit oneself representing sequence respectively, used the classification feature inside QIIME, according to The representative sequence of Greengene database classification OTU, obtaining Qi Jie, door, guiding principle, mesh, section, genus is that microorganism respectively. Classification analysis result obtains 29 can be categorized into the microorganism dominant genera belonged to and more than 0.001 (0.1%), and paints according to result Fig. 9 microorganism percentage ratio thermal map and block diagram and Figure 10 microorganism richness block diagram are made.

According to the analysis result of classification analysis, data go to show by more intuitive method.

According to Fig. 9, microorganism percentage ratio thermal map, can more preferably observe the microorganism difference condition in different sample:

According to sample clustering situation it is known that than sample just carry the similarity of clostridium difficile Patient Sample A AB1.1 and AB1.2 The situation of ordinary person NC (blank) closer to, and can substantially observe the microorganism degree difference at different sample rooms Not.

According to Figure 10, microorganism richness block diagram, 33 microorganism dominant generas in three groups of samples can be clearly apparent and account for The situation accounting for percentage ratio of superiority bacteria spp in the situation of sample percentage ratio, preferably expression sample.As shown in Figure 10, Ruminococcus (about 9%-25%), Corynebacterium (about 3%-9%), Enhydrobacter (about 2%-5%), Clostridium (about 2%-7%), Enterococcus (about 1%-2%) are 5 bacterium of advantage respectively in 3 samples Belonging to, the Clostridium (fusobacterium) that wherein this experiment is paid close attention to the most degree in three groups of samples is NC respectively Sample is 2.65%, and AB1-1 sample is 5.61%, and AB1-2 sample is 7.04%.

As can be seen here, clostridium difficile belongs to patient and substantially increases than ratio contained by Normal human patients, its Bacterial community dynamic Also there is somewhat difference.

Claims (9)

1. the method setting up faecal microbiota gene library based on high flux gene sequencing, the method uses " nest-type PRC " Method carries out Enrichment Amplification to 16S rDNA, is combined by V3 and V6 simultaneously, carries out specific amplified and carry out large-scale parallel order-checking, Obtain the target gene sequence storehouse of flora.

Method the most according to claim 1, wherein the method for " nest-type PRC " includes carrying out 16S from total genomic dna RDNA total length Enrichment Amplification, the primer sets used by amplification is made up of 3 pairs of PCR primer, and these 3 pairs of primer sequences are as follows:

1st pair of PCR primer sequence is:

SEQ ID NO.1:5 '-AGAGTTTGATCCTGGCTCA-3 '

SEQ ID NO.2:5 '-GGTTACCTTGTTACGACT-3 '

2nd pair of PCR primer sequence is:

SEQ ID NO.3:5 '-CTCCTACGGGAGGCAGCA-3 '

SEQ ID NO.4:5 '-ATTACCGCGGCTGCTGG-3 '

3rd pair of PCR primer sequence is:

SEQ ID NO.5:5 '-CAACGCGAAGAACCTTAC-3 '

SEQ ID NO.6:5 '-GGGTTGCGCTCGTTG-3 '.

3. the method setting up faecal microbiota gene library based on high flux gene sequencing, the method comprises the steps:

Step one, extraction faecal microbiota total genomic dna；

Step 2, with above-mentioned 1st pair of primer total genomic dna carried out 16S rDNA total length Enrichment Amplification, and use 16S rDNA V3 district and the 2nd pair, V6 district primer and the 3rd pair of primer carry out specific amplified respectively；

Step 3, carry out large-scale parallel order-checking, set up library.

Method the most according to claim 3, wherein in step 2, carries out the enrichment of 16S rDNA total length to total genomic dna The method of amplification is " nest-type PRC three one-step circulation method ".

Method the most according to claim 4, wherein the operating procedure of nest-type PRC three one-step circulation method is:

First diluting 100 times with the antibacterial total genomic dna of 100ng/ μ l, taking 1 μ l is the template template as amplification for the first time, Carry out PCR amplification with the 1st pair of primer, obtain the PCR primer of 16S rDNA；Second time amplification, with the 16S of its amplification enrichment of 1ul It is template that the PCR primer of rDNA dilutes 50 times, and primer is the 2nd pair of primer and the 3rd pair of primer, with identical dosing system and reaction bar Part carries out secondary amplification.

Method the most according to claim 5, wherein the reaction system of nest-type PRC three one-step circulation method is: PCR is that 50 μ l are overall System, specifically 2 × Taq PCR StarMix with Loading Dye 25 μ l, Primer-F/Primer-R 2 μ l, concentration It is diluted to the DNA profiling of 1ng/ below μ l, supplies 50 μ l systems with aquesterilisa.

Method the most according to claim 5, wherein the reaction condition of nest-type PRC three one-step circulation method is: 95 DEG C of denaturations 3min, then 95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 30sec, altogether circulation 28 times, and last 72 DEG C re-extend 5min。

Method the most according to claim 3, wherein the operating procedure of step 3 is as follows:

(1) end-filling DNA, purification reclaim purpose fragment: utilize Backman brand AgencourtAMPure magnetic bead by sample Purpose fragment is reclaimed with 1.5 times of volumes of magnetic bead volume (75 μ L) purification；

(2) jointing, nick translation, purification recovery purpose fragment: utilize Backman brand AgencourtAMPure magnetic bead Purpose fragment is reclaimed with 1.2 times of volumes of magnetic bead volume (120 μ L) purification by sample；

(3) amplified library, purification reclaim purpose fragment: utilize Backman brand AgencourtAMPure magnetic bead by sample and magnetic Bead is amassed 1.2 times of volumes (120 μ L) purification and is reclaimed purpose fragment；

(4) library Quality Control and decision template dilution gfactor:

Detectable concentration: take the library after 2 μ l amplifications, library is diluted to the dilution gfactor of final concentration 100pM, passes through Qubit 2.0HS DNA luciferase assay reagent carries out Concentration Testing；

Detection fragment distribution: according to Qubit detectable concentration, testing sample is diluted to 1ng/ μ l, by High Sensitivity DNA Assay test kit chip detection；

(5) prepared by template: use Ion OneTouch 200Template Kit OT2 test kit and OneTouch instrument to carry out Emulsion-based PCR；Use MyOne beads magnetic bead and Ion OneTouch ES instrument to carry out ISPs enrichment, finally give 3 ' ends with The positive template library of magnetic bead；

(6) order-checking and sequencing result analysis: by the library for preparing at the Ion PGM of Life technologies company Checking order on sequencer high-flux sequence instrument, sample generation sequencing reading length is at 200bp, and depth requirements is at more than 100X, number According to amount at more than 0.05M reads；Through FastQC-3.4.1.1 analyze sequencing quality control, use QIIME software, by with data base Comparison, can obtain the ecological species composition of flora, ratio.By obtaining the ecological species composition of flora, ratio judges advantage Inferior position bacterium situation.

9. carrying out the primer sets used by 16S rDNA total length Enrichment Amplification from total genomic dna, this primer sets is right by 3 PCR primer forms, and these 3 pairs of primer sequences are as follows:

1st pair of PCR primer sequence is:

SEQ ID NO.1:5 '-AGAGTTTGATCCTGGCTCA-3 '

SEQ ID NO.2:5 '-GGTTACCTTGTTACGACT-3 '

2nd pair of PCR primer sequence is:

SEQ ID NO.3:5 '-CTCCTACGGGAGGCAGCA-3 '

SEQ ID NO.4:5 '-ATTACCGCGGCTGCTGG-3 '

3rd pair of PCR primer sequence is:

SEQ ID NO.5:5 '-CAACGCGAAGAACCTTAC-3 '

SEQ ID NO.6:5 '-GGGTTGCGCTCGTTG-3 '.