CN108070643A - The construction method of microorganism 16S rDNA single molecules level sequencing libraries - Google Patents
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Abstract
The construction method of microorganism 16S rDNA single molecules level sequencing libraries of the present invention is related to technological field of biochemistry.Its purpose is to provide a kind of construction method of microorganism 16S rDNA single molecules level sequencing libraries at low cost, easy to operate.The structure method of the present invention comprises the following steps：Collecting sample extracts DNA；Amplification；Purifying；It is quantitative；Sequencing, bioinformatic analysis.The construction method of the microorganism 16S rDNA single molecules level sequencing libraries of the present invention is at low cost, easy to operate, can simplify experiment flow and reduce sequencing cost.
Description
Technical field
The present invention relates to technological field of biochemistry, and chemicals storehouse is established with biochemical method more particularly to a kind of
Method.
Background technology
16S rDNA sequencings refer to carry out environmental samples 16S rDNA hypervariable regions PCR amplification and one kind of high-flux sequence
Technology can effectively identify the microbe species and abundance of bacterium under specific environment and ancient bacterium.By detecting 16S
The sequence variations and abundance of rDNA reflect the classification of bacterium and abundance in environmental samples.16S sequences are by 9 hypervariable regions (V1-V9)
Composition, centre are interspersed with conserved region, and to 16S rDNA sequencings, there are mainly three types of methods at present：Ce Dan V areas (V3/V4/V6) survey double
V areas (V3-V4 or V4-V5) survey three V areas (V1-V3, V5-V7, V7-V9) or survey entire 16s rDNA complete sequences.V4 at present
Area it is each level on species identification accuracy highest, but double V areas than single V region sequences reading it is long longer, V3-V4 areas be it is all can
Become longest in area, adding up length has 459bp, and information contained amount is maximum, and V3-V4 areas primer binding specificity is good, in bacterium
High with the coverage rate in ancient bacterium, once sequencing can the Distribution center of detection bacterium and ancient bacterium, and for bacterium point simultaneously
It is horizontal that Lei Shuan V areas have been accurate to category even kind, it is not necessary that surveys entire 16s rDNA genes.So 16S for comprehensive
The sequencing of rDNAV3-V4 areas is the optimal selection of microbial species identification.
The grand gene order-checking of 16S rDNA sequencings or Illumina Hiseq platforms based on Illumina Miseq,
The parallel sequencing of multiple samples can be disposably completed, environmental samples species taxonomy, species abundance, population structure, system are provided
It evolves, group such as compares at all multi informations.Sequencing at present builds the method in storehouse mainly based on two-step method and one-step method, one-step method should
With now more and more, advantage includes operating simpler, and reduces step and can reduce due to operating the error brought and shadow
It rings, also one-step method is more cost-effective from the perspective of costs.But existing one step amplification does not account for amplification in itself
To the influence that result is brought, because the preference sex chromosome mosaicism of amplification, causes homogeneity poor, it is impossible to accurately react the rich of objective flora
Degree.And the advantage of unimolecule 16S rDNA sequencing detections is exactly that can be as accurate as single grand genomic templates, can be accomplished definitely
Quantitative flora enrichment analysis, existing unimolecule 16S rDNA sequencing technologies are mainly to be put down using the sequencing of ion torrent
Platform, the sequencing principle of ion torrent is inherently wrapped up single template by micro-droplet of oil and is expanded, therefore can accomplish list
Molecule rank.The illumina microarray datasets being more commonly used at present can't realize the sequencing inspection of 16S rDNA unimolecule ranks
Survey analysis.
Therefore, exploitation one kind is needed to can be as accurate as based on illumina platforms on the basis of one-step method advantage is kept
16s rDNA unimolecule ranks build storehouse sequencing approach.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of microorganism 16S rDNA unimolecules at low cost, easy to operate
The construction method of horizontal sequencing library.
The present invention relates to a kind of construction method of microorganism 16S rDNA single molecules level sequencing libraries, the described method includes
Following steps：
(1) normal person's fecal sample is gathered, 1mL excrement is deposited in and preserves mixing in liquid (can be preserved 15 days with room temperature), adopt
With the fecal microorganism DNA extraction kit (Quick-DNA of ZYMO companies of Tian Mo companiesTM Fecal Microbe
Microprep Kit), to specifications the step of extract excrement macro genome DNA；
(2) macro genome DNA after extracting uses the concentration and purity of micro UV spectrophotometer measuring sample；
(3) multiplex amplifing reagents (the Multiplex PCR master of Invitrogen of hero company are used
Mix first step amplification) is carried out；Sense primer REP1 and anti-sense primer REP2 sequences such as SEQ ID NO.1 and SEQ ID NO.2 institutes
Show；As shown in Figure 1；
First step amplification using fecal microorganism DNA for the cycle of template amplification two, ensure two sections be connected with connector completely and
Single library fragments generate；
(4) using the product expanded for the first time as template, P1 the and P2 short primers for adding in both ends are expanded for second step amplification
30 Xun Huan amplified signals；The sequence of sense primer P1 and anti-sense primer P2 are as shown in SEQ ID NO.3 and SEQ ID NO.3；
(5) 10uL products therefroms after the amplification of two steps is taken to be detected into row agarose gel electrophoresis；Testing result is shown in Fig. 2.
V3-V4 areas clip size is 455bp, along with two end connectors and sequence label, comes to 641bp.Electrophoretogram shows that sample expands
Increase stripe size with being expected to be consistent, and band becomes clear no non-specific amplification band.
(6) after the amplification of two steps products therefrom carried out with the AMPure XP magnetic beads of Beckman＆Coulter companies it is pure
Change：75uL suspension containing magnetic beads are added in into reaction system, mixing is fully blown and beaten, is placed at room temperature for 5min；Supernatant waste is drawn, is used
80% ethyl alcohol is washed twice, is placed at room temperature for 5min and is dried magnetic bead, and the low TE elution magnetic beads for adding in 20ul obtain survey after purification
Preface storehouse；
(7) sequencing library after purification carries out accurate quantification respectively with Qubit 3.0 and Agilent 2100 and segment is big
Small analysis, as shown in figure 3, the sharp clip size of main peak is correct, no other segments pollution；
(8) dilution sequencing library is required according to microarray dataset, using result quantitative Qubit 3.0 as standard and according to data
Amount needs to carry out sample mixing, is finally sequenced using Illumina HiseqX or Miseq；
(9) bioinformatic analysis is carried out to sequencing result, that is, completes microorganism 16S rDNA single molecules levels sequencing text
The structure in storehouse merges the reads that all both ends labels repeat and sequence repeats, and single-ended label does not repeat after merging, Miseq
Sequencing PE300reads longer can be spliced, but hiseqX PE150 sequencing reading length it is shorter cannot survey logical target fragment,
Both ends reads needs separately to be analyzed.
Preferably, the micro ultraviolet specrophotometer model Nanodrop 2000.
Preferably, 23 μ L PCR reaction systems of step (3) first step amplification are as shown in table 1：
23 μ L PCR reaction systems of 1 first step of table amplification
| Into being grouped into | Volume/amount of DNA |
| Multiplex PCR master mix (Multiplex amplifing reagents) | 11.5ul |
| Sense primer REP1 (10uM) | 1ul |
| Anti-sense primer REP2 (10uM) | 1ul |
| Microbe genome DNA | 3ng |
| Water | Supply 23ul |
First step amplified reaction program is as shown in table 2：
2 first step amplified reaction program of table
Preferably, 50 μ L PCR reaction systems of step (4) the second step amplification are as shown in table 3：
50 μ L PCR reaction systems of 3 second step of table amplification
| Into being grouped into | Volume/amount of DNA |
| First step amplification system | 23uL |
| Multiplex PCR master mix | 25uL |
| Sense primer P1, concentration 10uM | 1uL |
| Anti-sense primer P2, concentration 10uM | 1uL |
Second step amplified reaction program is as shown in table 4：
4 second step amplified reaction program of table
The construction method difference from prior art of microorganism 16S rDNA single molecules level sequencing libraries of the present invention exists
In：
1st, the primer that the present invention designs is the long primer comprising random tags, can ensure a label by the amplification of two steps
Correspondence is uniquely amplified 16S rDNA templates, finally with regard to that can be accurate to unimolecule template when the analysis of sequencing result
Level can more accurately calculate the flora abundance and composition of 16S rDNA.
2nd, the two-step amplification method of unique design of the invention, can be to the 16S rDNA of sample to be tested by first step amplification
Template carries out adjunction head, and both ends mark unique label simultaneously, and second step amplification is effective to be expanded signal but do not increase number of tags
Amount, convenient for subsequent sequencing and analysis.
3rd, the present invention is realized through illumina microarray dataset sequencing analysis unimolecule 16S rDNA, has been broken originally
The situation of unimolecule 16S rDNA can only be analyzed by ion torrent microarray datasets, simplify experiment flow and reduce sequencing into
This.
4th, the target fragment that long primer provided by the present invention is expanded is the V3-V4 areas of 16S rDNA, with only expanding 16S
The single sections of rDNA compared to expanding fragment length it is longer, comprising information content it is more, resolution ratio higher, convenient for distinguish kind between
Difference.
Description of the drawings
Fig. 1 is to draw in being expanded for the first time in the construction method of microorganism 16S rDNA single molecules level sequencing libraries of the present invention
The sequence chart of object, wherein, N：Uncertain base, index：Split sequence label；UID：Random tags；16S rDNA primer：
16S rDNA primer sequences；
After Fig. 2 is expands using two steps in the construction method of microorganism 16S rDNA single molecules level sequencing libraries of the present invention
Agarose gel electrophoresis figure, wherein left side for two steps amplification after product electrophoretogram, right side be DNA marker；
Fig. 3 is 16S rDNA single molecules level sequencing libraries 2100 Quality Control peak figure after magnetic beads for purifying；
Fig. 4 is composition and ratio on the enteric microorganism 16S rDNA of checking test sample belong to horizontal.
Specific embodiment
By following embodiment and checking test to microorganism 16S rDNA single molecules level sequencing libraries of the invention
Construction method is further described.
Embodiment 1
The construction method of the microorganism 16S rDNA single molecules level sequencing libraries of the present embodiment carries out according to the following steps：
(1) normal person's fecal sample is gathered, 1mL excrement is deposited in and preserves mixing in liquid (can be preserved 15 days with room temperature), adopt
With the fecal microorganism DNA extraction kit (Quick-DNA of ZYMO companies of Tian Mo companiesTM Fecal Microbe
Microprep Kit), to specifications the step of extract excrement macro genome DNA；
(2) macro genome DNA after extracting uses the concentration and purity of micro UV spectrophotometer measuring sample, micro
Ultraviolet specrophotometer model Nanodrop 2000；
(3) multiplex amplifing reagents (the Multiplex PCR master of Invitrogen of hero company are used
Mix first step amplification) is carried out；23 μ L PCR reaction systems of first step amplification are as shown in table 1, and first step amplified reaction program is such as
Shown in table 2, primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2；As shown in Figure 1；
First step amplification using fecal microorganism DNA for the cycle of template amplification two, ensure two sections be connected with connector completely and
Single library fragments generate；
(4) using the product expanded for the first time as template, P1 the and P2 short primers for adding in both ends are expanded for second step amplification
30 Xun Huan amplified signals, 50 μ L PCR reaction systems of second step amplification are as shown in table 3, second step amplified reaction program such as table
Shown in 4；The sequence of P1 and P2 short primers is as shown in SEQ ID NO.3 and SEQ ID NO.3；
(5) 10uL products therefroms after the amplification of two steps is taken to be detected into row agarose gel electrophoresis；Testing result is shown in Fig. 2.
V3-V4 areas clip size is 455bp, along with two end connectors and sequence label, comes to 641bp.Electrophoretogram shows that sample expands
Increase stripe size with being expected to be consistent, and band becomes clear no non-specific amplification band.
(6) after the amplification of two steps products therefrom carried out with the AMPure XP magnetic beads of Beckman＆Coulter companies it is pure
Change：75uL suspension containing magnetic beads are added in into reaction system, mixing is fully blown and beaten, is placed at room temperature for 5min；Supernatant waste is drawn, is used
80% ethyl alcohol is washed twice, is placed at room temperature for 5min and is dried magnetic bead, and the low TE elution magnetic beads for adding in 20ul obtain survey after purification
Preface storehouse；
(7) sequencing library after purification carries out accurate quantification and clip size point with Qubit 3.0 and Agilent 2100
Analysis；
(8) dilution sequencing library is required according to microarray dataset, using result quantitative Qubit 3.0 as standard and according to data
Amount needs to carry out sample mixing, is finally sequenced using Illumina HiseqX；
(9) bioinformatic analysis is carried out to sequencing result, that is, completes microorganism 16S rDNA single molecules levels sequencing text
The structure in storehouse merges the reads that all both ends labels repeat and sequence repeats, and single-ended label does not repeat after merging.
Embodiment 2
The construction methods of the microorganism 16S rDNA single molecules level sequencing libraries of the present embodiment it is different from embodiment 1 it
Be in：It is sequenced in step (8) using Miseq, step (9) continues to carry out biology using method same as Example 1
Bioinformatics analysis completes the structure of microorganism 16S rDNA single molecules level sequencing libraries.
Checking test
1, normal person's sample is collected, is tested by this method process described above, passes through Hiseq Xten platforms
Sequencing obtains sequencing result and is analyzed, GC between 51-53%, the reads numbers of bidirectional sequencing R1 and R2 3,000,000 or so,
It is respectively that 600,000 and the tag repetitive rates of 900,000, R1 are slightly above R2 after tag duplicate removals.The comparison rate of two-way 16S sequence libraries is all
Compare high by more than 90%.Because it is the sequencing of Hiseq platforms, the shorter 120bp of length of clean reads or so, if made
It can be surveyed with Miseq platforms logical, then two-way 300bp splices.The cluster quantity in units of category after cluster has more than 600
A, specific data are as shown in table 5.
5 checking test analysis result of table
From data in table 5, what wherein ratio was most is that Prevotella Pseudomonas accounts for the 37% of total amount, behind be successively
Through mucus Eubacterium Blautia (6.7%), Eubacterium Eubacterium (6.5%), Faecalibacterium
(4.0%), fusobacterium Clostridium (3.2%), Coprecoccus Coprococcus (2.6%), Bacteroides
Bacteroides (2.4%), Ruminococcus Ruminococcus (2.1%), remaining Pseudomonas are below 2% due to ratio
Do not list, but remaining Pseudomonas ratio summation can account for 34.6%, as shown in Figure 4.
With pertinent literature or audit report it was found that content of the Prevotella Pseudomonas in enteron aisle is obtained in this research
Result it is low, the ratio of some content rareness floras (eg. Bifidobacteriums) raises instead, thus it is speculated that may be due to conventional amplification
Method is further amplified flora ratio due to taking turns amplification Preference more, and rare flora is since ratio is low, competitive disadvantages
It is more and more lower that ratio instead must not expanded effectively, so unimolecule amplification method can more objectively react microorganism in actual enteron aisle
The truth of composition.
Although specific embodiments of the present invention have been described above, it will be appreciated by those of skill in the art that these
It is merely illustrative of, protection scope of the present invention is defined by the appended claims.Those skilled in the art is not carrying on the back
On the premise of from the principle and substance of the present invention, various changes or modifications can be made to these embodiments, but these are changed
Protection scope of the present invention is each fallen with modification.
Sequence table
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Claims (4)
1. a kind of construction method of microorganism 16S rDNA single molecules level sequencing libraries, it is characterised in that：The described method includes
Following steps：
(1) normal person's fecal sample is gathered, 1mL excrement is deposited in and preserves mixing in liquid, using the fecal microorganism of Tian Mo companies
DNA extraction kit, to specifications the step of extract excrement macro genome DNA；
(2) macro genome DNA after extracting uses the concentration and purity of micro UV spectrophotometer measuring sample；
(3) first step amplification is carried out using the multiplex amplifing reagents of hero company；
Using fecal microorganism DNA as two Xun Huans of template amplification, it is complete and single that two sections of guarantee is connected with connector for first step amplification
Library fragments generate；Sense primer REP1 and anti-sense primer REP2 sequences are as shown in SEQ ID NO.1 and SEQ ID NO.2；
(4) using the product expanded for the first time as template, P1 the and P2 short primers for adding in both ends carry out amplification 30 for second step amplification
Cycle amplified signal；The sequence of sense primer P1 and anti-sense primer P2 are as shown in SEQ ID NO.3 and SEQ ID NO.3；
(5) 10uL products therefroms after the amplification of two steps is taken to be detected into row agarose gel electrophoresis；
(6) products therefrom is purified with the AMPure XP magnetic beads of Beckman＆Coulter companies after the amplification of two steps：To
75uL suspension containing magnetic beads are added in reaction system, mixing is fully blown and beaten, is placed at room temperature for 5min；Supernatant waste is drawn, uses 80%
Ethyl alcohol is washed twice, is placed at room temperature for 5min and is dried magnetic bead, and the low TE elution magnetic beads for adding in 20ul obtain sequencing library after purification；
(7) sequencing library after purification carries out accurate quantification with Qubit 3.0 and Agilent 2100 and clip size is analyzed；
(8) dilution sequencing library is required according to microarray dataset, using result quantitative Qubit 3.0 as standard and according to data volume need
Sample mixing is carried out, is finally sequenced using Illumina HiseqX or Miseq；
(9) bioinformatic analysis is carried out to sequencing result, that is, completes microorganism 16S RDNA single molecules level sequencing libraries
Structure merges the reads that all both ends labels repeat and sequence repeats, and single-ended label does not repeat after merging.
2. the construction method of microorganism 16S rDNA single molecules level sequencing libraries according to claim 1, feature exist
In：The micro ultraviolet specrophotometer model Nanodrop 2000.
3. the construction method of microorganism 16S rDNA single molecules level sequencing libraries according to claim 2, feature exist
In：23 μ L PCR reaction systems of step (3) first step amplification are as shown in table 1：
23 μ L PCR reaction systems of 1 first step of table amplification
First step amplified reaction program is as shown in table 2：
2 first step amplified reaction program of table
4. the construction method of microorganism 16S rDNA single molecules level sequencing libraries according to claim 3, feature exist
In：50 μ L PCR reaction systems of step (4) the second step amplification are as shown in table 3：
50 μ L PCR reaction systems of 3 second step of table amplification
Second step amplified reaction program is as shown in table 4：
4 second step amplified reaction program of table
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| WO2019085546A1 (en) * | 2017-10-31 | 2019-05-09 | 南京格致基因生物科技有限公司 | Method for constructing microbial 16s rdna single-molecule level sequencing library |
| WO2020164015A1 (en) * | 2019-02-13 | 2020-08-20 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing library construction, and library construction method, sequencing method and library construction kit therefor |
| CN113025761A (en) * | 2021-05-27 | 2021-06-25 | 广州赛哲生物科技股份有限公司 | Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification |
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| CN108070643A (en) * | 2017-10-31 | 2018-05-25 | 南京格致基因生物科技有限公司 | The construction method of microorganism 16S rDNA single molecules level sequencing libraries |
| CN107937582A (en) * | 2017-12-29 | 2018-04-20 | 苏州普瑞森基因科技有限公司 | A kind of primer sets and its application for being used to analyze enteric microorganism |
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- 2018-07-13 WO PCT/CN2018/095582 patent/WO2019085546A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106497926A (en) * | 2016-11-03 | 2017-03-15 | 承启医学（深圳）科技有限公司 | A kind of amplicon primer and construction method for building microbial bacterial 16s rDNA variable regions sequencing library |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019085546A1 (en) * | 2017-10-31 | 2019-05-09 | 南京格致基因生物科技有限公司 | Method for constructing microbial 16s rdna single-molecule level sequencing library |
| CN108866220A (en) * | 2018-08-15 | 2018-11-23 | 博奥生物集团有限公司 | A kind of method and detection kit for nasal cavity bacteria detection |
| CN108866220B (en) * | 2018-08-15 | 2022-03-04 | 博奥生物集团有限公司 | Method and detection kit for detecting nasal flora |
| WO2020164015A1 (en) * | 2019-02-13 | 2020-08-20 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing library construction, and library construction method, sequencing method and library construction kit therefor |
| CN113166756A (en) * | 2019-02-13 | 2021-07-23 | 武汉华大医学检验所有限公司 | Fusion primer for third-generation sequencing and library building, library building method, sequencing method and library building kit |
| CN113166756B (en) * | 2019-02-13 | 2023-10-13 | 武汉华大医学检验所有限公司 | Fusion primer for three-generation sequencing library construction, library construction method, sequencing method and library construction kit |
| CN113025761A (en) * | 2021-05-27 | 2021-06-25 | 广州赛哲生物科技股份有限公司 | Multi-amplification matched high-throughput sequencing method and kit for pathogenic microorganism identification |
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