CN104263726A - Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library - Google Patents
Primer applied to amplicon sequencing library construction and method for constructing amplicon sequencing library Download PDFInfo
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Abstract
The invention discloses a primer applied to amplicon sequencing library construction and a method for constructing an amplicon sequencing library. The primer consists of a forward primer and a reverse primer, and the forward primer and the reverse primer comprise a joint sequence, a sequencing primer sequence and a target fragment amplification primer sequence from a sequence from the 5' end to the 3' end. According to the primer provided by the invention, the joint sequence, the sequencing primer sequence and the target fragment amplification primer sequence are integrated on a pair of primers, so that library construction can be finished in one step by utilizing the primer. The sequencing library quality and library construction efficiency are improved, the constructed amplicon sequencing library can perform high-throughput sequencing due to the joint sequence and the sequencing primer sequence used on a conventional sequencing platform by utilizing a common reagent for on-board sequencing, and the phenomena that an extra sequencing primer is provided and the mixed sequencing primers of the PHiX library are changed are avoided.
Description
Technical field
The present invention relates to high-flux sequence field, be applicable to the primer of amplicon sequencing library structure and the construction process of amplicon sequencing library in particular to a kind of.
Background technology
Amplicon order-checking is checked order to the PCR primer of length-specific or the fragment of catching, and mainly comprises that 16S rDNA checks order, 18S rDNA checks order, ITS order-checking and functional gene detection etc.Adopt the sequence of certain hypervariable region of 16S/18S/ITS of Illumina MiSeq s-generation high-flux sequence platform assay, carry out the difference of reaction environment sample in bacterium, fungi, archeobacteria classification between species, important directive function is configured with to the microorganism in the environment such as research ocean, soil, enteron stool; Equally, also by the order-checking to some functional gene fragment, excavate more biological information.
16S rDNA is the DNA sequence dna of coding prokaryotic organism small subunit ribosome rRNA, adopts MiSeq sequenator, checks order, to analyze the diversity of the bacterium in environmental microorganism or archeobacteria further to certain hypervariable region of 16S rDNA.
For completing the order-checking of 16S rDNA, first need to carry out corresponding library construction based on Illumina sequenator, the banking process at present based on Illumina order-checking platform mainly contains two kinds, comprises two steps amplification banking process and step amplification banking process.The first banking process is banking process comparatively early, is that storehouse is built in two step amplifications, namely uses two pairs of primers to carry out two-wheeled PCR and build storehouse.Second method is the step amplification method that Illumina official is recommended, and the method uses relatively more extensive at present, has namely been increased by a step and has built storehouse process.
In above-mentioned two kinds of banking process, although the reagent that two step amplification methods can directly use Illumina Miseq to match checks order, but build in the process of storehouse and need through twice amplification, step is comparatively loaded down with trivial details, and more through the pollution of twice amplification introducing, follow-up accuracy of analysis is reduced.Although a step amplification method comparatively can increase conveniently by One_step PCR and build storehouse, but the method on carrying out machine order-checking time, the reagent that Illumina Miseq can not be used to match checks order, need the sequencing primer of self-defined object fragment and the index sequencing primer of each sample, and, also need to change the corresponding sequencing primer of the λ-DNA library be mixed into (PhiX library) just can check order simultaneously.
Therefore, still need to improve existing banking process, to provide a kind of amplicon sequencing library construction process quick, of reduced contamination, and follow-up upper machine sequencing steps is also more convenient.
Summary of the invention
The present invention aims to provide and is a kind ofly applicable to primer that amplicon sequencing library builds and the construction process of amplicon sequencing library, to solve complex steps existing in amplicon sequencing library construction process in prior art or easily to pollute and the also inconvenient defect of follow-up upper machine order-checking.
To achieve these goals, according to an aspect of the present invention, provide a kind of primer being applicable to amplicon sequencing library and building, this primer is only made up of pair of primers, and upstream primer in pair of primers and downstream primer include joint sequence, sequencing primer sequence and object fragment amplification primer sequence according to from 5 ' end to the 3 ' order of holding.
Further, joint sequence in upstream primer is P5 end connector sequence, joint sequence in downstream primer is P7 end connector sequence, and the sequencing primer sequence in downstream primer also comprises sequence label, the sequence that sequence label is formed by different permutation and combination for 6 ~ 10 bases.
Further, primer is for building the amplicon sequencing library of 16S rDNA v4 district, 18S rDNA, ITS or functional gene.
Further, when primer is for building the amplicon sequencing library in 16S rDNA v4 district, primer comprises: the upstream primer shown in SEQ ID NO.1, and SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, arbitrary downstream primer shown in SEQ ID NO.23 and SEQ ID NO.24.
Further, when primer is for building the amplicon sequencing library of 18S rDNA, ITS or functional gene, the object fragment amplification primer sequence in primer is the conserved sequence on 18S rDNA, ITS or functional gene.
According to a further aspect in the invention, additionally provide a kind of construction process of amplicon sequencing library, this construction process obtains amplicon sequencing library by utilizing above-mentioned pair of primers through a step amplification step.
Further, construction process, after a step amplification step, also comprises: mix amplified production, obtains biased sample; Purified in electrophoresis is carried out to biased sample, obtains amplicon sequencing library.Further, the QIAquick Gel Extraction Kit of Qiagen company is adopted to carry out purifying in Purified in electrophoresis step.
Further, after obtaining amplicon sequencing library, and before carrying out high-flux sequence, also comprise the step of amplicon sequencing library being carried out to quality inspection.
Further, amplicon sequencing library utilizes Illumina Miseq matched reagent to carry out high-flux sequence.
Apply technical scheme of the present invention, by by joint sequence, sequencing primer sequence and these three kinds of sequences of object fragment amplification primer sequence are incorporated into, on the pair of primers of downstream amplification, make to utilize that this primer is enough have been increased library construction by a step, not only improve sequencing library quality and build storehouse efficiency, and the amplicon sequencing library of structure gained is because having joint sequence and sequencing primer sequence that conventional order-checking platform uses, make this sequencing library that the conventionally machine reagent used that checks order can be utilized to carry out high-flux sequence, and without the need to additionally providing sequencing primer and changing the sequencing primer in be mixed into PHiX library.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the primer construction schematic diagram provided in a kind of preferred embodiment of the present invention; And
Fig. 2 show the embodiment of the present invention 1 by one step amplification after obtain the result of amplicon sequencing library through electrophoresis detection.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
As background technology part mentioned, there is complex steps or easily pollute and be inconvenient to the defect operated the computer in the construction process of the amplicon sequencing library existed in the prior art.In order to overcome above-mentioned defect, in a kind of typical embodiment of the present invention, provide a kind of primer being applicable to amplicon sequencing library and building, as shown in Figure 1, this primer is only made up of pair of primers, and upstream primer in this pair of primers and downstream primer include joint sequence, sequencing primer sequence and object fragment amplification primer sequence according to from 5 ' end to the 3 ' order of holding.
Primer provided by the present invention, by by joint sequence, sequencing primer sequence and these three kinds of sequences of object fragment amplification primer sequence are incorporated into, on the pair of primers of downstream amplification, make to utilize that this primer is enough have been increased library construction by a step, not only improve sequencing library quality and build storehouse efficiency, and the amplicon sequencing library of structure gained is because having joint sequence and sequencing primer sequence that conventional order-checking platform uses, make this sequencing library that the conventionally machine reagent used that checks order can be utilized to carry out high-flux sequence, and without the need to additionally providing sequencing primer and changing the sequencing primer in be mixed into PHiX library.
In above-mentioned primer of the present invention, joint sequence in upstream primer is P5 end connector sequence, joint sequence in downstream primer is P7 end connector sequence, and the sequencing primer sequence in downstream primer also comprises sequence label, the sequence that sequence label is formed by different permutation and combination for 6 ~ 10 bases.Adopt above-mentioned joint sequence and sequence label to make to utilize above-mentioned primer of the present invention a step can complete the structure of amplicon sequencing library, and constructed amplicon sequencing library can support the use and carry out high-flux sequence with the check order reagent of platform of Illumina MiSeq.
Above-mentioned primer of the present invention, can be used for the amplicon sequencing library building any object fragment.Both can be to embody the 16S rDNA v4 district of species diversity, the amplicon sequencing library in 18S rDNA, ITS region, also can be the amplicon sequencing library of some functional gene interested to researchist and gene fragment thereof.Different amplicon sequencing libraries, only needs the primer object sheet degree amplimer in above-mentioned primer being replaced to interested gene fragment.Such as, when primer for build 16S rDNA v4 district, 18S rDNA, ITS or functional gene amplicon sequencing library time, the object fragment amplification primer sequence in primer is the conserved sequence on 16S rDNA v4 district, 18S rDNA, ITS or functional gene.
In a kind of preferred embodiment of the present invention, when above-mentioned primer is for building the amplicon sequencing library in 16S rDNA v4 district, primer comprises: the upstream primer shown in SEQ ID NO.1, and SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, arbitrary downstream primer shown in SEQ ID NO.23 and SEQ ID NO.24.
Utilize the primer of the above-mentioned amplicon sequencing library for building 16S rDNA v4 district provided by the present invention, the amplicon sequencing library in 16S rDNA v4 district can not only be made to be completed by a step amplification step, and constructed library can utilize the matched reagent of Illumina MiSeq order-checking platform to carry out high-flux sequence, do not need the change carrying out sequencing primer and PhiX library, the sequencing library that completes making 16S rDNA v4 district amplicon more convenient builds and upper machine sequencing procedure.
In the another kind of typical embodiment of the present invention, additionally provide a kind of construction process of amplicon sequencing library, in this construction process, utilize above-mentioned pair of primers to complete the structure of amplicon sequencing library through a step amplification step.Construction process of the present invention primer used further comprises in the upstream of object fragment amplification primer sequence when subsequent high pass measures sequence for sequencing primer used when the two ends adapter-primer sequence of amplified library and order-checking, thus the primer used by subsequent step is just completed by means of only the step that increases to object sheet degree, but also can support the use by other reagent used with the order-checking platform of routine, not only complete the structure of amplicon sequencing library efficiently, quickly, and be convenient to follow-uply to operate the computer, improve the whole efficiency of amplicon order-checking.
In above-mentioned construction process of the present invention, after a step amplification step, the mass discrepancy (comprising the difference of amplification efficiency and amplification purity etc.) according to the amplicon sequencing library of gained determines whether be necessary to carry out purification step.In the present invention, in order to confirm the size of the amplicon sequencing library of gained further and improve the purity of amplicon sequencing library further, in a kind of preferred embodiment of the present invention, above-mentioned construction process is after a step amplification step, also comprise: amplified production is mixed, obtain biased sample; Purified in electrophoresis is carried out to biased sample, obtains amplicon sequencing library.
Above preferred embodiment mixes the amplicon sequencing library obtaining different fragments after increasing to goal gene fragment, because the sequence label in different sample amplification product is different, and the amplicon library of fragment can adopt general adapter-primer sequence and sequencing primer sequence to carry out the high-flux sequence of follow-up upper machine, therefore product can be mixed; Then carrying out Purified in electrophoresis is the fragment of non-specific amplification got rid of from object fragment by electrophoresis, thus makes amplicon library purity to be checked order higher, and the sequencing data quality obtained is also better.
In above-mentioned Purified in electrophoresis step of the present invention, the present invention preferably adopts the QIAquick Gel Extraction Kit of Qiagen company to carry out purifying, and this kits effect compares other similar purification kits, and purity is higher.
The same with other high-throughput sequencing library construction steps at above-mentioned construction process of the present invention, after obtaining amplicon sequencing library, and before carrying out high-flux sequence, also comprise the step of amplicon sequencing library being carried out to quality inspection.Whether the clip size in the library constructed by being detected by quality inspection is suitable, and whether concentration reaches confidential asking, and determines according to the size of concentration the concentration that upper machine uses.
In above-mentioned construction process of the present invention, the amplicon sequencing library obtained, owing to having the follow-up order-checking of Illumina MiSeq order-checking platform amplified library primer used and sequencing primer, thus can utilize Illumina Miseq matched reagent to carry out high-flux sequence.
Below in conjunction with specific embodiments beneficial effect of the present invention is described.
Be applied to the construction process of the 16S rDNA v4 district amplicon sequencing library that Illumina MiSeq checks order, comprise following experimental procedure:
(1) 16S rDNA DNA concentration per sample, uses sterilized water by diluted sample to 1ng/ μ l, pollutes if there is a large amount of RNA in sample, dilutes after needing first to use RNase to digest again.
(2) pcr amplification.Synthetic primer shown in use table 1 increases, wherein SEQ ID NO.1 is the upstream primer of amplification, wherein, 1st ~ 21 bit bases are upstream joints sequence, 22nd ~ 58 bit bases are upstream sequencing primer sequence, and 59th ~ 77 bit bases are upstream object fragment amplification primer sequence.
SEQ ID NO.2 to SEQ ID NO.25 is downstream primer, wherein, 1st ~ 24 bit bases are downstream tap sequence, and 25th ~ 30 bit bases are sequence label, 31st ~ 64 bit bases are downstream sequencing primer sequence, and 65th ~ 84 bit bases are downstream object fragment amplification primer sequence.
Above-mentioned arbitrary downstream primer and the SEQ ID NO.1 upstream primer primer pair 16S rDNA v4 district that partners increases.
Table 1:
| Numbering | Primer sequence |
| SEQ?ID?NO.1 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGCCAGCMGCCGCGGTAA |
| SEQ?ID?NO.2 | CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.3 | CAAGCAGAAGACGGCATACGAGATTCATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.4 | CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.5 | CAAGCAGAAGACGGCATACGAGATTGCTGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.6 | CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.7 | CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.8 | CAAGCAGAAGACGGCATACGAGATGATCGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.9 | CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.10 | CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.11 | CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.12 | CAAGCAGAAGACGGCATACGAGATCTTGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.13 | CAAGCAGAAGACGGCATACGAGATAGTCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.14 | CAAGCAGAAGACGGCATACGAGATAGTTCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.15 | CAAGCAGAAGACGGCATACGAGATATGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.16 | CAAGCAGAAGACGGCATACGAGATCCGTCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.17 | CAAGCAGAAGACGGCATACGAGATGTAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.18 | CAAGCAGAAGACGGCATACGAGATGTCCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.19 | CAAGCAGAAGACGGCATACGAGATGTGAAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.20 | CAAGCAGAAGACGGCATACGAGATGTGGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.21 | CAAGCAGAAGACGGCATACGAGATGTTTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.22 | CAAGCAGAAGACGGCATACGAGATCGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.23 | CAAGCAGAAGACGGCATACGAGATGAGTGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.24 | CAAGCAGAAGACGGCATACGAGATGGTAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
| SEQ?ID?NO.25 | CAAGCAGAAGACGGCATACGAGATACTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT |
Reaction system is as follows:
16S V4 region PCR reaction system (30 μ l):
Response procedures is as follows:
(3) electrophoresis detection PCR primer.3 μ l are got to each sample and carries out product detection, detect as shown in Figure 2.
In Fig. 2, from left to right respectively represent SEQ ID NO.1 and SEQ ID NO.2 to SEQ ID NO.25 increase obtain product.Wherein, the band (marker) of DNA molecular size is indicated to be respectively from the bottom up: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1500bp.The size in normal 16S rDNA V4 district is about 300bp, and upstream and downstream primer is always about 160bp, and therefore, the overall length obtained that increases should be about 450bp.
PCR primer size shown in Fig. 2 is about about 400-450bp, visible, and the size utilizing above-mentioned primer of the present invention to draw together the amplicon sequencing library in the 16S rDNA v4 district that amplification obtains meets expection.
(4) PCR primer is mixed sample and is carried out Purified in electrophoresis, completes the structure of object frag-ment libraries.Quality is carried out etc. to PCR primer and mixes sample, carry out electrophoresis, cut object fragment, use the QIAquick Gel Extraction Kit of Qiagen company to reclaim, after recovery, can library construction be completed.
(5) utilize Agilent 2100 and the amplicon sequencing library of realtime-PCR to gained carries out object clip size, library concentration carries out quality inspection, Miseq after qualified, can be used to carry out upper machine order-checking.
(6) information analysis is carried out to the data obtained.
From above description, can find out, the above-mentioned primer that the above embodiments of the present invention provide according to thought of the present invention, completed rapidly by a step amplification and build storehouse process, and the reagent that constructed library can directly use Illumina Miseq to match carries out upper machine order-checking, do not need to carry out the synthesis of sequencing primer and the change in PhiX library, make 16S rDNA v4 district and more convenient the completing of other amplicons order-checking build storehouse and upper machine sequencing procedure.
These are only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the primer being applicable to amplicon sequencing library and building, it is characterized in that, described primer is made up of a upstream primer and a downstream primer, and described upstream primer and downstream primer include joint sequence, sequencing primer sequence and object fragment amplification primer sequence according to from 5 ' end to the 3 ' order of holding.
2. primer according to claim 1, it is characterized in that, joint sequence in described upstream primer is P5 end connector sequence, joint sequence in described downstream primer is P7 end connector sequence, sequencing primer sequence in described downstream primer also comprises sequence label, the sequence that described sequence label is formed by different permutation and combination for 6 ~ 10 bases.
3. primer according to claim 2, is characterized in that, described primer is for building the amplicon sequencing library of 16S rDNA v4 district, 18S rDNA, ITS or functional gene.
4. primer according to claim 3, is characterized in that, when described primer is for building the amplicon sequencing library in 16S rDNA v4 district, described primer comprises:
Upstream primer shown in SEQ ID NO.1; And
SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, arbitrary downstream primer shown in SEQ ID NO.23 and SEQ ID NO.24.
5. primer according to claim 3, it is characterized in that, when described primer is for building the amplicon sequencing library of 18S rDNA, ITS or functional gene, the described object fragment amplification primer sequence in described primer is the conserved sequence on 18S rDNA, ITS or functional gene.
6. a construction process for amplicon sequencing library, is characterized in that, described construction process obtains described amplicon sequencing library by utilizing the described primer any one of claim 1 to 5 through a step amplification step.
7. construction process according to claim 6, is characterized in that, described construction process is after a described step amplification step, further comprising the steps of:
Amplified production is mixed, obtains biased sample;
Purified in electrophoresis is carried out to described biased sample, obtains described amplicon sequencing library.
8. construction process according to claim 7, is characterized in that, adopts the QIAquick Gel Extraction Kit of Qiagen company to carry out purifying in described Purified in electrophoresis step.
9. construction process according to claim 7, is characterized in that, after obtaining described amplicon sequencing library, and before carrying out high-flux sequence, also comprises the step of described amplicon sequencing library being carried out to quality inspection.
10. construction process according to claim 6, is characterized in that, described amplicon sequencing library utilizes Illumina Miseq matched reagent to carry out high-flux sequence.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008093098A2 (en) * | 2007-02-02 | 2008-08-07 | Illumina Cambridge Limited | Methods for indexing samples and sequencing multiple nucleotide templates |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
| CN103045726A (en) * | 2012-11-20 | 2013-04-17 | 南方科技大学 | Method and apparatus for gene sequencing of multiple mixed DNA or RNA sequences |
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN103981259A (en) * | 2014-05-06 | 2014-08-13 | 山西晋城无烟煤矿业集团有限责任公司 | Analysis method for diversity of microbes and abundance of species in coal seam water |
-
2014
- 2014-09-25 CN CN201410499918.5A patent/CN104263726A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008093098A2 (en) * | 2007-02-02 | 2008-08-07 | Illumina Cambridge Limited | Methods for indexing samples and sequencing multiple nucleotide templates |
| CN102560688A (en) * | 2010-12-15 | 2012-07-11 | 深圳华大基因科技有限公司 | Novel library construction method based on illumina sequencing platform |
| CN103060924A (en) * | 2011-10-18 | 2013-04-24 | 深圳华大基因科技有限公司 | Library preparation method of trace nucleic acid sample and application thereof |
| CN103045726A (en) * | 2012-11-20 | 2013-04-17 | 南方科技大学 | Method and apparatus for gene sequencing of multiple mixed DNA or RNA sequences |
| CN103981259A (en) * | 2014-05-06 | 2014-08-13 | 山西晋城无烟煤矿业集团有限责任公司 | Analysis method for diversity of microbes and abundance of species in coal seam water |
Non-Patent Citations (3)
| Title |
|---|
| J.GREGORY CAPORASO ET AL.: "Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample", 《PNAS》 * |
| JAMES J.KOZICH ET AL.: "Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the Miseq Illumine Sequencing Platform", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| 邓冠华等: "高通量16SrRNA标签测序法比较人与不同动物肠道微生物组多样性", 《生态科学》 * |
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